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Scientific evidence and logic behind the claim that the Wuhan coronavirus is man-made

3/15/2020

2265 Comments

 
​There has been much controversy regarding the origin of the Wuhan coronavirus. It appears that both possibilities, naturally occurring or man-made, are legitimate enough to be debated fully. However, although voices on social media are equally strong from both sides, when it comes to written pieces, there is a predominance of scientific literature and other forms of writing that were produced to disapprove the “conspiracy theory”. In contrast, not nearly as much literature or other forms of substantial writing have been put out to describe or argue for the other possibility – this virus is man-made.  My goal here is to use scientific evidence and logical thinking to evaluate, and legitimate, the possibility that the Wuhan coronavirus (2019-nCoV, SARS2-CoV, etc.) is of non-natural origin. 
 
Importantly, I will base my reasoning on solid, credible evidence; I will exclude any unqualified evidence that may have been thrown in by the Chinese Communist Party (CCP) with an intention to disturb the “investigation” and thereby cover up the truth. My role or perspective here can be considered as a combination of a scientific reviewer, a detective, and a judge on a criminal trial. 
 
Why are people suspicious of the origin of the Wuhan coronavirus
 
This has a lot to do with how the sequence of this virus (in other words, its genome) compares with those of related coronaviruses. 
 
When comparing sequences, one can compare either gene sequences or protein sequences. For viruses, however, this makes almost no difference as the whole genome of a virus is practically translated into proteins (in fact, a virus typically produces a single polyprotein by translating its entire genome and then cuts this long polyprotein at specific places to produce a set of particular proteins for specific use). Here, we will compare different viruses only on their protein sequences. 
 
By doing such a comparison, one can see that the Wuhan coronavirus is about 86% identical to the SARS coronavirus, which caused a pandemic back in 2003. This level of sequence identity basically says that the Wuhan coronavirus could not have come from SARS, something the field agrees unanimously.  
 
At the same time, the Wuhan coronavirus is STRANGELY similar to two bat coronaviruses, ZC45 and ZXC21. Overall, the sequence of either of the two bat coronaviruses is 95% identical to the Wuhan coronavirus. In fact, for most part of the genome, such level of identity is maintained or even surpassed. The E protein, in particular, is 100% identical. The nucleocapsid is 94% identical. The membrane protein is 98.6% identical. The S2 portion (2nd half) of the spike protein is 95% identical. However, when it comes to the S1 portion (1st half) of the spike protein, the sequence identity suddenly drops to 69%. This pattern of sequence conservation, between either of the closely related bat coronaviruses and the Wuhan coronavirus, is extremely rare and strange!
 
This is extremely rare because natural evolution typically takes place when changes (mutations) occur randomly across the whole genome. You would then expect the rate of mutation being more or less the same for all parts of the genome. 
 
Could other forms of evolution lead to such a strange pattern of sequence identity? Yes, there is one evolutionary event that could lead to drastic changes in only one part of the genome. It is what is called “recombination”. We would defer to the next section to explain why recombination is also practically impossible in this case.
 
For now, let’s fix our eyes on the part that is seeing this sudden drop of sequence identity, the S1 portion of the spike protein. 
Picture
​Figure 1. Coronavirus particle with spike proteins (red) decorating its surface. Image from the CDC website (not a photo of a real virus, but a model generated based on scientific knowledge).
 
Spike proteins are the protrusions that you see on the outside of the virus particle (Figure 1). They are literally responsible for the name “corona” as they make the virus look like a “crown”. However, spike proteins are located here for reasons beyond decoration. They are actually the “key” that coronaviruses use to open the “lock” so that viruses can enter our (host) cells. 
 
Figure 2 shows the structure of the spike protein of the SARS virus (such structure images are as real as photos of actual people). Given the sequence similarity/conservation here, the spike protein of the Wuhan coronavirus would look pretty much the same, which is indeed confirmed by a recent publication (1). 
Picture
​Figure 2. Structure of the SARS spike protein and how it binds to human ACE2 receptor. Pictures generated using the published structure (PDB ID: 6acj) (2). A) Three spike proteins, each consisting of a S1 half and a S2 half, form a trimer. B) The S2 halves (shades of blue) are responsible for trimer formation, while the S1 portion (shades of red) is important for binding human receptor ACE2 (dark gray). C) Details of the binding between S1 and human ACE2. The part of S1 that is important and sufficient for binding are colored in orange, with most crucial amino acid sidechains shown as sticks. This orange piece is presumably what’s “taken out of” SARS spike and “inserted” into a bat coronavirus spike protein, thereby creating a novel human-infecting coronavirus.
 
Three spike proteins have to come together to function properly as the “key”. This three-protein assembly is what they call a “trimer”. To form this trimer, you would need the blue portion of the spike protein, which is referred to as S2 of spike. This S2 part can be regarded as the part of the “key” that you hold with your fingers; it does not actually go into the lock. However, for this “key” to work, S2 has to be there and has to preserve the ability of forming trimers. 
 
The other half of spike, the red portion or what is referred to as S1, is responsible for binding the host receptor. S1 can be considered as the portion of the “key” that literally enters the “lock”. It has to fit precisely to the delicate shape of the “lock” (host receptor) so that the “door opening” action can be accomplished. Whether or not a particular “lock” can be opened by a specific “key” is decided exclusively by this S1 part of spike. In other words, S1 of a coronavirus dictates which host(s) or cells the virus can infect.
 
Now you may be able to appreciate what I call extremely strange. While everything else of the Wuhan coronavirus remains almost identical to the two bat coronaviruses, the S1 portion, which dictates which host a coronavirus targets, has changed significantly from the two bat coronaviruses to the Wuhan coronavirus.
 
Let’s zoom in further (Figure 2C) and look at the exact part on S1 that dictates whether or not S1 binds a host receptor (in this case, the human ACE2 protein). This most critical part of S1 is a relatively small stretch of amino acids, labeled in orange in Figure 2C with important residues shown as sticks. This part includes everything needed for interacting with the human ACE2 receptor. You will see below how this segment, known to be unique to the SARS spike and sufficient for its interaction with human ACE2, is practically “copied” over by the Wuhan coronavirus.
Picture
​Figure 3. Sequence alignment of the spike proteins from relevant coronaviruses, including viruses isolated from current pandemic (Wuhan-Hu-1, 2019-nCoV_USA-AZ1), closely related bat coronaviruses (Bat_CoV_ZC45, Bat_CoV_ZXC21), and SARS coronaviruses (SARS_GZ02, SARS). Region marked by orange lines is the segment important for interaction with human receptor ACE2. Crucial residues for interaction are additionally highlighted by a red stick on top. Region marked by green lines is a furin-cleavage site that exists only in the Wuhan coronaviruses but not in any other beta coronaviruses. Alignment was done using the MultAlin webserver (http://multalin.toulouse.inra.fr/multalin/).
 
Figure 3 is the sequence alignment of the spike proteins from six coronaviruses. Two are viruses isolated from current pandemic (Wuhan-Hu-1, 2019-nCoV_USA-AZ1); two are closely related bat coronaviruses (Bat_CoV_ZC45, Bat_CoV_ZXC21); two are SARS coronaviruses (SARS_GZ02, SARS). By glancing through this figure, you can easily tell that the second half of spike (690 and beyond), namely S2, look pretty much the same for all six viruses. The difference is in the front half (1-~690), or the S1 portion. Now if you look at the top four sequences — the two Wuhan coronaviruses and two bat coronaviruses, you can see that they are largely the same across the S1 half of spike. Only a couple of places are different. However, the details of these differences and the way the human and the bat viruses differ from each other here in S1, in my and many other people’s eyes, practically spell out the origin of the Wuhan coronavirus – it is created by people, not by nature. 
 
First important difference is what is highlighted in between two orange lines in Figure 3. Clearly, this part of the Wuhan coronavirus spike differs significantly from those of the bat virus spikes, despite the overall high identity between them. Intriguingly, this same segment of the Wuhan coronavirus resembles, on a great deal, the corresponding piece on the SARS spike protein. Indeed, this is precisely the region highlighted in Figure 2C in orange. As we have pointed out earlier, this segment contains everything needed for human ACE2 interaction. Here, it seems that this critical piece was “copied” from the SARS spike protein and then “pasted” into a bat coronavirus. 
 
There are of course differences between these two, which may make it seem unlikely a direct “copy and paste”. However, careful examination shows that all residues essential for binding (orange sticks in Figure 2C and residues highlighted by red short lines in figure 3) are either precisely preserved or substituted with residues of similar properties. At the same time, differences lie mostly at residues non-essential for binding ACE2. Judging from this observation, one can safely envision that not only Wuhan coronavirus spike will bind ACE2 but also it will bind ACE2 exactly the same way that SARS spike does (Figure 2BC).
 
For the two bat coronaviruses here, given how they lack many of the key residues (what is marked by red sticks in Figure 3) for binding human ACE2, it is easy to predict that these two bat viruses would not be able to infect human.
 
The Wuhan coronavirus, while being almost identical to their bat relatives (ZC45 and ZXC21) everywhere else, has somehow “inherited” the critical, short piece from SARS spike to replace the incompetent piece in the bat coronavirus spike. As a result of this miraculous “replacement” in S1 — all key residues preserved and many non-essential residues changed, the Wuhan coronavirus has practically “acquired” the ability to infect humans, something its closest bat relatives do not have.
 
Could natural evolution achieve something this precise and, at the same time, this deceptive???
 
If you have not been “awed” enough, let’s move on to appreciate magic trick #2. Please look at the region marked by two green lines in Figure 3. Here only the Wuhan coronaviruses contain an additional piece, SPRRA. Importantly, this added piece allows the spike protein to be readily cleaved by a host protease enzyme – furin, a desirable property known to produce more infectious viruses in the case of influenza. Note that no beta coronaviruses in the same lineage (lineage B), except this new Wuhan coronavirus, contain such a furin-cleavage site. 
 
Further explanation on why these changes could not have come from nature
 
We have briefly explained why random mutations could not result in the weird pattern of sequence identity between the Wuhan coronavirus and related bat coronaviruses, ZC45 or ZXC21. Let’s dig a little deeper here. Although the spike proteins of different coronaviruses are more likely to differ, greater discrepancy in S1 may only be expected if two viruses have been long separated during evolution and have adapted, through random mutation, to their respective hosts for a long, long time. In that scenario, the overall sequence identity would be low as well. In the present case, however, the sequence identity between either of the bat coronavirus and the Wuhan coronavirus is over 95%, suggesting these two viral lineages must have diverged from each other fairly recently. Therefore, a sequence identity of 69% for the S1 portion of spike protein is simply insane. The S1 of Wuhan coronavirus could not have originated from the S1 of a bat coronavirus, a recent common ancestor that the Wuhan virus shares with ZC45 and ZXC21, through random mutations.
 
Now let me explain why recombination also could not be responsible for the observed pattern.
 
What happens in a recombination event is that one segment of a gene can be “replaced” by a similar segment from another gene. In evolution, recombination events happen much less frequently than random mutations. When recombination happens, however, it often brings abrupt changes to certain areas of the genome. 
 
If naturally-occurring recombination event(s) lead to the creation of the Wuhan coronavirus, how would it transpire? First, it would have to take place when an ancestor bat coronavirus, something very similar to ZC45 or ZXC21, co-existed with another coronavirus in the same cell of the same animal. Under extremely rare circumstances, recombination may occur, where a random piece in the ancestor’s genome is replaced by a similar but different piece from the other coronavirus. Importantly, to go from such ancestor to the Wuhan coronavirus, one combination event is not enough. What has to happen is that recombination has to take place twice during the evolution of the Wuhan coronavirus. In one occasion, the ancestor bat coronavirus would have to acquire, through recombination with a SARS-like coronavirus, the precise short segment of S1 that is responsible for human ACE2 interaction (region highlighted in orange in both Figure 2 and Figure 3). In another occasion, the “improved” bat coronavirus would further swap in a furin-cleavage site through recombination with yet another coronavirus that carries a furin-cleavage site between its S1 and S2 of spike. 
 
Also, again, given the overall high sequence identity (95%) between the bat coronaviruses and the Wuhan coronavirus, it is reasonable to believe that these two diverged from each other fairly recently. Therefore, both recombination events must have taken place fairly recently as well.
 
Now, we know that SARS crossing over to infect human is a very rare event. To have another SARS-like sequence exist in nature so that the ancestor bat coronavirus can do recombination with is a very unlike event. Not to mention that this SARS-like virus must have a spike that binds ACE2 the same way as SARS and yet the piece of S1 that is most critical for binding ACE2 would differ with that of SARS spike only at non-essential sites. On top of that, furin-cleavge site has not been observed in any beta coronaviruses in the same lineage so far. Although similar furin-cleavage sites have been observed in other coronaviruses, none of them contains the same exact sequence. Therefore, the chance that the furin-cleavage site in the Wuhan coronavirus was obtained through recombination with another furin-cleavage-site-containing coronavirus is very low.
 
Now, what are chances for both of these next-to-impossible recombination events to take place? My answer is NO CHANCE. This Wuhan coronavirus cannot be coming from nature.
 
Why some literature has to be excluded in the analysis
 
Someone who has been following the recent literature on this topic would point out that the above analysis failed to take into account some crucial evidence. Such evidence, coincidentally, supports a natural origin of the Wuhan coronavirus. Then how dare I leave it out in my analysis?
 
The short answer: that “evidence” was very likely fabricated. 
 
Please allow me to switch my mode now, from a scientist to a detective or a judge. If we consider this matter as a crime under investigation, then we have so far one big suspect, Dr. Zhengli Shi from the Wuhan Institute of Virology and the biosafety level 4 (P4) lab for virology research. As the top coronavirus expert in China, since the beginning of the outbreak, Zhengli Shi has been singled out as THE suspect who may have created this virus, which somehow leaked out of the P4 lab. Intriguingly, Shi published an interesting paper in Nature a couple of weeks ago (3). There she compared the freshly obtained sequence of the Wuhan coronavirus with those of other beta coronaviruses, which allowed her to delineate an evolutionary path of this new virus. All of a sudden, out of nowhere, she reported a bat coronavirus, RaTG13, which shares high sequence identity with the Wuhan coronavirus. Strikingly, between RaTG13 and the Wuhan virus, the mutational rate is low (or sequence identity is high, 98.5%) for all parts of the genome, including the spike protein. If we have questioned the origin of the Wuhan coronavirus because of the weird pattern of sequence conservation between the Wuhan coronavirus and the two bat coronaviruses, ZC45 and ZXC21, then RaTG13 does not show any concern in that regard. Here, the spike protein is just as conserved as other proteins. From the first look, it seems that RaTG13 belongs to the same small lineage as the Wuhan coronavirus and that the two must share a very recent common ancestor. Such finding strongly suggests a natural origin of the Wuhan coronavirus. This paper reporting the RaTG13 coronavirus (3) is the evidence that I “failed” to take into account in the earlier analysis.
 
According to credible sources, Shi has admitted to several individuals in the field that she does not have a physical copy of this RaTG13 virus. Her lab allegedly collected some bat feces about 7 years ago and analyzed these samples for possible presence of coronaviruses based on genetic evidence. To put it into plainer words, she has no physical proof for the existence of this RaTG13 virus. She only has its sequence information, which is nothing but a string of letters alternating between A, T, G, and C. 
 
Can the sequence be fabricated? It cannot be any easier. It takes a person less than a day to TYPE such a sequence (less than 30,000 letters) in a word file. And it would be a thousand times easier if you already have a template that is about 98% identical to the one you are trying to create. Once the typing is finished, one can upload the sequence onto the public database, without being really questioned for its authenticity or correctness. Once uploaded and released, such sequence data becomes public and can be used legitimately in scientific analysis and publications.
 
Then, can this RaTG13 sequence be used as evidence in judging the matter? Well, remember, a central part of the matter is whether or not this Wuhan coronavirus is engineered or created by ZHENGLI SHI. It is Shi, not anybody else, who is the biggest suspect of this possible crime that is grander than any other crime ever committed in human history. Given the circumstances, if the evidence she raised to prove herself innocent is nothing but a bunch of letters recently typed in a word file, should anyone treat it as valid evidence?
 
Unfortunately, in several recent reports, scientists indeed based their analyses on this RaTG13 sequence and thereby reached conclusions such as the Wuhan coronavirus is a result of natural evolution. I hope you now agree that these conclusions could not be trusted because they are based on data that is most likely FABRICATED BY SHI. 
 
Now, let us think about this from the other direction. The RaTG13 virus has a highly alarming sequence. Glancing at the sequence of the spike protein of this virus, any expert would immediately realize that this virus resembles SARS in its potential in binding human ACE2 and therefore may very likely be able to infect humans. Shi herself is such an expert. According to Shi, her lab studies bat coronaviruses so that they could someday predict novel coronavirus outbreaks and better prepare the public for such events. If her statement is true, then how could she possibly overlook this extremely interesting finding of RaTG13, something that clearly has the potential to infect humans? If this RaTG13 was discovered SEVEN years ago, why did Shi not publish this astonishing finding earlier? Why did she decide to publish such a sequence only when the current outbreak took place and people started questioning the origin of the Wuhan coronavirus? 
 
None of these make sense. All in all, these facts should make people question Zhengli Shi even more in terms of her possible involvement in the matter; she either was directly involved in the creation of this virus/bioweapon, or helped cover it up, or both. Of course, these facts also speak the necessity to exclude this RaTG13 sequence from any scientific analysis.

The same goes to the notion that pangolins might be the intermediate host responsible for transmitting the virus from bats to humans. In early Feb, a press conference was held, where three researchers from South China Agriculture University (SCAU) claimed that their recent findings point to Pangolin as a possible intermediate host. First of all, the timing of the press conference is interesting – just when people are saying that bat viruses cannot directly infect humans and an intermediate host must exist (where the viral spike protein would “learn”/adapt to bind an ACE2 similar to human ACE2). When something MUST exist to favor the side of the CCP, this something always miraculously appears, just like Shi’s RaTG13. This time, it is the Pangolin coronavirus. Before even publishing the paper, these researchers showed their evidence – sequence of the receptor binding domain of the pangolin coronavirus that looks almost identical to the Wuhan coronavirus. Again, no live virus exists here, just the sequence (not even released back then). It is the same deal as the RaTG13 case; a person can literally type this sequence out in a few minutes. Therefore, for similar reasons, one has to be extremely cautious and alert that this may again be fabricated by the CCP with an intention to help cover up the truth. 
 
Fortunately, the field seems to have excused pangolins. The pangolin coronavirus sequence that was finally released by the SCAU group and another research group in Hong Kong fell short in convincing people about pangolin’s role as an intermediate host (4, 5). This is in part because, according to its sequence, the pangolin coronavirus also does not have the furin-cleavage site. 
 
Nonetheless, like RaTG13, these recent papers claiming the role of pangolin as an intermediate host should be discarded (4, 5). In fact, very recently, these SCAU researchers admitted to the press that, upon further analysis of the complete sequence of the Pangolin coronavirus, they also do not believe Pangolin is a possible intermediate host of the Wuhan coronavirus. 
 
Some scientific literature that deserves the spotlight
 
We have just laid out the reasons why certain “scientific evidence” should be excluded. Now let us switch over to see why some other scientific evidence deserves our complete attention. 
 
First, the two bat coronaviruses, ZC45 and ZXC21, that are STRANGELY CLOSE to the Wuhan coronavirus were collected by a military research lab of the CCP. They published the finding and the sequences of these two viruses back in 2018 (6). I want to emphasize two facts here: 1) if the Wuhan coronavirus was man-made, then it must have been created using ZC45 or ZXC21 as a template; 2) nobody in this world has these bat coronaviruses, except for the CCP as evidenced by this publication.
 
Second, Zhengli Shi co-authored a paper in Nature Medicine back in 2015 (7), where she collaborated with Ralph Baric at the University of North Carolina to show that replacing the spike protein of a non-human-infecting coronavirus with a spike protein capable of binding human ACE2 led to a novel coronavirus that gained the ability to infect humans. Now, what is happening in the Wuhan coronavirus essentially follows the same scheme; the changes, although minimal, are sufficient to turn the bat coronavirus into a virus that can infect humans. The only difference is that, when changes are this subtle, tracing the origin of the virus becomes much difficult.
 
Third, a 2006 publication showed that inserting a furin-cleavage site in the junction region of S1 and S2 of spike of the SARS coronavirus led to much enhanced membrane fusion activity of the virus (8). Although viral infectivity enhancement was not observed in their study using pseudo viruses, presence of such furin-cleavage sites is known to be associated with high pathogenicity in influenza virus infections. Miraculously, this is precisely what is observed in the Wuhan coronavirus (region marked by two green lines in Figure 3). Furthermore, influenza viruses containing such furin-cleavage sites often infect a greater variety of cells and are therefore more likely to target organs in addition to the lung. Now you should recall multiple recent reports describing that the Wuhan coronavirus infects multiple organs, including lung, heart, blood vein, liver, central nerve system, etc. 
 
Simple and yet clear logic on how this Wuhan coronavirus may be made by the CCP
 
If you put the pieces together, you should be able to appreciate how easily this virus can be created by the CCP. Obviously, the starting virus template used here, either ZC45 or ZXC21, is owned only by the CCP (6). What they would do then was to modify things such that this bat coronavirus, non-infectious to humans, could be converted to a novel coronavirus that infects humans with high efficiency. They did so by following two published concepts (7, 8): 1) they converted the crucial spike protein to something that follows the scheme of the SARS spike protein so that the virus can target human ACE2; 2) they inserted a furin-cleavage site in between S1 and S2 of spike, which may make the virus more pathogenic. These two concepts are the only ones out there to get such a job done. Yet, miraculously, they are being followed precisely here. If it were mother nature who has created this virus, then mother nature must have studied recent scientific literatures very carefully and followed these key findings faithfully in her work (2, 6-8).
 
Also, let’s go back a little and think why they spend so much time fetching coronaviruses all over the place. Is it really like what they claimed – to understand the potentials of coronaviruses and therefore better predict future emerging coronaviruses? Why didn’t they put as much effort on vaccine research or drug discovery targeting a function/protein conserved in most coronaviruses then? The latter is not only more beneficial to the public but also way easier than predicting emerging viruses. 
 
Another possibility, of course, is that they are collecting these things to create coronavirus-based bioweapons. What is the truth? You can make up your own mind.
 
As of me, I am fully convinced that this is a bioweapon made by the CCP.
 
Given all the facts and the logic connecting them as laid out above, it is completely reasonable to argue that, unless the CCP can prove otherwise, the world has all the right to believe that the Wuhan coronavirus was made by the CCP.
 
 
Reference
 
1.         Daniel Wrapp NW, Kizzmekia S. Corbett, Jory A. Goldsmith, Ching-Lin Hsieh, Olubukola Abiona, Barney S. Graham, Jason S. McLellan. Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation. Science. 2020.
2.         Song W, Gui M, Wang X, Xiang Y. Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog. 2018;14(8):e1007236.
3.         Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020.
4.         Kangpeng Xiao JZ, Yaoyu Feng, Niu Zhou, Xu Zhang, Jie-Jian Zou, Na Li, Yaqiong Guo, Xiaobing Li, Xuejuan Shen, Zhipeng Zhang, Fanfan Shu, Wanyi Huang, Yu Li, Ziding Zhang, Rui-Ai Chen, Ya-Jiang Wu, Shi-Ming Peng, Mian Huang, Wei-Jun Xie, Qin-Hui Cai, Fang-Hui Hou, Yahong Liu, Wu Chen, Lihua Xiao, Yongyi Shen. Isolation and Characterization of 2019-nCoV-like Coronavirus from Malayan Pangolins. bioRxiv. 2020.
5.         Tommy Tsan-Yuk Lam MH-HS, Hua-Chen Zhu, Yi-Gang Tong, Xue-Bing Ni, Yun-Shi Liao, Wei Wei, William Yiu-Man Cheung, Wen-Juan Li, Lian-Feng Li, Gabriel M Leung, Edward C. Holmes, Yan-Ling Hu, Yi Guan. Identification of 2019-nCoV related coronaviruses in Malayan pangolins in southern China. bioRxiv. 2020.
6.         Hu D, Zhu C, Ai L, He T, Wang Y, Ye F, et al. Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats. Emerg Microbes Infect. 2018;7(1):154.
7.         Menachery VD, Yount BL, Jr., Debbink K, Agnihothram S, Gralinski LE, Plante JA, et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med. 2015;21(12):1508-13.
8.         Follis KE, York J, Nunberg JH. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry. Virology. 2006;350(2):358-69.

2265 Comments
Polo
3/27/2020 01:50:44 am

If true..then dna sequencing can help gene editing to get cure...let us not worry as detective has found answer..all go back home and use this evidence to get cure..bye all and dont worry..thank you detective/judge/biologitst/darwin

Reply
Nerd has power
3/29/2020 07:42:51 pm

Well, it is true that this has nothing to do with how to cure someone with coronavirus infections. But keeping a blind eye and not taking actions may have consequences. If you know someone in your neighborhood is a serial killer, would you feel comfortable giving him freedom and keeping him active? If CCP got away this time, you bet they will throw another one at you next time around. Things can be much worse than coronaviruses, and CCP definitely has more than one thing in their pocket.

Reply
Alberto Rubio Casillas
5/22/2020 08:08:05 am

Dear NERD HAS POWER. I have important information to share with you.
Please contact me to send it.
Best regards

Henri
5/23/2020 04:57:38 pm

Why don't you submit a letter to Cell detailing your analysis? The Editor should send the letter onto Zhengli Shi, allow a reply and publish both together in a future issue.

Adrian Gibbs link
5/23/2020 06:03:26 pm

I see that you are concerned that the sequences being discussed are merely sequences obtained by analysing materials(eg bat poo), and no live virions or viral nucleic acid are available. In fact much of virology is being done in this way nowadays - the sequences are called metagenomes, they are assembled in computers from short sequences read from various materials - even water.

Kumar
7/6/2020 03:21:00 pm

WHO Visit to China to inquire into Virus Origin (gleaned from reading detailed Research Papers by an anonymous 'Nerd has Power')

Kumar

Questions to WHO citing weblinks to your two Articles.

1 Abrupt variance in S-1 (Key that binds with Human Cells) in nCoV w/ Bats' ZC45 and ZXC21, while other portions match 94-100%

2 Similarity of S-1 in nCoV w/ SARS, which allow Bat Viruses to infect Human

3 Appearance of small String allowing Cleavage of S-1 by Furin -not in other Bat Coronas

4 VLow Possibilty of Double Recombination Event in Nature

5 Zhangli Shi of Wuhan Inst Vir published fictitious RaTG13 seq in Jan 2020 of a Virus collected in 2013 w/o Virus Physical Copy to divert attention from Gain Function

6 To explain pangolin as int host a fictitious binder seq w/o virus physical copy published by S. China Agri Univ in Feb 2020

7 Zhangli paper in 2015 how to replace spike protein to make virus to infect Human

8 2006 paper showed cleavage at S1-S2 can make 'em more pathogenic

9 Is Mother Nature reading Nature/ Virology Jls and recombining Viruses?

David Rivard link
7/7/2020 11:23:53 am

Good comments, but this is the moment in history that such research have been speculating about..in situ! Keeping the world's best minds in synch, and in science in a no blame environment is critical. Apart from that, Congress needs to explore EcoHealth.org's publically available 1090's and their auditor's required supporting docs to help unravel their research. This science would help with vaccines and therapeutics.

David Rivard link
7/16/2020 09:49:22 am

There have been a few apologetic comments whose authors note; "but I realize this is beyond the scope of this scientific piece." In these historic times, however, our analysis could benefit now and then from a Voltaire who could see that we are living in an age of survival where his "all things are related" thesis might now have a deeper relevance (with certainly the original authors of this blog). It might be that if we now and then see a purpose to function we can develop new relevant sets of discoveries that can further assist our virology, especially now with an additional overlay personal survival but on a shared massive scale. Its an awareness of purpose that comes from our own personal (and according to Voltaire, relevant) level of consciousness.

If the science overturned on this site gains wider acceptance, we all realize that it will open another door that will inescapably be policy driven.

coolbean
8/2/2020 06:00:38 pm

Nerd Has Power,

Well, it looks like you were right that RaTG-13 is fake.

According to the Chinese Whistleblower, Li-Meng Yan, she said in a recent interview that SARS-CoV-2 was lab made by the PLA, and RaTG-13 was faked by the WIV. The closest relative to the virus actually did not come from Yunnan Province, it came from Zhoushan Island in Zhejiang Province instead.

https://youtu.be/WUXm0PepVUQ?t=194

https://youtu.be/w6lNtUBiqAw

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135831/

Craig Paardekooper
10/5/2020 07:55:18 am

When you know someone in your neighbourhood is a serial killer....

When we hear that SARS_COV_2 mutates, does that mean it mutates naturally, or does it mean that bio-weapons scientists are still tweaking it to perfect its effects. Do recent mutations indicate Gain of Function or Loss of Function?

It is quite possible that the outbreak was only version 1, and will be regularly updated to achieve its intended purpose.

Vivian Wang
10/21/2020 08:15:21 pm

Hi there, I'm a New York Times reporter hoping to reach you about your assessment here. If you're willing, would love to hear from you. Thanks very much.

新新
6/7/2021 02:18:44 am

Even if it is a human-made virus, it is not necessarily made by the Wuhan Virus Laboratory. Before the epidemic of the new crown virus, Wuhan held a military games. Who can say that it is not a virus released by the relevant personnel? This is not the first time the United States has done such things, and it cannot be the last. At least the Americans have done this, and personally will not be surprised. The fake is planted and the thief shouts about the thief,

Rascal
9/4/2021 02:54:42 pm

I would like to understand why it is reasonable to believe ZC45 & ZXC21 are both natural taking from your article;
"What is inconsistent with this trait is the fact that ZC45/ZXC21 and the Wuhan coronavirus, while significantly distant from each other in evolution, share 100% identity in E proteins. Again, in no way this could be a result of natural evolution. This further supports the claim that the Wuhan coronavirus is made in a lab by following ZC45/ZXC21 as a template."
If both so identical in this ?
I have no genetics training or detail knowledge so please excuse any lacking....
I am an engineer so inquisitive....

Errata
9/6/2021 01:41:46 pm

@Rascal

Why should ZC45 & ZXC21 be accepted as natural?

CoV2 must have been built around a natural virus backbone. Shortly after the CoV2 outbreak began, Chinese scientists published a flurry of virus sequences. The raw data looks fake and the pattern of mutations between them looks unnatural. ZC45 & ZXC21 date from before the outbreak, their raw data looks normal, and the comparison of ZC45 to ZXC21 shows no anomalies. The collection of ZC45 & ZXC21 was in 2015-2017 with publication in 2018 before the current spat of fraudulent papers. Although ZC45 might be fake or it might only be a cousin of the ancestral lineage, it is the best candidate for a natural virus backbone donor from among the published viruses in the CoV2 family tree.

Another reason is that the existence of ZC45/ZXC21 is awkward for the CCP's narrative. The publication by Zhengli Shi of RaTG13 came out simultaneously with the publication of another paper giving ZC45/ZXC21 for precursors of CoV2. From the 1st Yan Report: "the Shanghai Public Health Clinical Centre, which published a Nature article reporting a conflicting close phylogenetic relationship between SARS-CoV-2 and ZC45/ZXC21 rather than with RaTG13, was quickly shut down for 'rectification'. It is believed that the researchers of that laboratory were being punished for having disclosed the SARS-CoV-2 — ZC45/ZXC21 connection."

Adrian Gibbs
5/12/2020 04:05:10 pm

I am a long retired virologist living in Canberra Australia. Is there any chance of getting that alignment from you as I would like to have a look at it, but, as a very elderly person, I baulk at the task of assembling and aligning so many long sequences.

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Adrian Gibbs
5/12/2020 04:10:02 pm

Apologies. I am interested in obtaining the nucleotides not the amino acids. Adrian

charly
5/18/2020 03:31:03 am

you can find all published sequence on websites like genbank, eg: https://www.ncbi.nlm.nih.gov/nuccore/MT439597.1 for your concern

Nerd has power
5/18/2020 07:39:45 am

Thank you, chary, for the help.

Hi Adrian, sorry for the delay. I'm a little occupied at the moment and could not keep up with all the discussions. I have listed either the unique names or the accession numbers of these sequences in the figure legends. Please go to the Genbank and search with these info. They should lead you to the correct sequences. Sorry that I won't be able to list every sequence here as there are too many. If there is anything you could not find with the Genbank search, please let me know and I can help you with individual cases. Thank you and take care!

Geo
8/6/2020 10:36:59 am

The point is if they can make this virus that is hard to detect in who made it and created a weapon. It shows the CCP is making illegal biological weapons of mass destruction. Can you imagine if they did the same thing with small pocs?

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Dave Romain
6/1/2021 07:11:38 am

PLEASE send me a copy of your 'proof' that RATG13 is fake and permission to use it. I intend to include it in a self-published book's chapter on the |Cov2 virus.

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Chris
3/27/2020 06:55:03 pm

about the publications 3,4,5 even if I understand that the dates of publications arrive at the right time just when it was necessary to justify the theory of natural genetic evolution, do you have more convincing elements which could accredit the thesis cheating?

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Na
3/29/2020 05:54:10 am

These papers were published by the Chinese (government) and used as evidence to discredit this theory. 1. These papers came after the initial accusation 2. They are taken by most scientists as evidence to disprove the man made theory. That's all he is telling you - paper was written claiming this was man made. Fake reports were then published by China to disprove this theory, scientists keep citing these papers as a reason this theory is wrong. He is telling you these papers need to be discounted as a reason, plus he is asking why would they have been necessary, all published by CCP scientists.

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Nerd has power
3/29/2020 08:01:13 pm

Technically, I do not have more elements. A lawyer or a judge don't have to show proof to discredit certain evidence, right? It's common sense that the defendant's own words don't count. I am simply saying, given the circumstances (timing of publication being only one, but not the only one), the RaTG13 sequence cannot serve as credible scientific evidence. The same goes to the other two papers on pangolin coronaviruses (there are odd things in the way they acquired their samples, BTW. I can't spend the time describing this though). The CCP should show proof on RaTG13 and pangolin coronaviruses. Again, when the evidence against them is strong, they cannot prove themselves innocent only with their own "words" (sequences).

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Nerd has power ---- corrections made on 3/29/2020
3/29/2020 08:17:42 pm

I updated some things in the article. Someone kindly pointed out two errors in my earlier version. First, in the 2006 paper (reference 8), they actually did not see enhanced viral infectivity. My memory fooled me during my writing. I have corrected this mistake and clarified that insertion of Furin cleavage site lead to greater infectivity and greater cell tropism in INFLUENZA virus, not the pseudo virus in this 2006 paper.
Second, I originally wrote that Furin-cleavage site does not exist in any beta coronaviruses. That is not correct (you can tell that I am not a coronavirus expert). Such site is not seen in any lineage B beta coronaviruses, but similar ones (not the exact sequence) are observed in lineage A and other coronaviruses (such as MERS). I have corrected this also. I have to admit that this change weakens my argument (that acquiring this Furin cleavage site through recombination is extremely low) slightly. I have changed my tone accordingly. However, I don't think such change makes any fundamental difference in the overall picture. I remain fully convinced that this Wuhan coronavirus is of non-natural origin.

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Debbie
5/15/2020 05:19:56 pm

Dr. Judy Mikovits confirms much of your evidence, and I hope you have watched her interviews online - thank you for your great courage and integrity.

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Andrew M
5/16/2020 01:42:28 am

https://www.snopes.com/collections/plandemic/
https://www.sciencemag.org/news/2020/05/fact-checking-judy-mikovits-controversial-virologist-attacking-anthony-fauci-viral

Pete Ross
5/21/2020 10:57:02 am

Dr. Mikovits does not refer to the analysis here.

She only makes the assertion.

About her credibility, follow HousatonicLive, YouTube

a
4/1/2020 08:08:29 pm

Interesting article.

Could you provide more details on where you obtained genetic sequences, and what arguments to use for analyzer?

Pictures are too small, and impossible to inspect

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Nerd has power
4/6/2020 09:19:13 pm

These sequences are all on the public database (https://www.ncbi.nlm.nih.gov). I will list the accession numbers and you can use them to retrieve the actual sequences of these viruses.
For comparing sequences, you may use Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or play with the server listed in Figure 3 (http://multalin.toulouse.inra.fr/multalin/).

Wuhan-Hu-1 NC_045512

2019-nCoV/USA-AZ1 MN997409

CoV_ZC45 MG772933.1

CoV_ZXC21 MG772934

SARS_GZ02 AY390556

SARS_CoV NC_004718.3

MERS_CoV NC_019843.3

Bat_RaTG13 MN996532

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Piguin
4/6/2020 08:01:13 pm

Nicely written and very hard to dismiss.
My only question is how do you think Shi did it?
There are existing papers claiming that with known methods, it is easy to detect if a virus is artificially made.

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Nerd has power
4/6/2020 09:10:52 pm

I have some thoughts on what methods Shi or others might have used to create it. I might put it into another writing. I know what paper you are referring to though. It's the nature medicine paper published by Andersen, right? His statement that it is easy to detect if a virus is artificially made is laughable. He is either deliberately helping cover things up (his co-author Lipkin is a longterm partner with the CCP) or is simply ignorant about current tools for cloning. Nowadays you can easily edit genes without leaving a trace. Even if it is tedious to remove the trace, wouldn't you do all you can to achieve so when you are making a bioweapon? That nature medicine paper is a shame.

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J17
5/8/2020 06:28:56 pm

Which nature medicine article "The proximal origin of SARS-CoV-2"??? I have computer modeling background as well as virus/bacteria manipulation. I can't believe that made it into nature.

I talked to my lab guy and he said he could do it in both RNA & DNA form. Crispr has a method for RNA

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239212/

Chris Martinsen from Peak Prosperity talks about infectious DNA clones which makes it even easier. There is also a paper on converting a RNA virus (HIV) into a DNA clone.

https://pubmed.ncbi.nlm.nih.gov/10505681/

Nerd has power
5/10/2020 04:25:36 am

Thank you for sharing these info. Yes, we were referring to that Nature Medicine paper.

What might be the best way to deceive the world and cover this up? My answer would be to have a renowned team of scientists, all with non-Chinese names, publish that kind of an article on a most renowned scientific journal. If you look a little carefully at their backgrounds, almost all authors on that paper had close ties with China. An interesting question is who orchestrated the collaboration as these individuals don't seem to have close ties with one another in the past.

Hugo S. L.
5/10/2020 07:09:02 am

Could you explain if Kristian Anderson's paper is full of flaws then why no other academic papers challenging him and why Dr. Fauci quoted it? A statement made by Anderson to dismiss the lab-manipulation is that the RBD of SARS-CoV-2 is not optimized for affinity with ACE2. I found that argument is somewhat vulnerable if someone only aimed to using lab-manipulation to create a artificial chimeric virus from pangolin and RaTG13(let's ignore its authenticity in nature for now). I found several other weakness in his argument especially when he was interviewed by NYTimes because he could not articulate a persuasive argument to explain how the evolution process could have produced SARS-CoV-2 from pangolin and RaTG13. So I am wondering why there is no academic paper to challenge Anderson's view? Does that mean the whole academics are corrupted because, for example, they all want to find grants from Dr. Fauci or someone else who has supported gain of function research in China? Why did not Ralph Baric say something about it as he was the one who helped China to gain the genetic technology to manipulate virus genes in the lab?

John F Signus
5/10/2020 08:54:39 pm

In fact, when you look at the RBD domain, the underlying nucleotide sequence diverge significantly from the pangolin sequence. Which rule out recent recombination as a possible source of the RBD assuming that sequence was real. (BTW. the "new" Nature paper was a reprint of the 17/02/2020 paper where MP789 first came out, and none of the pangolin strains were submitted to GenBank before 08 Feb 2020. They likely waited for the E gene of SARS-CoV-2 to come out, before using it to finalize the "reconstruction" of the genomes. which explained the fact that they does not show up in an "identical protein report" from the E protein data of any of the SARS-CoV-2 E proteins.)
If the recombination was not recent, such a virus (RaTG13-like with the RBD of SARS-CoV-2, but without Furin cleavage site) should be already detected in nature, due to the high infectivity of the SARS-CoV-2 RBD. The outbreak should have started approximately 19.8-55.4 years before the RBD nucleotide sequence have drifted into the sequence we seen here. A period that is so long, we should have already seen the trace.
Also, a pair of new restriction sites found in neither RaTG13 nor MP789 (NbvI and YwpI) can be found flanking the RBD of SARS-CoV-2. The restriction sites was not found in either RaTG13 or MP789. Similar new restriction sites also show up around the S1-S2 junction with the PRRA insert. (the BspMI and BsrDI site), with corresponding removal of a Conserved Bse1I/BsrI/BseNI/BseSI site between the sites, which was found in both RaTG13 and MP789 but not SARS-CoV-2.
All these restriction sites and the fact that the inserted RBD differs greatly from the MP789 ("pangolin coronavirus" in both papers) on the nucleotide level, points toward the fact that the RBD sequence seen in SARS-CoV-2 was likely synthesized from the Amino Acid sequence, optimized to match the codon reference to the rest of SARS-CoV-2, before being put into there via molecular cloning, the new sites being introduced via PCR before a traditional restriction digestion and ligation process was used to splice the construct into the genome.
The same thing also point toward the S1-S2 junction, with the large number of synomynous substitutions and the differing internal restriction sites within the region also point toward the S1-S2 junction, with the furin cleavage site insert PRRA, was likely a synthetic construct that was cloned into the SARS-CoV-2 Genome, using the new BspMI and BsrDI sites that were likely introduced using PCR.

John F Signus
5/10/2020 08:57:11 pm

For the reference on the Restriction sites, go here
https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

Nerd has power
5/18/2020 08:26:17 am

Thank you, John! Powerful analysis and reasoning as always. I do see restriction sites flanking the RBD, even before I read Yuri's article. I agree that there is a very good chance that the RBD was cloned in using the traditional method of cloning.

Hi Hugo, I think John's analysis is very powerful on the science part. I will share my view on why no academic people came out and publicly opposed it.

I don't think all people are corrupted. I think more than a handful of people were pissed when Andersen said that, if the virus is man-made, you will definitely see traces. However, academic people are also miserably busy and not all had the time or interest to dig into it (even though they should because this thing affects everyone).

More importantly, Andersen used RaTG13 as the main evidence to support the notion that the virus is of natural origin. If you don't question that (most people usually don't when it's a Nature publication), then Andersen has no major holes to cover in his claims. Most academic people, even if they are paying some attention to this topic, must feel that, given the "existence" of RaTG13, the Wuhan coronavirus can easily come from Nature.

Finally, for people who are convinced that it is man-made, they can't argue for this claim without pointing out that people may have fabricated things like RaTG13 and Pangolin coronaviruses. Articles like mine here have too much politics involved, which makes them not suitable for pretty much all scientific journals. But in reality, it's the CCP that is putting politics into this in the first place. The cover-up with RaTG13 or pangolin viruses is more of what the CCP needs than Shi's personal interests.

Sorry if this sounds frustrating. If you are convinced with these articles, please help spread them around so that more people are aware. Things will change because truth will prevail, gradually but surely.

Nerd has power
5/18/2020 08:52:27 am

Forgot that ioderESTEl shared this on May 5th:

"Some other science community commentary on the original Nature Andersen, et al paper.

https://www.gmwatch.org/en/news/latest-news/19387-another-expert-challenges-assertions-sars-cov-2-was-not-genetically-engineered

This in particular cites a method for creating a large number of spike protein candidates using a “phage display library.”

https://gmwatch.org/en/news/latest-news/19383

Note how Nature refused to publish the challenge."

David Rivard
7/16/2020 09:54:15 pm

Aside from the etiology, but perhaps supporting your general thesis, is the continued forward thrust of information focused upon mortality. WHO is still echoed by most other national authorities. Perhaps the most important information that the public should be focused upon is morbidity. Other than delayed reporting, WHO continues to: 1) Relate C-19 to the common flu. This influenced much of the global public to take it both lightly and politically. 2) Emphasis on deaths, rather than morbidity presented to policy makers to distill the danger, while emphasizing alleviation of respiratory conditions as defining the cure. Note the tens of thousands, and at least 50% of those testing positive, have actually been on their own "recuperation" phase, some since the disease was revealed. There have been support groups established in almost every country. No centralized, (WHO??), or local national authorities, are cataloging long term outcomes (gathering and sharing local epidemiologic data). This population is complaining about being much less vital. Additionally, the only measurable morbidity would be if they were athletic and had past performance or work levels they could measure against (in te scientific world). In fact, a perfect bio-weapon would make populations less vital, with an albeit inevitable, but much smaller but consequential death rate. In concourse it is truly amazing that every country on earth is following the only effective treatment, the science of avoidance, and there is such a dearth of information shared with the public.

Haha
4/7/2020 01:55:50 pm

But Shi's research on collecting virus all over China is funded by ecohealth alliance, via the PREDICT program funded by USAID.

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Nerd has power
4/9/2020 04:44:16 am

Ecohealth alliance may be one of her funding sources. But researchers receive funding from many sources. Shi is funded much much more by the CCP. She is the top expert in virology and leads the top lab at the top facility in China. You can find a bunch of titles that the CCP has given her. If you think she is not under the control of the CCP, then you probably don't really know how things are in China. USAID might have given her some money, but that doesn't mean that they get to watch what her team is doing every minute. However, the CCP can and will. Has Shi obeyed and served the CCP? These titles of hers may tell you a bit on that.

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List Mist link
4/11/2020 10:13:12 am

Well written. I am not a virologist. But can you take a look to see if ihttps://www.pnas.org/content/early/2020/04/07/2004999117 is valid? 70 cent army paid by CCP posted this link almost everywhere in YouTube. Here is what I replied to them:

A paper based on an incomplete virus sample set is invalid. Haven't all virus samples already been destroyed by CCP by January 3rd? I can easily find materials stating that CCP virus is made by CCP: https://nerdhaspower.weebly.com/ Further, if CCP can corrupt U.S. media like VOA, CNN, Washington Post, Wall street Journal, what else cannot they corrupt? Which costs CCP a whole lot less? Buying a few authors to fool the world or paying the world for their inhuman crime? Surely, the former.

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Chris
4/11/2020 11:17:15 am

Do you have the YouTube Links ?

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Nerd has power
4/11/2020 11:22:05 am

I saw this paper. You have made a very good point that the sample size in this PNAS paper is too modest. Furthermore, although I'm not a huge expert in bioinformatics, I can say that the way they analyzed these sequences (tracing individual mutations) is very old fashioned and not powerful in today's standards. The combination of these two already diminished their conclusions. But the biggest problem is that they did their analysis by assuming that the RaTG13 virus is the origin of the Wuhan coronavirus (reference 7 in their paper). Their conclusion is reached based on which sequence resembles the RaTG13 sequence more. As you probably know, I am convinced that any scientific analysis based on this RaTG13 sequence should be discarded.
I completely agree with your other point too. The CCP always gets its way by corrupting a few individuals. I'm not saying the authors of this PNAS paper are also puppets of the CCP, but many people out there are, including the president of the WHO. US pays big dollars for the organization, while the CCP still made WHO work for them by corrupting the one(s) in power. It is the typical approach of the CCP.

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List Mist
4/11/2020 12:15:23 pm

Thank you for your explanations! Well said! Here are the videos:

https://www.youtube.com/watch?v=3bXWGxhd7ic

https://www.youtube.com/watch?v=Mg5budPRY1Q&lc=Ugz3ew_4EdozAqOqeqR4AaABAg.975XmdSc5DM97JHddv_GEM&feature=em-comments

Jim Low often picks the comments that have most thumbs up to speak in favor of CCP .with his links.

Shaun link
4/12/2020 04:03:56 am

This paper/blog is in *serious* need of an abstract. Please consider.

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Shaun
4/12/2020 12:25:49 pm

This is the most insane paper. I've read the whole thing from top to bottom and have shared it with the world. I am also convinced that this is a bioweapon. The timing is too perfect, everything from the furin-cleavage site to the S1 protein being a SARS variant, and the fact that these "recombination" techniques were previously used by the "Bat Lady" in research *specifically* for simulating potential human coronaviruses.

And she warns, "there's more where that came from" - because the binding site won't be a target

But a random question. Weren't there two coronavirus strains that came about "magically" at one point? And that one "suddenly appeared" somewhere?

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Nerd has power
4/12/2020 02:54:07 pm

Thank you for sharing it with others. The matter itself is insane and everybody deserves to know what's really going on.
I don't think I quite understand your question. Could you rephrase it a little bit? Are you asking whether recombination between two coronaviruses as magical as this one has been evidenced before?

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Shaun link
4/14/2020 02:08:25 am

Referring to the confusion that arose towards the very beginning of the epidemic re: Seattle coronavirus vs. Wuhan

Like there were two different strains or something

If this were the case, if there were in fact two different strains of coronavirus circulating, this would speculate further into the nature of the potential "release" scenario.

There was some "mystery" towards the beginning of the outbreak with regards to two different strains.

Shaun link
4/14/2020 02:19:42 am

*Furthermore* if the "recombination" event (i.e. addition of SARS-like spike protein) happened on two separate bat coronaviruses, that would almost *solidify* the fact that it were not by natural selection if those SARS-like spike proteins were similar.

Of course, this all pending the level of expertise one could have with creating new SARS-like viruses by testing binding affinities to ACE2

What an extraordinarily dangerous time we're living in. Who needs nuclear weapons when you can build genetically-engineered viruses

You have to consider, and wonder, if this was just a nuclear test.

Nerd has power
4/14/2020 09:33:19 pm

Thank you for the clarification. I see your point now. As far as I know, the evidence predominantly supports a single origin of the virus. There appear to be questions of where it originated (although there is actually no doubt that it came from Wuhan), but I don't think any scientific literature really hinted about different strains initiating independently at different places. Again, I think the evidence strongly support a single origin of the pandemic.
And, yes, extraordinarily dangerous time indeed. Nuclear weapons don't spread like this. Nobody is not affected by it. The world needs to wake up to the fact that communism and dictatorships are enemies of the entire human race. If we don't get them and instead allow them to gather money and resources, they will get us. The CCP has to be taken down and eradicated from this world.

Jim jones
5/8/2020 04:31:17 am

Communism is great and has nothing to do with dictatorships. Capitalism dictates. You gave the game away with your scientific bias there. This appears to be about your personal fears and political predudices.You haven't even given a motive for creating this virus or how it benefuts china. Viruses and RNA sequence evolution are far more complex than you make out. Just like your politics.

Nerd has power
5/10/2020 04:38:44 am

Thanks, Jim Jones, for sharing your thoughts. You and I apparently have different views on communism. I just want to bring up one fact. Many countries have converted from communism to capitalism. Have you seen much complaints or people there wanting to go back? I never said capitalism is perfect, but in my opinion it is far more superior than communism. Also, if Kim Jong Un can be interested in making nuclear bombs, we will have to admit that communism is not against having massively disruptive weapons.

David Rivard
7/18/2020 11:44:19 am

I participate in several scientific blogs regarding C-19. Our office also gets up-to-date information from the executive offices of many different countries (typical of many executive offices) and the Health Minister then produces health policies regarding C-19. I have reached my own conclusions from my experiences so far.

Lacking an invitation, the lab door has been opened by many scientists outside of the scientific establishment. Defenders of the door have been the most prominent academic publishers, such as Lancet, Science, Nature et al, not to mention the CCP, unwitting UN organizations and even our own CDC and popular media. Gradually, academic publishers just started to assist in opening the door with a few journalists in the popular media.

What was behind the door? A completed bio-weapon, requiring no passaging, that is extremely adept at using many delivery pathways.

The unintended, but inescapable, effect was to cause (relatively) few deaths. The design effect; de-vitalizing individuals, infrastructure, cultures and economies until they finally succumb. The design effect is well underway.

On an individual level, the CDC and WHO's continued focus on mortality rates to define the effects of the disease, comparing it to a severe flu, rather than morbidity rates lulls a younger public into thinking that getting the disease is simply like the flu and if contracted they will simply develop immunity. Additionally, mentioning possibilities of "herd immunity" and vaccine development offer public optimism for politicians but is counterproductive to other recommended efforts to take the disease more seriously. The only proven science we have thus far to combat C-19 is to practice disease avoidance.

However, C-19 has an effect upon the immune system more akin to HIV. Those contracting the disease are initially alarmed by respiratory symptoms and seek health care. When they get the benefit of a test or when they are determined positive symptomatically they are cataloged. These patients leave the hospital because the respiratory symptoms get better. When they get two consecutive negative PCR tests, (that measure the virus in the upper respiratory tract), they are officially pronounced cured. The tens of thousands of those previously infected, and, so far, over half of those who tested positive, even those that were initially deemed as "asymptomatic" or "recovered", are increasingly found in support groups where they seek advice about non-acute but lingering and worsening health problems (and their information is still relegated to anecdotal forums where data is not collected).

The disease seems to maintain a chronic state even when acute symptoms subside. Some have "caught" it twice, where it seems to be worse. Many have had persistent symptoms over many months and complain about getting worse. They do not feel like they have completely recovered. Some, through their personal physicians, have discovered that a stool sample is far more accurate in measuring viral loads than the T cell (antibody) tests that are so unavailable.

The next door to open should be easier, ironically because the timing was too perfect. Policy makers must now determine if it was an intentional release.

sam
5/8/2020 05:20:13 am

Did Zhengli Shi say, "There's more where that came from"? Can you show me where she says that?
Thanks

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Angel link
7/16/2020 03:05:45 pm

I'm relieved that so many have been eyeballing this virus' origin question! I am not a scientist or biochemist in background but have spent some years learning bits and pieces. My educational tool through this Pandemic has been MedCram.com. They blessedly covered biochemistry basics along this journey as well as what the virus DOES and HOW it is mutating. My husband has been my captive audience throughout our SIP as we both have underlying conditions of concern. I mentioned to him in February-March about how this virus didn't seem to be "acting" like a normal virus we see. I started watching this one in DECEMBER 2019. Our other viruses I've glanced at and just went over "laundry list" of "do's" and on with life. Not so for this one; how would our household prepare and meet this one. I told husband at different times that "this one acts engineered".
Regular searching led to regular mainstream sources-"Bah!!!" You have all reassured me that I am not wrong. My next question: is anyone working on bringing this thing to a stand still? Too may lives have been lost already, and hate to see more! An in self-quarantine right now. Guy sneezed on me--allergies, cold, Covid-19? Sometimes I wish there were a specific sign that shows up--purple ears, green little toe? Something? The stress is getting to many, myself included! But I really do thank you for your conversation here and your "Doing" whatever you can to help the world.
Can you imagine if this had been meant for spreading at OLYMPICS, but escaped early?

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Seek The Truth
4/13/2020 07:19:26 pm

Fascinating...I notice that the comments are "split" (no pun intended)...between here and the Chinese version...I noticed some interesting comments from Elannor D Allens (and your responses) in the Chinese comments section....could you do an 'edit' to incorporate newly provided info/discovery from Elannor D Allens here as well? (I noticed from your comments here...English section...that you made updates as made aware by the commenters)...I think that inclusion of Elannor D Allens' additional info to your write-up would strengthen your assertion considerably...

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Nerd has power
4/14/2020 09:38:37 pm

Great suggestion! I completely agree that comments made by Elannor D Allens are very powerful and supportive of the conclusion here. I will work on translating these over to this English section soon. Thank you!

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Chris
4/16/2020 03:44:13 pm

Yes please translate this comments by Elannor

Greg Felton
8/26/2020 04:37:30 pm

Who is Elannor (Eleanor?) Allens and where did she publish her findings about those 79 codons?

Greg Felton
8/27/2020 07:53:59 am

Who is “Elannor D Allens” and is that spelling correct? a google search turned up nothing. Is it supposed to be Eleanor D. Allen?

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Greg Felton
8/29/2020 10:16:00 pm

STT:
I am trying to find Elannor D. Allens and her comments about the syn/non-syn ratio, but her name does not turn up on any search. It seems as though she may be Chinese and "Elannor" (odd spelling) is her English name.

Can you point me to where I can find her and her evidence? I can read Mandarin.

Thanks

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CCP is NAZI
8/29/2020 10:24:25 pm

Search "Elannor D Allens" in the next door for Chinese comments

Nerd has power
4/16/2020 03:56:59 pm

As noted by Seek The Truth in the comment above, Elannor D Allens made an excellent point in the Chinese comments section next door, which strongly supports the claim that the Wuhan coronavirus is man-made. I have looked into this aspect myself and found it very convincing. Below is this finding and its implications in English. Please note that it is not an exact translation of the comments made by Elannor, although the contents are largely the same.

This concerns the S2 portion of the spike protein (blue labeled parts in Figure 2AB). More specifically, here, we are comparing the Wuhan coronavirus and the RaTG13 virus (the sequence of which I believe is fabricated by Zhengli Shi) on the sequence between 647 and 1124 of the S2 protein. Note that this part of the protein typically sees mutations as evidenced in Figure 3. However, interestingly, between Wuhan coronavirus and the RaTG13 virus, not a single amino acid has changed in this region. What is striking is that, at the gene level and within the same region, the two differ 79 times.

When a gene (composed of nucleotides) is being translated into a protein (composed of amino acids), every three consecutive nucleotides constitute a codon and each codon corresponds to a particular amino acid. On the other hand, an amino acid typically corresponds to four codons, although some amino acids have one or two more and some one or two less (you can learn more about it here: https://passel2.unl.edu/view/lesson/3ccee8500ac8/6). What does it mean? It means that, when a nucleotide has changed (or in other words a single mutation has occurred) in the gene, the codon is certainly altered but the corresponding amino acid may or may not change. This is because the new codon may encode the same amino acid as the old codon did. In evolution, on average, every nine nucleotide changes result in the change of one amino acid.

Now, let’s take a look at our case here: 79 nucleotides are different and yet not a single amino acid has changed. Remember, again, we are looking at a region of the virus that can easily tolerate changes. The above pattern is, therefore, EXTREMELY STRANGE! According to Elannor’s calculations, the chance of this happening in nature is 1/9,526,094 or 0.000000104974819 (Elannor listed an equation for this, which I’m not expert enough to comprehend unfortunately). Furthermore, the nucleotide changes almost exclusively took place at the third position of the codon (77 out of 79), which tend to lead to no change in amino acid. Such a strong bias to the third position, over the first and second positions, is a strong indication of artificial manipulation – can nature be so selective in carrying out RANDOM mutations?

What do these observations mean? They mean that, between the Wuhan coronavirus and the RaTG13 virus, at least one is non-natural. If one is natural, then the other one must be not. Of course, the other possibility also exists – neither of them came from nature.

Elannor seems to believe that RaTG13 does exist and was used as the template to create the bioweapon, the Wuhan coronavirus. When those devils worked on creating this bioweapon, they followed the same exact amino acid sequence of S2 of RaTG13 but may have intentionally changed its nucleotide sequence, without affecting any amino acid identity, to make the bioweapon look different from RaTG13 (The two nucleotide sequences being exactly the same would be a clear indication that the bioweapon is made out of RaTG13).

I tend to believe the other possibility: the RaTG13 virus does not exist and its sequence was fabricated to make people believe that there is a natural origin of the Wuhan coronavirus. In this scenario, when they were fabricating the RaTG13 sequence, they wanted to make sure that the amino acid sequence of S2 does not change much from that of the Wuhan coronavirus because S2 is typically conserved as seen in other coronaviruses. At the same time, they wanted to make sure that the gene sequence of RaTG13 differs just the right amount from the Wuhan coronavirus so that the two look like very close relatives with each other, thereby deceiving people to believe an evolutionary origin of the Wuhan coronavirus. The thing they forgot to take care of here, however, is the correlation between the number of nucleotide mutations and the number of amino acid mutations. Their product, the RaTG13 sequence, now exhibits an extremely unnatural disproportion of 79 nucleotide changes vs. zero amino acid change, when compared with the sequence of the Wuhan coronavirus at this part of S2. This, together with the bizarre bias of “mutations” occurring predominantly at the 3rd position of the codons, adds tremendously to my already well-supported notion that the RaTG13 sequence was very likely fabricated.

Whichever scenario is true, this finding undoubtedly strengthens the assertion (thank you, Seek The Truth, for these words) that the Wuhan coronavirus is

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Nerd has power
4/16/2020 03:58:21 pm

a bioweapon made by the CCP.

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Ilius
4/24/2020 07:51:18 am

It appeared that there have already been amino acid mutations, acquired during the spread of this virus in humans, that are located at this particular part of the S protein, found in the later samples of this virus which have mutated in humans since it’s contnued spread about a month ago— further indication that such a part of the S protein can indeed tolerate changes and have started to change once it have been spreading intensively in humans. Further proof of the unnatural origin of the original SARS-COV-2 genomes, of which the protein sequences here are all identical with RaTG13.

Nerd has power
4/25/2020 06:12:21 am

Thank you so much for sharing your finding. It is very powerful and strongly supporting the claim that the virus is man-made.

Annette
5/12/2020 04:49:01 pm

A very interesting article and I will need to read it again. I was most interested in the E proteins as they to my understanding cause the immune reaction and possibly the cytokine storm. Please can you comment on the use of E proteins in SARS Cov 2? https://thenativeantigencompany.com/products/sars-coronavirus-envelope-protein-e-coli-2/

Nerd has power
5/18/2020 08:39:52 am

Hi Annette, my knowledge on the E protein came largely from reading this review:

https://virologyj.biomedcentral.com/articles/10.1186/s12985-019-1182-0

I wouldn't be able to explain things any better. Hope you find the answers you are looking for in there.

john kelleher link
5/8/2020 02:36:22 pm

Thank you for your work.The youtube channel Peak Prosperity has backed up some of your conclusions. He is a trained virologist.
As you probably know the scientific community has been a target over the climate change issue. Trump is heavily involved with this and is continuing to ignore science on other issues. I think his comments concerning Covid-19 being made in a lab in Wuhan will go nowhere. He has many agendas. Thanks, John.

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Nerd has power
5/10/2020 05:05:15 am

Thank you for the comments, John. I have seen one video from Peak Prosperity. It was fantastic. I think it's a believable source of scientific insights into this issue.

I happen to support President Trump's claim that Covid-19 is made in a lab. Truth will come out, with more solid evidence backing it up. Once that happens and everyone is convinced that it was a bioweapon made by the CCP, I don't see any reason for anybody not to be angry about it. For many people, Trump might not be their most favorable person. But he didn't create this most horrible Covid-19 and let it spread to every corner of the world. If he is telling the truth here and the CCP did all that, I actually can picture how people from left and right all join him to fight that increasingly-visible enemy. It would be for the good of the USA and world. My two cents.

Alesh Aras
5/27/2020 06:42:05 am

Where did you get the info that the Peak Prosperity guy is a “trained virologist?” Below is is the public profile I could get from his website:

“I’m a trained scientist, having completed both a PhD and a post-doctoral program at Duke University, where I specialized in neurotoxicology. I tell you this because my extensive training as a scientist informs and guides how I think. I gather data, I develop hypotheses, and I continually seek to accept or reject my hypotheses based on the evidence at hand. I let the data tell me the story.”

https://www.peakprosperity.com/about/

Greg Felton
8/19/2020 09:26:28 pm

Hi Nerd:
Is there any way you can point me to the source of Eleanor Allens' analysis. I can read Mandarin though I will likely need a dictionary for technical terms.

Thanks,

Greg

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Greg Felton
8/30/2020 12:21:11 am

Sorry, CPP is Nazi:
I found it.

List Mist link
4/16/2020 06:31:03 pm

Beijing tightens grip over coronavirus research, amid US-China row on virus origin: https://www.cnn.com/2020/04/12/asia/china-coronavirus-research-restrictions-intl-hnk/index.html. Exactly, what are CCP trying to hide? The more CCP are trying to hide, the more they are actually exposing the origin of this lab made CCP virus. We definitely need to investigate this thoroughly and hold CCP accountable for their virus. Otherwise, we don't even know how we die one day in the future with more evil virus CCP are so eager to develop!

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Nerd has power
4/17/2020 05:56:14 am

Completely agree. When we say the CCP is anti-human, we risk of understating its evilness. It used to be that not everybody believes this -- because people are generally nice and probably never even tried to comprehend things of such evil nature. But now is the time that everybody be brought to the same page. The CCP must be held accountable. The world cannot afford having communism around any longer.

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Chris
4/17/2020 03:26:39 am

Can I spread your text in France ?

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Nerd has power
4/17/2020 06:10:11 am

Please do! I was disgusted by how some high-profile scientific publications misled people, intentionally or not. That's why I wrote this. I just want more people to know the truth. The more the better. Thank you for your efforts. We are doing the same things.

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Pete Ross
5/21/2020 11:35:23 am

There are experts, including Luc Montagnier, who assert that the SARS-CoV-2 genome contains HIV-like sequences. Thoughts?

Also, to date, the pathophysiology of COVID appears to be best characterized as a coagulopathy, either though disruption of free radical suppression pathways causing injury to the endothelium and thus release of pro-clotting factors (i.e. Von Willibrand factor). Alternatively or in addition, is anyone making a search for pro-clotting factors and/or pro-free radical factors in the SARS genomes, i.e. SARS-1, CoV-2, etc?

Nerd has power
5/23/2020 09:07:18 am

Hi Pete, I have said a few times in the comments that I'm not convinced that there are insertions of HIV sequences into the SARS-CoV-2 genome. Please scroll down to find such comments if you are still interested.

In terms of pathophysiology, I must admit that I'm completely unqualified to comment on that. I'm also not aware of any research looking for pro-clotting factors in these viral genomes.

Patric Hausammann
4/17/2020 10:11:36 am

I have checked your work by comparing it to the published sequences on "ncbi" sources of the viruses and I couldn't find any mistake. I think the virus was pretty sure lab made. The mutations do not look natural at all, and I guess some virologes should study more, or they should not take money from the CCP.
The shown and marked sequences at "fgure 3" are striking. But there seem to be more obviouse mutations. Here a link: https://photos.app.goo.gl/1WcGJhwY4sWaggzh6
I marked the strangest mutations in violet.

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Nerd has power
4/18/2020 09:39:00 am

Thank you very much for your efforts. There are indeed more mutations than what I have focused on describing. The reason I didn't discuss about all mutations is because I wasn't sure how the rest of them might be involved in the actual function. I'm pretty sure about the mutations I did talk about and highlight in terms of how they are important in function and might have been manipulated. There could be more manipulations than what I was able to detect, which may involve the additional strange mutations that you've marked out. But, again, I wanted to focus on things that are more solid and well supported. I don't want to include any less-than-convincing arguments because they may take advantage of those arguments to trash the whole article.

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Patric Hausammann
4/22/2020 01:57:08 pm

Thank you for your appreciated answer. I absolutely agree to use your suggested sequences in your article to prove the claim of man made manipulations. Maybe, there comes out some evidence about the other "strange" changes later.

Greg Felton
8/26/2020 12:23:00 pm

Nerd: I have read that you oppose the HIV origin of the PRRA insert at aa 681. What is the reason for your doubt, and where do you think the insert came from?

Thanks.

Annette
12/12/2021 01:54:52 am

Looks like the discussions on HIV and sARS Cov-2 lab chimera is not rearing its ugly head blaming SA and some sick man as patient ) for omicron! Has anyone explored the Omi genome yet?
https://www.theguardian.com/world/2021/dec/11/why-uncontrolled-hiv-may-be-behind-the-emergence-of-omicron?utm_term=61b5ac0bb546408905fd390aae42f99f&utm_campaign=GuardianTodayUK&utm_source=esp&utm_medium=Email&CMP=GTUK_email

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Patric Hausammann
4/17/2020 04:58:09 pm

Here I have a link to the uploaded first 1501 positions of the sequences of WUHAN HU-1, 2019-nCoV, bat CoV ZC45, bat CoVZXC21, RaTG13("invention"), SARS, SARS_GZ02 and MERS in comparison to eachother.

https://drive.google.com/file/d/178M3rj9OdfWS-5zxp1QKz_PG56ljroLk/view?usp=sharing

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Jean Claude perez
4/20/2020 09:02:47 pm

> Jean-Claude PEREZ. (2020). “WUHAN COVID-19 SYNTHETIC ORIGINS
> AND EVOLUTION.” International Journal of Research - Granthaalayah, 8(2), 285-324.
> https://doi.org/10.5281/zenodo.3724003.

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Visitor
4/21/2020 07:12:02 am

Your article appeared to have been deleted. Could you please upload it again?

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Perez
4/21/2020 07:48:22 am

> Jean-Claude PEREZ. (2020). “WUHAN COVID-19 SYNTHETIC ORIGINS
> AND EVOLUTION.” International Journal of Research - Granthaalayah, 8(2), 285-324.
> https://doi.org/10.5281/zenodo.3724003.

Patric Hausammann
4/22/2020 01:43:41 pm

I couldn't open the link to the article of Mr. Perez too. But I think I've found a working link to his work.
Here it is: https://zenodo.org/record/3724003#.XqCrjplCSUl

Nerd has power
4/23/2020 07:01:19 am

Dear Mr. Perez, thank you very much for sending your published work. I have to admit that the mathematics involved in your study is way beyond me. Although I am in agreement with your conclusion that the Wuhan coronavirus is a result of human manipulation, I'm completely disqualified to comment on this part of your study.
I did look into the sequence similarity between certain Spike regions of the Wuhan coronavirus and different HIV/SIV sequences that you identified. However, unfortunately, my blast search did not return confident results (mostly because the sequences are too short). I am inclined to believe that such level of sequence similarity is not a strong evidence of HIV/SIV sequences being inserted into the Wuhan coronavirus genome. Of course, my judgement here is completely up for debate.

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Simen D arreighher
4/21/2020 02:20:53 am

This is something that you may wan’t to see.
https://harvardtothebighouse.com/2020/01/31/logistical-and-technical-analysis-of-the-origins-of-the-wuhan-coronavirus-2019-ncov/
Also this.
https://harvardtothebighouse.com/2020/03/23/no-monkey-ever-reheated-a-frozen-burrito-what-the-expanse-tells-us-about-the-covid-19-pandemic/
I found that the references quite convincing. Though some of the references these two articles used have yet again forced to be withdrawn.
CCP pressure, perhaps?
https://www.cell.com/pb-assets/journals/research/cell-host-microbe/PDFs/chom_2285_preproof.pdf
It appear that one the only animals that have been proven to produce viable infections of Covid-19 that is not a human are ferrets and tree shrews—both lab animals. And it require lab conditions for the animals to be infected in the first place, as there were no evidence anywhere a wild animal, or any animal that is outside a lab, to be confirmed to be infected at all.
Which confirms the findings of https://www.biorxiv.org/content/10.1101/2020.04.04.025080v1
Apparently undergoing edits to incorporate more data from more animals, that confirms that the best animal hosts for the virus were indeed ferrets and tree shrews. And that all coronaviruses that target the human ACE2 receptor, won’t be able to form a good bond with the pangolin ACE2 receptor— indicating that such an RBD is incapable of infecting pangolins at all.

https://pubmed.ncbi.nlm.nih.gov/22723413/

Also backed up by

https://academic.oup.com/nsr/advance-article/doi/10.1093/nsr/nwaa036/5775463

Which suggest the “similarity” are at best the result of convergent evolution, and a recombination of RaTG13 and one of the pangolin CoVs would be at least “19.8 to 55.4 years ago”. At least two times the evolutionary distance of RaTG13 to Covid-19, an estimate of 9~10 years. Meaning that the alleged similarity was extremely unlikely due to the virus passing through pangolins at all.
Serial passage within ferrets have been used to make the deadly H5N1 bird flu airborne in mammals—And the commenter suggest that all viruses that have been made to perform airborne transmission in ferrets, are capable of equally powerful airborne transmission in humans—further proof that the only human-like ACE2 receptors are found in the lab animals ferrets and tree shrews.

https://www.sciencemag.org/news/2011/11/scientists-brace-media-storm-around-controversial-flu-studies

Sialiation of the proteins may also play a role—as suggested by

https://www.nature.com/articles/s41586-020-2179-y

which proves that one of the sialic acid residues on the Human ACE2 receptor were used in the interaction between Covid-19 and ACE2.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1075706/

It appear that the withdrawals were due to Nature killing articles that disfavors the CCP, though the accuracy of this is yet to be confirmed.

https://www.insidehighered.com/news/2018/10/03/book-publishers-part-ways-springer-nature-over-concerns-about-censorship-china

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Nerd has power
4/23/2020 07:25:05 am

Thank you for your work. What you have posted are all quality info in my opinion. Among them, the most intriguing is the link to ferrets and tree shrews. These really may have been the lab animals that they used in the course of selecting and improving viral strains, which should have followed their initial manipulation of the genome via molecular cloning.

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John F Signus
4/25/2020 04:45:51 am

The nucleotide sequence coding for the E protein in Bat-Cov-ZC45,ZXC21 and RaTG13 are exactly the same, indicative of an extremely well conserved gene that have not seen a single mutation during the entire 5 years of divergent evolution across the two very distantly related viruses(as indicated by the vast differences in the S protein) —but curiously, the first sample of SARS-CoV-2 show 3 nucleotide substitutions within this gene (without changing the amino acid sequence), and newer examples of SARS-CoV-2 have shown amino acid substitutions within up to 4 different locations within this protein, in merely 3 months of human-to-human transfer. An indication of an extremely high mutation rate within the E gene, and the permissivity of the E protein toward changes in it’s amino acid sequence.
The E protein of Coronaviruses is on the inside of the viral envelope and is a structural protein— it can not even make contact with host receptors and does not partition in interaction with host cellular proteins since it’s role is to line the inside of the mature virions—a place that is devoid of any host proteins.
This mean, that the E gene play absolutely NO role in host selection and virulence in specific hosts, and the mutation rate within this particular gene should be relatively constant across all coronaviruses. A survey of bat coronaviruses confirmed that this protein in deed tolerate large amount of changes across both bat hosts and human hosts(SARS).
So how did such a gene manage to not change a single nucleotide across the very distantly related ZC45/ZXC21 and RaTG13, Code for the exact same protein in the very first sample of SARS-CoV-2, yet suddenly started to change in both the nucleotide sequence and the amino acid sequence it codes for once it’s in a human host? Remember that the E protein in Bat coronaviruses varies greatly across different strains—which mean that such changes could easily happen and be tolerated in a bat host. (That mean that the mutation rates of the E gene are similar in both bats and humans, and this gene should not be as conserved as indicated by the sheer evolutionary distance between ZC45/ZXC21, RaTG13 and for the protein, SARS-CoV-2.)
Or alternatively, this feat could also easily be explained via molecular cloning of the ZX45/ZXC21 E gene into the RaTG13 sequence, with subsequent codon optimization to generate the SARS-CoV-2 E protein.
Sequences to look for and MultiAlin to confirm this discovery:
Wuhan-Hu-1
ZC45
ZXC21
AP040581.1
RsSHC014
SC2018
NP_828854.1
SARS_GD01
BtRs-BetaCoV/HuB2013
SARS_ExoN1
BM48-31/BGR/2008
SARS_TW-GD1
SARS_Sino1-11

QHZ00381.1
QJA42107.1
QIS60608.1
QIZ14355.1
QIU81527.1

Look for the full GenBank of the protein sequences and find the corresponding nucleotide sequence, in order to get the nucleotide sequence of the E genes for these viruses.

Nerd has power
4/25/2020 06:09:47 am

Wow!!! Powerful evidence! Thank you so much for sharing. I completely agree with your analysis. The E protein being identical among ZC45, ZXC21, RaTG13, and initial samples of SARS-CoV-2 is highly suspicious to begin with. Now amino acid changes in E protein have already been observed in such a short period of human-to-human transmission. It's just such a strong proof.

John F Signus
4/25/2020 10:30:36 am

Closer analysis of RaTG13 and ZC45 have also revealed that the identicality of the E gene on these two viruses being highly suspicious.
The BLAST result page.
Bat coronavirus RaTG13, complete genome
Sequence ID: MN996532.1Length: 29855Number of Matches: 2
Range 1: 1 to 21563GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score Expect Identities Gaps Strand
26679 bits(14447) 0.0 19227/21597(89%) 80/21597(0%) Plus/Plus
Query 16 CTTCCCAGGTAACAAACCAACTAACTCTCGATCTCTTGTAGATCTGTTCTCTAAACGAAC 75
||| ||||||||||||||||| ||||||||||||||||||||||||||||||||||||||
Sbjct 1 CTTTCCAGGTAACAAACCAACGAACTCTCGATCTCTTGTAGATCTGTTCTCTAAACGAAC 60

From the comparison analysis, the similarity between RaTG13 and ZC45 is about 89% in the part of the viral genome where a match have been found. There were parts that were not matched between these two viruses—let’s ignore all if these.—the most conservative estimate.
The # of base pairs that were different=21597-19227=2370
The total query length is 29855.
The length of the E protein gene=75AA. There is no W within the gene, and the first codon AUG can’t mutate. All other codons have an alternative codon that codes for the same AA at the third location. Two of the Amino acids, L and R, can accept mutations at the first position without change of the AA sequence.
Let’s assume that the E protein somehow reject all changes within it’s amino acid sequence. This gives a total of 74 positions where a substitution could land on.
The chance of a single mutation from ZC45->RaTG13 to not land on one of these silent locations=(29855-74)/29855=0.99752135.
The total chance of ALL mutations from ZC45 to RaTG13 to not land on one of this sites=0.99752135^2370=0.00279008 < 0.3%.
This indicate that there ls less than 0.3%(at most 1.8% if the kind of nt substitution is considered) of a chance that the nucleotide identity of ZC45 and RaTG13 was the result of natural evolution.
Which mean that this identity is extremely unlikely to be fortuitious.

Nerd has power
4/28/2020 08:20:31 am

Thank you again for these excellent analyses. I looked at the nucleotide conservation myself for the E proteins. There are actually two single nucleotide substitutions between ZC45/ZXC21 and RaTG13, although they did not lead to amino acid changes. It's a small error, which of course does not alter your whole logic or conclusion.
I am preparing an updated version of my article, which will likely be posted on a different platform. I would like to incorporate your finding on the E protein in that version. Please let me know whether it is okay. Of course I would acknowledge you for observing this phenomena and providing your insightful analysis. Thank you!

John F Signus
4/29/2020 01:59:56 am

Did you use ZXC21? I am not aware of that sequence. I did a Multalin on the sequence ZC45 and RaTG13 and the nucleotide sequences were exactly the same. If the 2 substitutions on the ZXC21/ZC45-RaTG13 were on the ZXC21 sequence, then it will be a more solid evidence that the RaTG13 N protein gene was a direct clone from ZC45. Further evidence that RaTG13 was unnatural.

John F Signus
4/29/2020 02:47:45 am

Sorry for the mistake. I think I compared the wrong sequence.
With two nucleotide substitutions, with the “strict” standard on mutation rates the chance of ZC45/ZXC21 to RaTG13 mutation being natural becomes 0.0021805 and 0.02067 assuming all other mutations are freely available. Considering the levels of protein sequence conservation across the rest of the sequences (which was to assume that the number of variable positions within the rest of the viral sequence being similar to that of the variability of the E protein itself as seen in other bat coronaviruses), however, this chance pummels to 0.9948692^1563=0.00032228, about 0.04%.

Nerd has power
4/30/2020 07:37:39 am

Thank you for the update. Please don't apologize. Your analysis was brilliant. I'm still working on that updated version of the article. I will show a sequence alignment/comparison by following your suggested strains. That, in combination with what you have described for E proteins, is very convincing.

Silverfox
4/22/2020 01:27:10 am

I I have read your analysis carefully and I do find that it stands completely.
A few days ago, a new study appeared in preprint.
https://www.medrxiv.org/content/10.1101/2020.04.14.20060160v1.full.pdf

In this study by Chinese researchers (sic), it appears that the virus undergoes significant mutations at a fairly short rate. Some of these mutations appear to increase the aggressiveness of the virus. As I understand it, Li's team has detected more than 30 mutations. Of these, 19 mutations - about 60% - were new.

They discovered that some of these mutations could cause functional changes in the virus’s advanced protein, the unique structure above the viral envelope allowing the coronavirus to bind to human cells. A computer simulation predicted that these mutations would increase its infectivity.

According to the words of Professor Luc Montagnier, 2008 Nobel Prize in Medicine, if the virus is a virus manipulated in the laboratory, it will quickly mutate on the portions of its RNA that have been modified because "nature does not accept any changes ". He considers this behavior to be further proof that the virus would have been manipulated.

In view of this last study, what are your interpretations in relation to the analysis that you have carried out? Does this support your hypotheses?

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Nerd has power
4/23/2020 07:37:36 am

Thank you for your comments. I was made aware of Dr. Li's work, although I did not get to read it in full. I think her work is very relevant in developing therapeutic and treatment strategies, which may or may not hint the origin of the virus. My two cents.

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Pete Ross
5/21/2020 01:47:51 pm

Anyone address this type of contention?

http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384

"We know from influenza H1N1, for which we have serial isolates from 1918 to the present, that wobble base mutagenesis occurs at a rate of 0.95% per decade. This permits an estimation of the TMRCA of the two sequences nCoV2019 and RaTG13 of 69.5 years ago – roughly 1950 +/- 10 years or so.

RaTG13, or anything nearly identical to it at the RNA level, simply could not be a proximal source of nCoV2019. It just LOOKS like it might be…at first glance.

Given that furin cleavage signals are present in other coronaviruses at exactly that point in the S1/S2 boundary region, it only LOOKS unusual, especially against the backdrop of SARS. The preponderance of evidence, coupled with Ockham’s razor (that the simplest explanation is preferred) dictates that the PRRA sequence has been conserved in nCoV2019 from a long ago ancestor virus. It is not of suspicious origin. The closest bat virus sequence is really not close at all.

RNA don’t lie.

Bill Gallaher"

Lois Geerhardt
4/8/2021 07:04:46 pm

Hi,
I'm late to this, but, if you have updated your very helpful analysis, if you would email I will be busy passing it on with proper attribution. I'm not savvy enough to find it if it is here on this site.
THANK YOU for writing this!

RaffRag link
4/23/2020 01:14:26 am

Sir, you have only shown that a stretch of amino acids of S1 looks like “copied” from the SARS spike protein and then “pasted” into the Wuhan coronavirus. Don't you think that, to confirm your hypothesis, you should verify that the same does not hold true for the S1 segment from other species?

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Nerd has power
4/23/2020 08:03:53 am

Thank you. It's a very good question. Most coronaviruses don't contain this "copy/pasted" piece in their S1. The exceptions are SARS (of course) and about two SARS-like bat viruses. However, none of these SARS-like viruses "copy/paste" this critical piece as precisely as what's seen in the Wuhan coronavirus (residues important for SARS spike/ACE2 interaction all preserved/conserved and changes only occur at non-essential locations).
More importantly, this "copy/paste" is striking because it is in stark contrast with how the rest of the genome match between Wuhan coronavirus and the "template" coronaviruses (ZC45 and ZXC21). Such level of genome-wide similarity is not observed between Wuhan coronavirus and any of the SARS-like coronaviruses that contain a similar "copy/pasted" critical piece. Again, the "template" coronaviruses are discovered, published, and owned only by the military research lab of the CCP. Hope I'm making myself clear.

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MZ1234
4/24/2020 12:40:39 am

A leading Russian microbiologist has claimed the coronavirus is the result of Wuhan scientists doing 'absolutely crazy things' in their laboratory.

World renowned expert Professor Petr Chumakov claimed their aim was to study the pathogenicity of the virus and not 'with malicious intent' to deliberately create a manmade killer.

Professor Chumakov, chief researcher at the Engelhardt Institute of Molecular Biology in Moscow, said: 'In China, scientists at the Wuhan Laboratory have been actively involved in the development of various coronavirus variants for over ten years.

'Moreover, they did this, supposedly not with the aim of creating pathogenic variants, but to study their pathogenicity.

'They did absolutely crazy things, in my opinion.
'For example, inserts in the genome, which gave the virus the ability to infect human cells.
'Now all this has been analysed.
'The picture of the possible creation of the current coronavirus is slowly emerging.'
He told Moskovsky Komsomolets newspaper: 'There are several inserts, that is, substitutions of the natural sequence of the genome, which gave it special properties.

'It is interesting that the Chinese and Americans who worked with them published all their works in the open (scientific) press.

'I even wonder why this background comes to people very slowly.
'I think that an investigation will nevertheless be initiated, as a result of which new rules will be developed that regulate the work with the genomes of such dangerous viruses.
'It's too early to blame anyone.'

He said the Chinese scientists created 'variants of the virus … without malicious intent' possibly aiming for an HIV vaccine.

Reply
List Mist
4/24/2020 11:08:13 pm

Well, https://www.newsmax.com/navrozov/china-biological-russia/2009/09/17/id/335042/ written over 10 years ago speaks well regarding whether the Chinese scientists created variants of the virus with or without malicious intent.

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Nerd has power
4/25/2020 05:46:13 am

Thank you! I obviously agree that this virus is a result of human manipulation. However, I don't think this Russian expert should so confidently say that they did it "without malicious intent". In my opinion, the evidence speaks the opposite.

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RaffRag link
4/24/2020 01:38:05 pm

Thank you, sir. Anyway, I have found this recent article: https://www.nature.com/articles/s41586-020-2179-y. What do you think about?

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Nerd has power
4/25/2020 06:33:27 am

Thank you for bringing up this article. It actually proves two of my predictions. First, it proves that the S1 of the Wuhan coronavirus (SARS-CoV-2 as everyone else prefers) binds ACE2 the same way as SARS S1 does. I predicted this by just looking at the sequence comparison. As I mentioned in the article, there appears to be a well-plotted "copying" of a critical piece in S1 of SARS and then "pasting" it into a template virus to create the S1 of SARS-CoV-2.
Second, this Nature paper proves that the S1 of the RaTG13 virus can indeed bind ACE2. So the RaTG13 virus, if indeed exists, should be able to infect humans. I said in my article that Zhengli Shi needed to take one little peek at the sequence of RaTG13 and realize at once that this virus has the potential to infect humans. There is no reason that she should let this thing sit for 7 years and only decided to publish its sequence once the outbreaks took place.
Note: the RaTG13 S1 gene used in this Nature article was synthesized, not obtained from Zhengli Shi (they have been collaborators in the past), a proof of my other claim --- Shi does not have a physical copy of the RaTG13 virus.

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NotA~A
4/25/2020 02:51:35 am

From the comment section of this article: https://www.virology.ws/2020/02/20/pangolins-and-the-origin-of-sars-cov-2-coronavirus/

------

Regarding the mysterious sequence RaTG13, and the hint that I should look closer at the sequence KP876546 that is cited in the article:
Ge, X., Wang, N., Zhang, W. et al. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft. Virol. Sin. 31, 31–40 (2016). https://doi.org/10.1007/s12250-016-3713-9

The sequence KP876546 in NCBI is very short (only 370 bp) and is defined as Rhinolophus bat coronavirus BtCoV/4991 partial RNA-dependent RNA polymerase. This sequence is also analysed in the article Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats https://doi.org/10.3390/v11040379.

I blasted the KP876546 sequence in NCBI and I got 100% identities with RaTG13 and 99% identities with MT039890 Severe acute respiratory syndrome coronavirus 2 isolate SNU01, complete genome (South Korea). Next closer sequence not from SARS-CoV2 is the pangolin sequence MT084071.
To my opinion the sequence KP876546 could be the first evidence of the RaTG13 sequence or a sequence even closer to SARs-CoV2. In Ge et al., it is stated that the 370 bp sequence was prolonged of 816 bp and the spike protein was sequenced but this information for this sample has been not made public.

I have found a publication on the comparison of KP876546 with SARS-CoV2 before that RaTG13 was submitted to NCBI:

Liangjun Chen, Weiyong Liu, Qi Zhang, Ke Xu, Guangming Ye, Weichen Wu, Ziyong Sun, Fang Liu, Kailang Wu, Bo Zhong, Yi Mei, Wenxia Zhang, Yu Chen, Yirong Li, Mang Shi, Ke Lan & Yingle Liu (2020) RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak, Emerging Microbes & Infections, 9:1, 313-319, DOI: 10.1080/22221751.2020.1725399

The author writes that: “further sequencing of the corresponding PCR product (from SARS-CoV2) surprisingly suggested that the virus discovered is more closely related to BtCoV/4991” (KP876546) “(97.35%) but not SARS-CoV. The genomes of the 2019-nCoV were further analysed to determine its origin and evolutionary history. Full genome comparisons indicated that 2019-nCoV is close to CoVs circulating in Rhinolophus (Horseshoe bats). For example, it shared 98.7% nucleotide identity to bat coronavirus strain BtCoV/4991 (GenBank KP876546, only 370 nt sequence of RdRp gene) and 87.9% nucleotide identity to bat CoV strain bat-SLCoVZC45 and bat-SL-CoVZXC21, indicating that it was quite divergent from the currently known human CoV, including SARS-CoV (79.7%). The close relationship with BtCoV/4991 is quite essential in tracing the potential reservoir host of 2019-nCoV. Unfortunately, the BtCoV/4991 sequence was only partial (373bp in length) and thus no comparisons can be made for the rest of genomes.”

In the article:
Zhou, P., Yang, X., Wang, X. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). https://doi.org/10.1038/s41586-020-2012-7

where for the first time RaTg13 appears, it is written:

“We then found a short RdRp region from a bat coronavirus termed BatCoV RaTG13 which we previously detected in Rhinolophus affinis from Yunnan Province showed high sequence identity to nCoV-2019. We did full-length sequencing to this RNA sample. Simplot analysis showed that nCoV-2019 was highly similar throughout the genome to RaTG13, with 96.2% overall genome sequence identity.”

Interestingly, the article of Ge et al. is not part of the bibliography of this work. To my opinion further sequencing of KP876546 was so interesting that the results were kept secret and manipulations of this virus was carried over until the outbreak of SARS-CoV2.

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Nerd has power
4/25/2020 06:46:00 am

Thanks for sharing! Whoever made the comment has captured something very interesting, which successfully eluded the public's attention.There seems to be a reason that Zhengli Shi does not bring this up herself -- she would struggle to explain it one way or the other. Again, great catch!

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Hydride
4/25/2020 12:19:20 pm

bits and pieces. RaTG13 used for gene assembly, the optimize to get SARS-CoV-2. How could an RdRp from an alphacoronavirus (phylogenetic distance so high that even recombination (require homologous sequences flanking the RNA fragment)was considered impossible with betacoronaviruses.) wind up in a “Natural” bat Betacoronavirus?
Further evidence that RaTG13 was unnatural.

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Bryan
5/7/2020 01:56:43 pm

Great find. This is real evidence that RaTG13 did exist prior to 2020 and wasn't completely made up.

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Veltra
5/10/2020 10:31:55 pm

Did look at the publication--and they did not actually post any of the sequence data in regard to RaBt4991/RaTG13. Except a single figure of the phylogenetic tree. We can still not prove that RaTG13 is not manipulated.

Bryan
5/11/2020 07:45:16 am

It doesn't prove that the rest of the genome wasn't made up, but they did share at least part of the replicase gene in 2015 https://www.ncbi.nlm.nih.gov/nuccore/KP876546.1/

Biohazard0808
6/9/2020 03:30:05 pm

First of all, when looking at the "viruses" isolated from bats, it should be noted that no replication-capable viruses were actually sampled in most cases. As Shi's Science paper from 2005 describes, not a single active virus was isolated from the first 408 samples including blood, respiratory and faecal swaps. P.D. said in a recent interview that a total of 15,000 samples were taken to date just to find a few replicable viruses. Apparently, everything else was "reproduced" from RNA fragments, reverse transcribed into cDNA and re-assembled. In fact bat SL-CoV-WIV1 was the first viable CoV that was isolated in 2013 (described in nature paper). From only about 1.5% of the (mostly faecal samples) a positive RT-PCR was obtained according to the first paper in Science (although sampling methods have improved since then - in 2013 there were 23% that delivered at least a positive PCR result).

This outcome is not surprising, since RNA is extremely fragile and, in most cases, highly fragmented RNA was isolated from samples (e.g. from bat droppings), which can then only be reverse transcribed and amplified into pieces of cDNA with a length of just a few kbp. For most (partial) sequences published there has obviously no active replicating virus been isolated in the first place. However, R.B. special "no see’m cloning technology" allowed the cDNA pieces to be assembled "seamlessly" with each other, as he himself states about "novel means to recover previously uncultivable zoonotic coronavirus strains". Maybe these were also utilized in addition to the few active CoV they isolated to reinfect captured bats that were kept in cages. P.D. says in his interview (US Embassy KL Talks 42:50 min) after being asked if bats were immune to the CoV "We've shown that in the lab", i.e. that isolated (if not reconstructed) viruses were used in infection studies with bats (maybe even the ones which are kept at WCDC). Even if these viruses originated from nature, there is a certain probability that the dead specimens from bat droppings were brought from the caves into civilization.

Regarding sequence analysis: Of course, the observed genetic mosaic may be a result of a recent natural recombination event (otherwise you'd expect a higher divergence between the viruses back in Dec). RdRP copy choice errors in co-infected host cells as B.G. postulated earlier would also be possible. However, his hypothesis does not explain the accumulation of synonymous mutations within spike gene compared to the published bat CoV and the "normal" mutation rate within this "conserved" sequence since its first human-to-human transmissions. This many synonymous mutations within Spike compared to other genetic regions, however, could also point towards a viral barcoding method, similar as used by A. Russell, C. Trapnell and J.D. Bloom in 2018 instead of visibly tagging the sequences. This method was described in a paper titled “Extreme heterogeneity of influenza virus infection in single cells”. They used the technique for heterogeneous influenza virus populations in single cells to be able to differentiate the variants from each other after selection in vitro. It is also possible to distinguish whether a cell was initially infected by one or multiple viral particles or whether an observed viral heterogeneity resulted from "true infection" rather than "mRNA leakage" from lysed cells and perform a great statistical analysis of viral variability.

The purpose of this is to study how the outcome of infection varies between different cell types. To achieve that you must firstly create a library that is subsequently selected in whatever target cell lines (let's take for example Vero E6, Hela, CaCo-2, Calu-3 and 293T) using low MOI as they did with influenza pseudo virions. The artificially selected (still highly heterogeneous) populations are then evaluated by single cell sequencing using the viral barcoding method. Since you need to preserve the amino acid sequences in the barcoded region, you have to use synonymous mutations of course, which could explain the observation within the spike sequence. It is obvious that with a barcode system on a 2100 nt stretch in every third position of a codon you can tag an infinite number of genetic variants, which should not have too much impact on viral growth kinetics. RaGT13 may be just one of these variants from such a library. The spike gene is then used as a conserved sequence (maybe E-protein too), which you can find in extremely heterogenous populations using primers within S-gene without having to sequence the whole 30 knt genome. Although this observation is certainly no proof of a virus engineered in a laboratory, it adds to the evidence that libraries may have been used to test different variants of bat viruses in various cell lines, perhaps in mammals, possibly even in bats.

Reply
NotA~A
4/26/2020 07:54:47 am

something is moving:

https://project-evidence.github.io

https://osf.io/wy89d/

https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

Reply
Nerd has pwer
4/26/2020 07:47:23 pm

Thanks for sharing. The more we dig, the more people digging it, the sooner we will get to the bottom of this. We are moving along.

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RaffRag link
4/26/2020 08:06:03 am

Sir, thank you for your patience and consideration. I have a few questions. First, that the S1 of the SARS-CoV-2 binds ACE2 the same way as SARS S1 (or as the S1 segment from other species, not only bats, does) does not prove that binding is necessarily infectious. For example, does SARS-civet infect humans? Second, isn't the fact that the high level identity for most part of the genome with that of bat coronaviruses is not maintained in the S1 segment of the Wuhan coronavirus only a prove of how nature has learned to preserve the ability to bind ACE2, whatever random mutation or recombination may take place? Others seem convinced that SARS-CoV-2 might be a recombinant virus between viruses closely related to SARSr-Ra-BatCoV RaTG13 and pangolin-SARSr-CoV/MP789/Guangdong/2019. Indeed, coronaviruses have been shown to have a high frequency of recombination (Adv Virus Res, Volume: 48, Pages: 1-100, 1997). Their tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches, including natural recombination in coronavirus associated with human infection. Last, but not least, you suspect that a colleague of yours has only recently published a paper about RaTG13 infectiousness, of which she was aware SEVEN years ago, which has no other explanation than her involvement in spike protein engineering. How could she know that without having checked human infection?

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Nicholas V Thartkins
4/26/2020 09:31:18 am

(Translated from the Chinese section of this blog, originally by Dithionite)
The problem with MP789, GenBank MT084071.1, was that the MP789 sequence was incomplete--A quick check in the GenBank revealed 1872 nucleotides, 1872/27989=6.69% of total, that are marked as "N", or nucleotides that are unknown.
This incompleteness mean that actual, live examples of the MP789 coronavirus does not exist, and what they do have is just the incomplete metagenomic data, Submitted at 13-FEB-2020 and couldn't have actually been sequenced one month earlier , AFTER the outbreak. Impossible to rule out sample contamination, especially if SARS-CoV-2 was used as the template for it's reconstruction.
Secondly, There were the GX pangolin Covs, of which the complete sequence ARE available within GenBank, with RBDs of the same length as MP789 and SARS-Cov-2, but with different critical amino acid residues (Fig.3) on the place where the RBD binds ACE2, with a consensus sequence of
DALTGgNY__LYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV.
Especially for the four major difference between the two, SARS/MP789->GX_P1E/P5E/P4L/P5L/P2V
L(I)446X S(D)499R Q(Y)503H G509N of which will cause either Electrostatic repulsion or Steric clash that would prevent the proper binding of SARS-CoV-2 or MP789 RBD to the pangolin ACE2 receptor,
This sequence is only about 78% similiar with that of SARS-CoV-2, 76% similiar with RaTG13.
As substitution mutations are all that is needed to change the binding affinity for an receptor when the length of the RBD domain was the same, the existence of a Consensus sequence between actual pangolin CoVs that are very different with MP789 imply that the the Actual RBD sequences that can bind to the pangolin ACE2 receptor would be not very different form the Consensus sequence of the GX-pangolin-Covs for ACE2 RBDs of this length.
As the sequence we seen in MP789 or SARS-Cov-2 differs greatly from the consensus sequence between 5 different Complete pangolin CoV sequences that have actual samples of the virus, and since there were computational analysis,
https://www.biorxiv.org/content/10.1101/2020.04.04.025080v1.article-info
that have concluded that Coronavirus ACE2 RBDs that binds optimally to Humans, such like RaTG13 or SARS-CoV-2, binds to the pangolin ACE2 receptor very poorly (and much less than that of ferrets or tree shrews), we could safely say that the MP789 RBD will not bind optimally to the pangolin ACE2 receptor--implying that the MP789 sequence was either contaminated, or was the result of fabrication.

Sequences of SARS-CoV-2, MP789,RaTG13 and the Gx-pangolin-CoVs are available for Multalin analysis below
>SARS_COV_2
NNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
>MP789
NNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFHPTNGVGYQPYRV
>RaTG13
HIDAKEGGNFNYLYRLFRKANLKPFERDISTEIYQAGSKPCNGQTGLNCYYPLYRYGFYPTDGVGHQPYRV
>GX-P1E
DALTGGNYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV
>GX-P5E
DALTGDNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV
>GX-P4L
DALTGGNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV
>GX-P5L
DALTGGNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV
>GX-P2V
DALTGGNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRV

Reply
Nicholas V Thartkins
4/26/2020 10:03:15 am

Especially L449K, of which a major steric clash from the much longer Lysine side chain would shave off at minimum 150KJ/Mol of binding affinity of MP789/SARSCoV2 to the pangolin ACE2 receptor -- bringing the affinity from 699Kj/mol typically seen in Coronaviruses to it's native host ACE2, down to at most 550KJ/Mol.
Burying the charges at R449S and H503Q would knock dowwn another 20KJ/mol, Clash at H503Q would shave off another 50KJ/Mol and L/I446X would probably lose another 4Kj/mol, bringing the pangolin ACE2 Affinity down to about 480KJ/mol -- less than even the RaTG13, which have an affinity of 504.76KJ/mol and are considered not being able to use pangolins as a host In Vivo.
Not a single chance that the alleged MP789 or SARS-CoV-2 could possibly infect a pangolin at all.

Nerd has power
4/26/2020 08:31:24 pm

Thank you so much! Great translation and analysis!

Nicholas V Thartkins
4/30/2020 05:22:44 am

The units should be Kcal/mol not Kj/mol.

RaffRag link
4/26/2020 02:31:47 pm

Line 8 of my last comment
"prove" reads "proof"

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Nerd has power
4/26/2020 08:27:43 pm

I will try to answer each one of your questions.
First, binding data alone certainly does not mean that the RaTG13 virus is actually infectious toward human. But it speaks strongly for such a possibility or potential. That's what Shi was claiming to chase after -- things that have the potential to cross over to humans. The fact that the authors in this Nature paper tested the binding between RaTG13 RBD and human ACE2 indicated that this potential is nothing but legitimate.
Second, you are talking about convergent evolution leading to different viral strains (SARS-CoV-2 and SARS, for example) preserving the same critical motifs for binding human ACE2. It is a true phenomena in evolution. If we are only comparing SARS and SARS-CoV-2, we may very comfortably suggest that the two ended up having similar RBD motifs due to convergent evolution (other parts of the genomes differ quite a lot too between the two, which is consistent with convergent evolution). But here, we are comparing ZC45 and SARS-CoV-2 (Wuhan coronavirus), which share high sequence identity overall and clearly share a recent common ancestor. I'm saying the evolution events that separate these two lineages cannot be random mutation (convergent evolution occurs through random mutation, many many rounds of random mutation). Hope I'm making this point clear.
Now this brings to your next point, could recombination be responsible? I have detailed my reasoning about this in the article. However, you mentioned a scenario that I excluded from my analysis: recombination between RaTG13 and Pangolin coronavirus. Unfortunately, an important part of my article was to disapprove both of these as valid, credible scientific evidence. You may look back at the comments I made on Apr 16th to see additional evidence of RaTG13 being very likely fabricated. Nicholas also kindly translated info on some additional evidence against the credibility of the pangolin coronavirus.
Finally, I don't have any colleague who are involved in the publication. I meant the authors of the Nature paper that you brought up used to collaborate with Zhengli Shi. If RaTG13 is a real virus, these authors could very conveniently obtain a copy of the gene from Shi. However, they chose to synthesize the gene instead. Again, Shi does not need to know whether this RaTG13 can or cannot infect humans to decide to work on it. She only need to recognize such a potential, which is very apparent in this case.

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Nerd has power
4/26/2020 10:20:24 pm

Forgot to comment on the high frequency of recombination in coronavirus. Yes, it is a true fact. However, that does not mean that any type of change in coronavirus evolution is reasonable. I did the best I can to explain how I think recombination is unlikely to lead to the weird pattern of sequence similarity between SARS-CoV-2 and ZC45/ZXC21. Anyone can of course make up his/her own mind on this claim. Again, please check the comment made on Apr 16th. That one, in my opinion, is a very strong evidence.

Reply
Metabisulfite
4/26/2020 08:15:33 am

https://www.biorxiv.org/content/10.1101/2020.01.30.927871v1.full.pdf
The alleged claim of HIV GP120 and Gag inserts in SARS-CoV-2.
By performing a PDB analysis (3JWD, 4NCO) of the HIV GP120 protein, It seems that the first and second insert (V4 and V5 variable regions) formed the CCR5 binding site of the original HIV-1 GP120 protein. the same study have also indicated thet the three inserst are tightly clustered together on the "corners" of the S protein-- in a separate domain that is located before the ACE2 RBD. in addition, the high and uniform pI(11,10 and 10.84) of all three inserts appeared to indicate that these three inserts formed a binding site for a specific protein co-receptor. considered that the HIV V1 binds CD4, V4 and V5 binds CCR5, and the fact that this virus can enter immune cells, it appeared that these three inserts have in deed imparted HIV-1 like function to the SARS-CoV-2.
MAY NEED FURTHER VERIFICATION

Reply
Metabisulfite
4/26/2020 08:22:55 am

Also, leronlimab, a CCR5 antagonist, seems to work well for treating COVID-19.
https://www.clinicaltrialsarena.com/news/cytodyn-leronlimab-covid-19-second-trial/

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Nerd has power
4/26/2020 08:42:40 pm

Thank you for your comments. I have answered this in the Chinese section, I believe. I am actually skeptical whether there are any insertions of HIV sequences in the SARS-CoV-2 genome. I have read this preprint article when it first came out and was not convinced by the evidence within. According to my own blasting, those "inserted" pieces do not necessarily come from HIV.

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Kathy
4/26/2020 08:31:31 am

KP876546 is a SARS-related betacoronavirus

I guess RaTG13 = R = Rhinolophus A =affinis TG= Total Genomic 13= 2013 sequence

A interesting new article:
https://www.researchgate.net/publication/340924249_Is_considering_a_genetic-manipulation_origin_for_SARS-CoV-2_a_conspiracy_theory_that_must_be_censored

Got already a bad comment here:
https://flutrackers.com/forum/forum/the-pandemic-discussion-forum/824572-discussion-chinese-academy-of-sciences-cas-in-wuhan-has-been-working-with-bats-and-coronavirus-for-many-years-dna-manipulations-cloning/page4#post854139

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Nerd has power
4/28/2020 08:37:23 am

Thank you for sharing these!

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Silverfox
4/27/2020 11:42:20 pm

Hi again, I read every day comment on your article, and I realy appreciate your analyse. Then I would like to submit you a new article, from Jean-Claude PEREZ and Luc MONTAGNIER published in preprint on april 26.

https://osf.io/d9e5g/

This article seems to confirm, as the precedent article from PEREZ
https://www.researchgate.net/publication/339331507_Wuhan_nCoV-2019_SARS_Coronaviruses_Genomics_Fractal_Metastructures_Evolution_and_Origins

that there's less and less chances that this virus is natural. As predicted previously, the portions of the RNA code who are mutating faster than the rest of the genome are precisely the "Exogeneous Informative Elements"...

Did you read it ? What do you think ?

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Nerd has power
4/28/2020 08:46:53 am

Thank you for sharing these articles. Unfortunately, I have been a little busy the last few days and did not get to read them. Hopefully I will get to later this week. I may not be able to offer much insights though because I don't have the proper knowledge in Mr. Perez's research area and methods.

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Nerd has power
5/3/2020 06:34:02 am

Sorry for the delayed response. I did get to read Mr. Perez's work here. It's different from the one he shared with us earlier. Although I'm in agreement with Mr. Perez on the conclusion that the Wuhan coronavirus is of a synthetic origin, I again would have to admit that I'm not the best person to judge such an article. Based my limited knowledge, I would say that the sequences suspected as insertions are all on the shorter end, which tend to make things inconclusive in terms of where (HIV or other origins) they could be from. I did find the higher mutational rate of the EIEs over the whole genome very interesting. This could be a sign of artificial manipulation. On the other hand, it seems to me that these EIEs are located within Orf1b and the Spike regions (correct me if I'm wrong as I'm not 100% sure I have this right). I believe that these two regions of the coronavirus genome do tend to see higher frequency of mutations. This could just be a natural trait of coronavirus evolution. Of course, this may also mean that this bioweapon did not go through exhaustive optimization/passage before being put on the real mission and therefore is now adapting to the new host.

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Tholix
4/28/2020 04:15:24 am

https://www.researchgate.net/publication/340786428_COVID-19_Pandemic_Its_Origin_Implications_and_Treatments
Peer reviewed article about the origin of the coronavirus. The same author have purposed a working serum therapy that have saved lives during the early outbreak in WuHan. A therapy that includes the extraction of neutralizing polyclonal antibodies from recovered patients—which could be conceivably cloned to generate both a working vaccine and a viable cure.

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Nerd has power
4/28/2020 08:49:06 am

Thank you for sharing the info!

Reply
Ana Salinas
5/1/2020 05:32:23 am

Hi Nerd has Power, I would like to talk to you a little further. Would you give me your mail? thanks in advance!

Reply
Nerd has power
5/3/2020 06:46:53 am

Do you mind sharing your thoughts here in the comments section? I would prefer public discussions. The communications here have been great. Thank you!

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Edix
5/1/2020 07:58:27 am

I did the Homology based modeling analysis on the MP789 RBD and the pangolin ACE2 receptor.
In order to ensure the free energy calculations are limited to Binding energies only, Chimeric hACE2/pACE2 receptors are constructed using Homology Based Modeling, by swapping out the sequence of the part of the hACE2 protein that binds the ACE2 RBD with that of the pACE2 protein. Similarily, Chimeric MP789 RBD is constructed by swapping out the Receptor Binding Motif(RBM) of the SARS-COV-2 RBD with the sequence from MP789.
The Rossetta-based software, Foldit, was used for the docking and the modeling of the interaction. The proteins are docked and the free energies of the resulting complex were minimized, before a total free energy reading was taken.
The following are the result, in Rosetta Energy Units(REU),of the total free energy of three protein complexes.
SARS-COV-2-ACE2-RBD+hACE2=-522.530
Chimeric MP789-ACE2-RBD+Chimeric pACE2=-498.16
As a control, the total free energy of SARS-COV-2 and hACE2, when separated, is calculated to be -502.69 R.E.U.
Since the canonical binding free energy of the SARS-COV-2 RBD to hACE2 was determined to be -904.76Kcal/Mol,

https://www.biorxiv.org/content/10.1101/2020.04.04.025080v1

and the Rosetta Energy Unit scales only with total molecular mass and number of residues within a protein (of which were the same across two different experiments), The scale of the R.E.U for this particular experiment was determined to be -904.76/(-522.419-(-502.690))=45.85Kcal/Mol.
Using the scale obtained from the control experiment calculation, the binding energy between MP789-RBD and pACE2 was calculated to be (-498.16-(-502.690))=4.53 R.E.U =+207.7005+-500Kcal/mol, with a maximum binding affinity of -293.2995Kcal/mol and a minumum binding energy of +707.7005 Kcal/Mol. None of which could lead to In-Vivo infection as indicated with the same computational study using Bat_CoV as a control on the hACE2 receptor.

A positive binding free energy indicate that the proteins will not dock--the dramatically different binding residue pattern of the pangolin ACE2 receptor would have excluded the SARS-CoV-2 RBD, or even more so, the MP789 RBD as the hydrogen bonding between a binding side Aspartate residue with Q503 of SARS-COV-2 was abolished when the longer Glutamine side chain is relaced with the shorter Histidine side chain.
From closer structural analysis, it turn out that a major clash between Y513 of SARS-COV-2/MP789 and a histidine residue of the pangolin ACE2 receptor where a Glycine was present in the Human ACE2 receptor at the location, along with the abolishment of two(three if counting Q503H) of the four major interactions between the hACE2 and SARS-COV-2 inMP789/pACE2, completely abolishes binding of the MP789/SARS_COV_2 ACE2 RBD to the pangolin ACE2 receptor.



The pACE2 sequence used for the analysis:
>XP_017505752.1 PREDICTED: angiotensin-converting enzyme 2 [Manis javanica]
MSGSSWLLLSLVAVTAAQSTSDEEAKTFLEKFNSEAEELSYQSSLASWNYNTNITDENVQKMNVAGAKWS
TFYEEQSKIAKNYQLQNIQNDTIKRQLQALQLSGSSALSADKNQRLNTILNTMSTIYSTGKVCNPGNPQE
CSLLEPGLDNIMESSKDYNERLWAWEGWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEAE
GANGYNYSRDHLIEDVEHIFTQIKPLYEHLHAYVRAKLMDNYPSHISPTGCLPAHLLGDMWGRFWTNLYP
LTVPFRQKPNIDVTDAMVNQTWDANRIFKEAEKFFVSVGLPKMTQTFWENSMLTEPGDGRKVVCHPTAWD
LGKHDFRIKMCTKVTMDDFLTAHHEMGHIQYDMAYAMQPYLLRNGANEGFHEAVGEIMSLSAATPKHLKN
IGLLPPDFYEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFSGQIPKEQWMKKWWEMKREIVGVVEP
VPHDETYCDPASLFHVANDYSFIRYYTRTIYQFQFQEALCQTAKHEGPLHKCDISNSAEAGQKLLQMLSL
GKSKPWTLALERVVGTKNMDVRPLLNYFEPLLTWLKEQNKNSFVGWNTDWSPYAAQSIKVRISLKSALGE
KAYEWNDSEMYLFRSSVAYAMREYFSKVKKQTIPFEDECVRVSDLKPRVSFIFFVTLPKNVSAVIPRAEV
EEAIRISRSRINDAFRLDDNSLEFLGIQPTLQPPYQPPVTIWLIVFGVVMGVVVVGIVVLIFTGIRDRKK
KDQARSEQNPYASVDLSKGENNPGFQNVDDVQTSF
The hACE2 used for this analysis:
>AAQ89076.1 ACE2 [Homo sapiens]
MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWS
AFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQE
CLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVN
GVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYS
LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWD
LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS
IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP
VPHDETYCDPASLFHVSDDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLL

Reply
Exins FG Adam
5/2/2020 07:18:54 am

https://drive.google.com/open?id=1opowSQgcvpSb58piY1mvSf4AIGzpjssS
I have currently submitted this article to preprints.org and other biorxiv.org--Disproving both RaTG13 and MP789.

Reply
Chris
5/2/2020 10:52:18 am

Thank you. Tell us if the paper is accepted or not
Thanks

Reply
Exins FG Adam
5/2/2020 06:59:29 pm

Sorry but biorxiv.org have rejected my paper--citing that is should be sumbitted on a peer-reviewed journal instead of on BiorXiv.
Can you send the google drive file to luc.montagnier@hotmail.com so he could issue an review?

Nerd has power
5/3/2020 07:51:28 am

Thank you for posting it here too. Great work! Too bad that they turn it down at bioRxiv. I saw some other preprints on this place: zenodo.org. Maybe you can give it a try here?

Reply
Exins FG Adam
5/5/2020 04:30:13 am

https://zenodo.org/record/3786451#.XrFN4YijeUk
Thank you so much. Sorry that I can't show my name here.
This is now a preprint.

Nerd has power
5/7/2020 09:55:17 am

That's great! Congrats on the preprint! Thank you for doing it.

JC onabike link
5/2/2020 01:52:45 pm

I’d love to discuss this with you. I would love to make a video summarizing your take. It’s very similar to mine and others. Harvard2Thebighouse has done some of the first coverage in Jan.

I did some videos exploring papers cited by Dan and here we are now months later.

Please if you are interested in working with me, Ill be making a commentary on this work this week.

https://youtu.be/HmSCMb8Nds4

Be well,
J.C. on a bike

Reply
Nerd has power
5/3/2020 07:35:08 am

Great video! I love the concept of journal club on a bike. You properly shattered that Nature Medicine paper.

It would be great if you could summarize this write-up in a Journal club video. I also have an updated version with some significant additions. It's published on a platform named Gnews:

https://gnews.org/192144/

I will likely post the updated article here too as the one on Gnews had a little too many editing errors unfortunately.

Let me know if you want to discuss anything further. Thank you!

Reply
ioderESTEl
5/2/2020 10:09:02 pm

You may want to check this as well:

https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

In it you will find they linked a 2019 Chinese study that added furin cleavage sites through an off-the-shelf kit method. Apparently it is very easy to do.

The study:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832359/

Inserted into live chicken virus.

The medium post goes into great detail in showing that published research demonstrates the capacity to complete each form of edit necessary for this kind of lab based recombination.

Reply
Nerd has power
5/3/2020 08:05:49 am

Thank you for sharing these info. I love the article written by Yuri Deigin. It's so thorough, rich in information, and in the end convincing (although I'm already convinced). I learned quite a lot of details and side stories reading it. The only thing I would be picky about is that he was too scientific in his discussions and did not properly factor in how evil the CCP can be. It's certainly not Yuri's fault. The CCP certainly want to and can make bioweapons. They are also professional on covering things up, using all sorts of resources. Here they chose to use, among other things, fake science (RaTG13, Pangolin coronaviruses, Nature Medicine paper by Andersen and Lipkin, etc).

Reply
ioderESTEl
5/5/2020 02:00:39 pm

Some other science community commentary on the original Nature Andersen, et al paper.

https://www.gmwatch.org/en/news/latest-news/19387-another-expert-challenges-assertions-sars-cov-2-was-not-genetically-engineered

This in particular cites a method for creating a large number of spike protein candidates using a “phage display library.”

https://gmwatch.org/en/news/latest-news/19383

Note how Nature refused to publish the challenge.

Nerd has power
5/10/2020 05:43:18 am

Thank you so much for sharing these. So glad to see prominent scientists coming out publicly to challenge that Nature Medicine paper and the claim that the virus is of natural origin. I thoroughly enjoyed reading these two articles as they articulated many of the same things that have been in my mind for a long time. I focused on describing the logic and evidence of human manipulation, but I had similar thoughts on how they might have worked on creating this artificial virus. Directed evolution could easily be involved.

Nerd has power
5/3/2020 12:48:17 pm

As you may have noticed, there are new pages added to this site. I just published an updated version of the earlier article on GNEWS (a great news platform created by the heroic Miles Kwok). However, there were quite a few editing errors of my GNEWS article. So I'm posting this updated article here too (RATG13 IS FAKE). This new version benefited tremendously from the insightful comments made here, especially by Ellannor D. Allens and John. F. Signus. I have acknowledged their contributions at the end. Thank you!

Reply
Viennah K Erchus
5/3/2020 06:59:55 pm

A takedown on the recent study claiming that the furin site may have been from HKU9 through "Co-infection"
http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384/4
The HKU9 isolate were found in Guangdong province in 2011--thousands of miles away from Yunnan where they allegedly claim to have started. Bats does NOT migrate--no way that HKU9 could have traveled there.
The RBD from HKU9 belong to the kind that will not infect humans-- Not a chance that it could form a viable infection in humans for the recombination to occur.
About potential recombination--Should the HKU9 claim of a template selection error to be valid, especially since the non-orphaned random 11-mer of the claimed insert sequence are literally found all over biology, we should have seen Bat-borne coronaviruses of Lineage B with inserts at the S1-S2 junction all over the place in nature...... Mors specifically, a variant of HKU9 with the exact insert should have already been detected in bats, should such an insert confers evolutionary advantage in the Bat host.
But there were not a single bat lineage-B betacoronavirus that have been discovered with an insert at the S1-S2 junction, not a single lineage B isolate fron a reviously detected sorce that have a number of amino acids other than 6 between Y673 and R681/684, including RmYN02.
In addition, a Furin site have never been observed within the S1-S2 junction in lineage B betacoronaviruses other than SARS-CoV-2, even the kind that would arise from a single nucleotide substitution as in the Lineage A coronavirus, the MERS-CoV.
This proves that the Bat host, or any Wild animals in that matter, will not tolerate a sequence length at the Si-S2 junction other than 6 amino acids, and a Furin site is certainly not tolerated at the location for anything that exists in the wild.
One of the probable reason may be that such a cleavage would separate the S1 and S2 of the spike protein in a wild animal--the more robust immune system of wild animals, with the kind of antibody that were found in bats, (in comparison to the immunocompromised conditions found in cell or lab passage) will pry the S1 off the S protein shoult it exit the cell pre-cleaved, disabling the virus and stopping such lineages as soon as it emerged.
Such an observation would also be consistent with the general observation that no animals are capable of transmitting the virus outside of lab conditions, without intentional inoculation.
This would have excluded the bat/intermediate host-recombination theory--a recombinant virus with a furin site at the S1-S2 junction will not be able to survive in any animals other than a human, at most past a single host. a virus that can not survive in it's original host, without a rerfectly optimized RBD at least as good as in SARS-CoV, that would allow immediate direct human-to-human transmission (Since neither HKU-9 S1 nor RaTG13 S1 have been proved to show infectivity for humans In Vivo), such a virus is orphaned and will just fizzle out, without any possibility to transmit any further.
Also, dont forget, that they are only able to deduce 10 out of the 12 nucleotides within the insert--
HKU9 gcatttgta caga------cctcggcgggc ctctgt

CoV-2 tatcagact cagac ttg 'ct "cctcggcggg" ' c acgtagt
('' is what they claimed to be the insert in compared to RaTG13, which was very likely invalid, while "" is the part they have claimed to find in HKU9)
There were an entirety of 5 nucleotides, 2 of which were found in the PRRA insert, that were orphaned and NOT found in the HKU9 "source"!
So in order for this particular virus, which couldn't infect a bat any more due to the intolerance toward insertions at the S1-S2 junction in Lineage B betacoronaviruses in a Bat host, to even survive and then come up with a PRRA furin insert at the location, it would have to immediately find the other two missing nucleotides through insertion, an additional chance of 1/(29855^2*4^2)=1/14261136400 for a piece of RNA that were now broken and would be deader than a doornail in the bat cell that the "template selection error" allegedly happened.
So far we have excluded the intra-animal-recombination theory.
Now could this have happened in a co-infection within a human host?
The RBD of the Bat-HKU9 have not been proved to bind to the human ACE2 receptor, nor does it's sequence suggets that it could do, of which the only sequence that is capable of doing so for a bat virus is that of RaTG13, and the ACE2 receptor of a bat is very distant from that of a human--nomatter what kind of In Vitro data they may get for isolated (VERO E6, not even human) cells without an immune system, a bat virus can NOT infect a human In Vivo. Inoculation in the bloodstream to cause an immune response as in a vaccine, Yes. Actual infection as entry into human cells, No. And there were no concerns about a recombinant super-virus arising from a live vaccine inoculation because it can't enter human cells.
Could it be ZC45/ZXC21 and HKU9 in hu

Reply
Viennah K Erchus
5/3/2020 07:01:08 pm

Could it be ZC45/ZXC21 and HKU9 in humans?
According to Figure 3, the RBM of ZC45 and ZXC21 are broken--they will never be able to bind hACE2, and will not enter a human cell in the wild. so the other pieces of the puzzle, the only confirmed wild virus with the exact E protein as in SARS-CoV-2 other than RaTG13 are ZC45/ZXC21. From phylogenetic analysis we have already established that neither inserts nor furin sites at the S1-S2 junction is tolerated in lineage-B betacoronaviruses in a bat or a wild animal, so the only place this could have taken place is within a human. Human cells of which ZC45/ZXC21 can't get into. Again, not possible.
Could it be RaTG13?
According to the analysis done by Elennor D Allens and the newer article here, the sheer discrepancy of sense V.S. silent mutations between RaTG13 and SARS-CoV-2 mean that if one is natural, the other must not be. If RaTG13 is valid, then SARS-CoV-2 is DEFINITELY engineered from it, case closed. If RaTG13 is Invalid, then the only RBD in bat caves that could enter a human cell just vanished. Leaving any S1-S2 insert-and-Furin-site-containing reject Coronaviruses (that could no longer spread and evolve within it's original host unless it drops the insert immediately) without the necassary RBD for it to enter the only host where it can survive and thrive, the Humans.
Additional bat viruses to conferm that an S1-S2 insert is not tolerated in any lineage-B betacoronaviruses that uses bats or wild animals as a host.
AAS00003.1
AAV49723.1
AVP78042.1
AVP78031.1
ADE34766.1
ADE34755.1
AID16716.1
ADE34812.1
AAZ41329.1
ADE34812.1
ADE34722.1
ACU31051.1
ATO98169.1
ABD75332.1
ACJ60703.1
QDF43835.1
ACU31032.1
AIA62320.1

Reply
Nerd has power
5/4/2020 05:23:27 am

Thank you for the thorough and powerful analysis! I think you successfully dismissed all possible routes of its natural occurrence. However, they can always fall back to say that there might be a perfectly identical furin-cleavage site in a bat virus out there that we just haven't discovered yet (don't be surprised if one of the Chinese research labs will soon publish such a sequence). The burden is definitely heavier on our side, but it's becoming more apparent. The truth will come out.

Eitcherius F Asken
5/5/2020 03:55:23 am

https://www.youtube.com/watch?v=uZUJhKUbd0k
Well, someone in this have just pointed out a study that confirmed that neither furin sites nor inserts in the S1-S2 junction exist within any coronavirus spike proteins with sequence homology to SARS-CoV-2 higher than 40%. Confirming the finding that such a site is likely not tolerated in wild animals.
Without possibility of Furin-site persistence in the wild after an alleged recombinition, it becomes exponentially unlikely that the single virus, foolish enough to get this site or inserts at this location in a wild animal, to just jump to humans in the exact same time befor it gets eliminated. and even if this DO happen, the lack of a viable RBD within bat caves that constitutes in-vivo human infections, let alone human-human transmissible ones that is necessary for persistence of the hypothetical precursor in the human population, even after a furin insert, prevent any possible "spillovers" of this potential virus to the human population.
That is, unless cell culture were involved in it's passage, so the potential infection is not immediatedly eliminated by a human/animal-like immune system. However, with cell culture passage, this will be a clear lab-based scenario.

Nerd has power
5/5/2020 05:19:24 am

Thank you for posting this YouTube video. I listened to the whole thing. It's wonderful. I apparently agree with you and Chris in this video that the natural occurrence of the Furin-cleavage site is impossible. What I did not appreciate in full earlier was that all the viruses containing a similar (no one has the exact) sequence are less than 40% identical to SARS-CoV-2. That makes the chance of such a site arising from natural recombination close to zero. It's a great finding. Of course, that family tree, portraying SARS-CoV-2 being the one in that half of the branch that has a furin-cleavage site, is also very powerful. Better than what words can describe. Overall, the claim that this furin-cleavage site is a result of lab manipulation is becoming rock solid.

Ornius
5/3/2020 07:47:38 pm

Someone kindly reminds me that such co-infection experiments have already taken place in IMMUNOCOMPROMISED BAT CELL LINES by Zhengli Shi. So a recombination during cell passage may also be a plausible origin of the Furin site, in addition to deliberate insertion.
Smoking article:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178081/
2018.
"We believe that future studies should investigate coinfections in specific bat cell lines using a coronavirus similar to Ro-BatCoV HKU9 & the relevant orthoreoviruses from which it will be possible to reveal more of the underlying basis of heterologous coronavirus recombination"
Coinfection may be unnecessary in this case though, as chimeric Spike proteins with a SARS/MERS RBD and the C terminus gene, bearing the sequence claimed to be the source of the Furin site insert, derived from the very HKU9 coronavrus spike protein, were already patented by Ralph Baric in 2015.
Smoking patent:
https://patentimages.storage.googleapis.com/e9/b2/98/15d4808eff87c9/WO2015143335A1.pdf
If someone decided to perform cell or animal passage of a spike protein, constructed according to the patent, within lab condition, where Polybasic cleavage site inserts ARE tolerated for influenza viruses, The site could then arise from an Intragenomic recmbination event and be easily tolerated within the cell lines or intentionally-inoculated lab animals, where it manifests as an increase in pathogenicity. (bad in wild animals as this will likely either kill the host and destry the virus prior to transmission, or separate the S1 and S2 and deactivate the virus itself against a wild-animal immune system.) Increase of pathogenicity that would be then immediately recognized as a potential bioweapon.

Reply
Nerd has power
5/4/2020 05:39:12 am

Completely possible. They could introduce it in several different ways and animal passage might very likely be involved. The method that was actually used may only be revealed by the one(s) who did it.
The literature/patent evidence, if you dig, clearly says that the techniques and knowledge are all out there for someone to create, conveniently, a bioweapon-equivalent virus.

Viennah K Erchus
5/4/2020 10:57:40 pm

I have seen bad comments on your Gnews article.
CCould you also include an E alignment of Bat and SARS coronaviruses to show that this protein is indeed highly variable, even in bats, in your article?
SARS_CoV_2, ZC45/ZXC21, RaTG13, Bat and SARS sequences provided below.
>AA_Wuhan-Hu-1_E
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCC
NIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV
>AA_ZC45_E
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCC
NIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV
>AA_ZXC21_E
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCC
NIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV
>AA_RaTG13_E
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCC
NIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV
>AA_Bat_APO40581.1
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYIYSRVKNLNSSQGIPDLLV
>AA_RsSHC014
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSQGVPDLLV
>AA_SC2018
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTIYVYSRVKNLNSSEGVPDLLV
>AA_Bat_NP_828854.1
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV
>AA_SARS_GD01
MYSFVSEETGTLIVNSVLLFLAFMVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV
>AA_BtRs-BetaCoV/HuB2013
MYSFVSEETGTLIVNSVLLFVAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV
>AA_SARS_ExoN1
MYSFVSEETGTLILNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV
>AA_BM48-31/BGR/2008
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTFYVYSRVKSLNSSQEVPEFLV
>AA_SARS_TW-GD1
MYSFVSEETGTLINSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV
>AA_SARS_Sino1-11
MYSFVSEETGTLINSVLLFLAFVVFLLVTLAILTALRLCAYCCYIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV

Reply
Nerd has power
5/5/2020 05:24:27 am

I certainly can! Thank you for providing these sequences. I don't think I get to update the GNEWS article, but I can update the version here by adding an amendment. I will leave a comment on GNEWS and direct people to check it out on this blog. I hopefully will get it done by the end of tomorrow. Thanks again!

Reply
Nerd has power
5/10/2020 05:52:34 am

Hi Viennah, sorry for the delay, but I had finally updated the article per your suggestion. Thank you so much for retrieving these sequences and put them into the right format. It made creating the figure so much easier. I will leave comments under the GNEWS article and encourage people to migrate over here to see the updated info.

Reply
Kathy
5/6/2020 03:25:27 am

I have a question: the furin cleavage site sequence is inserted in SARS-CoV-2 in a way that produces a split of the codon for Serine in the pangolin sequence MP789 and Ratg13. Please see fig.1 here:

https://www.researchgate.net/publication/340924249_Is_considering_a_genetic-manipulation_origin_for_SARS-CoV-2_a_conspiracy_theory_that_must_be_censored/figures

I know that you consider Mp789 and RaTG13 not real, but could this insertion prove that the furin sequence was artificially inserted? I am wondering if natural recombination could result in such split. All the people I have asked so far do not know.

Reply
Firox TD Jackinson
5/6/2020 04:45:26 am

It maybe that the insert was made in that way. Site-directed mutagenesis experiments are very conservative about the number of nucleotides they can insert/delete, and would normally be designed in such way that it minimizes the change to the original genome while achieving the goal of the operation. Making the result look as natural as it can. So if the one who made the insert found out that by splitting the Serine codon one can minimize the overall change toward the entire genome and make it look more natural than just inserting in-frame, then it will be such an insert that ended up being chosen.
Also, It's unclear that the insert was actually out-of-frame, as https://www.youtube.com/watch?v=uZUJhKUbd0k pointed out, the insert can also be completely normal and in-frame, if a silent mutation was ended up being introduced into the overall sequence at the 3rd position of the S679 codon during the subsequent optimization process, Inserting/replacing 13 nucleotides in total. Which is likely the case since the new silent mutations introduced, considering the overall difference from RaTG13>SARS-CoV-2, was quite uniform across this region, Indicative of a round of optimization after insertion.
It may also be a distraction, should they decide to orchestrate the insertion/substitution in such a way that it would appear out of frame by +1. As this would make it look more "natural". This may be of significant consideration when designing a Bioweapon without leaving a trace--if they find out that the can shift the insert by +1 while preserving the overall sequence, they will surely do it in that way to minimize trace. 12 nucleotides are not that hard to design in this way.
In fact, due to S679's high variability, should the +1 out-of-frame insert be natural, then that paticular Serine will unlikely be preserved, as the first nucleotide, not in-frame to the PRRA, have a 50% chance of changing it to an Arginine. Which will make the Furin site canonical (RPRRAR, two cuts instead of just one) and dramatically increase the infectivity than even the current PRRA. So the only reason for such a choice is because that the entire sequence was first laid out in Amino Acids, then back-translated to nucleotides--of which the S->R mutation will not be considered when designing the Amino acid sequence alone.
Remember that neither RaTG13 nor MP789 were independently sequenced other than by their original publisher--and both of them were likely fabricated. So the RaTG13 and MP789 sequences themselves may well be made in such a way that it make the insert looks out-of-frame, hiding the traces of artificial insertion.
https://zenodo.org/record/3786451#.XrKaHoijeUm

Reply
Firox TD jackinson
5/6/2020 05:30:56 am

Looked up the codon table-- and the +1 insert is very easy to design. the codon for A/Alanine can have any nucleotide at the 3rd position, so just keep the 3rd position of the original S679 at the last 1bp of the PRRA. So leave it unchanged & save 1 nucleotide on your primer. The Codon for Serine TCA/TCT can also have any nucleotide at the 3rd location and it's not in-frame with the P-R-R-A. So just change it to anything else.
Sorry for the prior Arginine claim. But it demonstrates that is very easy to design the insert in such a way that it would appear +1 out-of-frame and look more "natural". Primers for Site-directed mutagenesis are very expensive and a thorough consideration for nucleotide design must have taken place prior to performing such an insertion. Depending on techniques used, Placing the insertion at frame +1 may actually reduce the # of nucleotides needed to be designed/synthesized to 11 instead of 12 (by reusing the end of the reverse primer, shifting it by -1). Which is unlikely to have slipped past the designer's eyes when every extra nucleotide count in such experiments. It may even look more natural if a template that may form such a sequence was already recorded in their databases--random 8-mers are not that hard to hunt down from GenBank, especially if they can fit the core NCCNCGNCGNGC(N) to some virus of bat origin, nomatter how strange or out-of-frame it may be on the "source". 2^8=65536--a suitable candidate could be found once in every 65536 nucleotides of random DNA. Both R in the P-R-R-A insert uses the codon that can have any nucleotide at the third, or Wobble position, CGG-CGG--and that is not by accident. So does the split Serine codon. It reduces the range one needed to search for a "natural" candidate down by as much as 2*2*4*4=64 times. Making a Cover-up much more easier, especially since this is a Bioweapon that needs to be covered up.
And it's not by accident that such a choice reduce the total number of "defined" nucleotides within this sequence to the absolute minumum--It was designed as such. To both save cost for Oligonucleotide synthesis, to minimize overall changes to the genome, and to aide in covering up the bioweapon.
The bottom line is that all such nucleotide-changing experiments are always carefully designed nucleotide-by-nucleotide because Oligonucleotide synthesis costs far more than computational screening/design of the sequences themselves. Modern tools have made optimizing the required changes cost next to nothing while Oligonucleotide synthesis still cost a lot and still require weeks to complete. Therefore, even a single nucleotide reduction in the length of sequence they needed to synthesize (which saves a fortune) matters enough to justify putting the entire insert +1 out-of-frame (Which costs next to nothing).
Especially if such a design choice makes covering-up the traces much more easier (which is one of the uttermost concern for designing a bioweapon).

Firox TD Jackinson
5/6/2020 05:42:26 am

4^8=65536.

Kathy
5/6/2020 07:52:31 am

Thank you very much for checking this and the links!

I think that here https://www.youtube.com/watch?v=uZUJhKUbd0k at 19:31 they did just a translation of the amino acid PRRA to show the nucleotide sequence of the furin site. My alignment of SARS-CoV-2 and RaTG13 is quite different and also here at a first look you think that it is frame, but you can see the shift checking the codons.

The real sequence inserted is ctcctcggcggg and not cctcggcgggca as they show.

Anyway, for the rest I liked very much. Mostly the comments about Andersen & co. eheheh

I agree that this sequence must have been designed to preserve as much as possible the original amino acid sequence. The change in the codon is not visible at a first look.

Nerd had power
5/6/2020 12:39:26 pm

Thank you for the analysis and explanation. I did not get to read the original article, but I don't think there is anything that you did not cover :). The Youtube video was excellent. It made it so crystal clear that the furin-cleavage site was not out of natural evolution.

Firox TD Jackinson
5/7/2020 05:49:54 am

Thank you for your analysis. Technically the insert could be either +1 or +2 out of frame, or is a 12nt in-frame insertion+1 silent mutation. With the +2 insert however, it would make the needed primer 2nt shorter than the in-frame 12, by shifting the reverse primer by -2. reducing the total absolute extra length need for synthesis to 10. Unlikely to have slipped past the eyes of the designer/software when designing the insert/primers.
Also, if the insert were indeed +2 and is natural, then there will be an extremely high chance (11 out of 12) that the Serine will not be preserved-- it may turn into F,Y, L, C or W. of which L is known to appear on the S1-S2 junction and was the go-to for most betacoronaviruses of Lineage B.
Technically, whether the insert was +1 or +2 was simply the result of the kind of algorithm you uses for alignment-- it depends on the "seed squence" that were used in this kind of analysis. Both +1 and +2 inserts are equally valid for a 12nt insert, and a 12nt in-frame insert with a silent mutation at the wobble position of the S679 Codon is still the most likely scenario.
With a +2 insert, however, it would likely save one extra nucleotide on the primers used and not change the total number of defined nucleotides that is needed to search for a cover-up. So maybe it was what that was used in the final design decision.
However, with the Ser and Ala codon both having C at the second position, Both a +1 and +2 primer design will be equally valid and will apear identical on the final sequence. There will be no distinctions. and a +1 primer is still the most likely scenario for the insertion, since it's easier to design.
Also, since the first 1, or 2 nt of the 12nt insert are both "Orphaned" on Genbank, the actual case for primer design may still be +1 instead of +2. as that will be the insert that is most similar to a sequence that is found on GenBank, instead of the +2 one.
However, since the +2 insert costs 1nt less for Oligonucleotide synthesis than the +1 one, it may be chosen due to economical reasons once they figured out that the cost of the +1 design could be further reduced by shifting the frame by a further +1 nucleotide on synthesis while preserving the original destination sequence.
However, since either design will end up with the exact same nucleotide sequence on the SPRRA junction, we may never know which was actually chosen. As both S679 and A683 have C on the second position for all codons that preserves the flanking sequence, There are no alternative forms of the +2 insert other than a simple frameshift of the +1 insert by +1 for S-P-R-R-A, hence both primer designs are equally valid.
Also, Even with a +1 insert, you still end up having to make up the first 1nt of the insert by an extremely unlikely insertion--a frameshift after recombinition is always lethal. As insertion rates are at a measly 10^-7 per site per cycle for even the most unstable retroviruses,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC253716/pdf/jvirol00087-0299.pdf
and that the recombination and the insertion would have to happen at the exact same cycle on the exact same RNA molecule, with recombinition rate of less than 1/1404.76 that of the substitution rate, The chance of such an insertion to happen at the exact same site as the recombinition, which was iteslf happen less than (120/1404.76)=0.085 per species per year globally, is less than 1e-7*0.085/(29855^2)=9.58*10^-18 times per species per year. Assuming every bat carries it's own species of Coronavirus (which was order of magnitudes higher than what may actually be since there were never observation of >30 species of Coronaviruses per cave, caves that hosts ~100 to ~3000 bats each), and all the bats are allowed to recombine with the one spicies that had this exact sequence, the smallest bat weights ~10g and the total mass of wild mammals being 3.8e+7 tonnes, and let's say all of these were bats (which was obviously not the case), it would still take at least 26841.63 years for such an event to happen in nature for Once.
And we know that Furin sites were not tolerated in any coronaviruses that had a Spike(S) protein gene sequence homology larger than 40% with SARS-CoV-2, meaning that such a site can never survive in a bat even if it could form, It would have to then enter humans at the exact same cycle as it was formed or else it will be eliminated.
As the closest Coronavirus to SARS-CoV-2, RaTG13, was allegedly first obtained in 2013 and does not have a furin site, This leaves the total time window for such an opportunity being at most 7 years where an emergent site of this strain could enter humans.
7/26841.63=1/3834.519. This is the chance that the Furin site found in SARS-CoV-2, so far being the only example (Species) of it's clade, being the result of a natural recombinition event, even assuming such recombinition could happen in the first place.
In the other way around, the chance of the Furin site insert within the S1-S2 junction of S

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Firox TD Jackinson
5/7/2020 06:02:41 am

Protein being of Non-natural origin is more than 3833/3834, or more than 99.9739%
Or, spoken in the other way. It is 3833.519 times more likely that the PRRA furin site insert of SARS-CoV-2 was the result of purposeful manipulation, instead of being the result of "natural recombinition".

NasCinx
5/6/2020 04:01:58 am

It seems that a lot of debate was going on a short 370bp partial RdRp sequence known as RaBtCoV/4991, claiming that it was the first sample of RaTG13. However, this does not remove any of the questions surrounding RaTG13.
First, if there was already RaBtCoV/4991, why they did not recognize and publish the RBD?
This will be a proof that the rest of RaTG13 was fabricated from the small sequence.
Second, Assume that RaBtCoV/4991 is indeed complete and is RaTG13, then it would be obvious that SARS-CoV-2 was made from it, as pointed out by Elannor D. Allens and your more thorough analysis on the identicality of E and unnaturally low sense/silent mutation rates of S2.
"Out of SARS-CoV-2 and RaTG13, only one could be natural."

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Nerd has power
5/6/2020 12:40:28 pm

Exactly!

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Kathy
5/9/2020 02:35:51 pm

Now we know it:

Peter Daszak confirmed that RaTG13 is BtCoV4991.

https://twitter.com/PeterDaszak/status/1259111768217063424

Link to the database:

http://www.mgc.ac.cn/cgi-bin/DBatVir/main.cgi?func=accession&acc=MN996532

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Nerd has power
5/10/2020 06:27:23 am

Thank you for sharing these, Kathy. I invited Peter under that tweet to come over and see how "RATG13 IS FAKE". I would love to see his comments coming up in this section.

RV
5/7/2020 12:10:59 pm

I find your hypothesis very compelling and I also find an article arguing the opposite very compelling. What do you think? You both argue your cases very well and as a layman I don't know who is right. I hope you will look at it and consider a rebuttal.

http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384

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Alix
5/7/2020 07:02:07 pm

See Viennah K Erchus’s comment on the takedown of that hypothesis.
Also, HKU9 is a guangdong strain while RaTG13 is a Yunnan strain. They are geographically too distant to be seen infecting the same cell of any animals. Bats included. Except in labs where “co-infection experiments” were performed in bat cell lines.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178081/
2018.
"We believe that future studies should investigate coinfections in specific bat cell lines using a coronavirus similar to Ro-BatCoV HKU9 & the relevant orthoreoviruses from which it will be possible to reveal more of the underlying basis of heterologous coronavirus recombination"

Also see Firox TD Jackinson‘s comments on why such an insert was ended up being chosen. Remember that this is a bioweapon and it needs Cover-up. The choice both eases cover-up , is more economical in term of Oligonucleotide synthesis and is very easy to design

Reply
Firox TD Jackinson
5/9/2020 09:07:03 pm

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075523/
Well, I just recieved a confirmation from Yuri Deigin that the HKU9 Spike will not bind ACE2, DPP4 nor CD147(which was on the NTD of SARS-CoV-2, the part where the "HIV inserts" were). The Spike protein is just too distant from either SARS-CoV-2, RaTG13 nor MP789 for them to even bind to the same receptor, let alone to the same cell in the same species.
Since HKU9 can't even infect the same kind of cells as RaTG13, MP789 or SARS-CoV-2, let alone the same species, Any recombinations with HKU9 can be solidly ruled out.
https://medium.com/p/98c838e8387e/responses/show

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Nerd has power
5/10/2020 10:24:32 am

Thank you, Alix and Firox. Very nice info and analysis!

Hi RV, this Youtube video explained the Furin-cleavage site issue very well. I highly recommend that you check it out:

https://www.youtube.com/watch?v=uZUJhKUbd0k

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NM
5/7/2020 01:26:08 pm

hopefully you have seen this ....
https://www.zerohedge.com/health/database-wuhans-batwoman-altered-48-hours-covid-19-samples-ordered-destroyed

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Rikaja
5/7/2020 01:58:00 pm

Amazing research. Please, try to publish in a peer-reviewed journal for scientists to see.

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Bryan
5/7/2020 02:10:06 pm

One thing that really throws off estimations of similarity between RaTG13 and SARS CoV2 is the weird mutation pattern. Almost all the SNPs are C<->T or A<->G changes. This is not the mutation profile of replication errors. A recent article makes a pretty good case that this could be the result of deamidation by host cell APOBEC or ADAR proteins.

https://www.futuremedicine.com/doi/full/10.2217/fvl-2020-0066

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Argin
5/7/2020 05:41:34 pm

Technically, this will not affect the mutation rate analysis as the same holds true for ZC45/ZCX21 and the rest of RaTG13/CoV2.
The Syn/Nonsyn subatitution rate data actually accounts for all possible RNA modification systems within host cells, as SNP polymorphisms like this are just equally as likely to cause AA changes as the “normal” kind.

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Bryan
5/9/2020 09:11:04 am

That's not quite right. I'd assume enzymes like APOBEC edit any position in a codon equivalently. But the type of mutations done by APOBEC or ADAR are transitions (APOBEC: T<->C and ADAR: A<->G). Generally in the 3rd, or "wobble" position of a codon, transitions do not change the encoded amino acid, whereas a transversion does. Transitions will still usually change the encoded amino acid if they occur in the 1st or 2nt position of a codon. Interestingly, I just did a quick analysis of the SARS-CoV2 spike protein and there is a somewhat higher percentage of Cs and T/Us in the 3rd codon position (62% C/T content) compared to the first (41%) or second (54%). If APOBEC randomly converts any C/T, it's more likely to occur an the wobble position just because of the abundance of C/Ts. Overall, this means that APOBEC mutations are much more likely to cause silent (synonymous) mutations than more random mechanisms (like coping errors).

Argin
5/10/2020 08:15:41 am

However, when you look at the mutation rates between the rest of RaTG13/CoV2, the Syn/Nonsyn ratio is completely normal near 5:1. If an RNA modification system was involved, why it would selectively modify just the S2 but not the rest, like ORF1ab, M or N?

Also, APOBEC and ADAR works on Cellular mRNA. they have a single reading frame and can be easily modified on the Wobble position only. Coronavirus Genomic RNA, however, contain multiple ORFs and the APOBEC and ADAR will make their modifications Out-Of-Frame, nullifying the "wobble position only" hypothesis.

The real problem is that the the nonsyn/syn ratio on S2 was unnaturally low between RaTG13/SARS-CoV-2 when compared to the rest of the sequence itself (which look completely normal) and the fact that they still manage to hit an overall 5:1 Syn/Nonsyn ratio on the entire S1+S2. (but bungled with the distribution of Syn/Nonsyn mutations)

The cover-up was what have exposed them.

Again, the real problem it not that the nonsyn/syn substitution rates on the S2 was unnaturally low, it was the fact that it was Unnaturally low When compared to the rest of the sequences.

Argin
5/10/2020 08:26:36 am

62% compared to (54%+41%)/2 was not that high. Assuming only conversions from APOBEA/ADAR possible, it will only cause a minor (6.526-to-1 instead of 5-to-1 "natural") deviation from the established pattern. Not the complely unnatural 44-to-1 ratio as we see here. Even factoring the RNA modification, this ratio still return a chance of 2.76e-6 for the entire S2 protein to be natural. a 1 in 361730 chance.

Argin
5/10/2020 04:54:37 pm

In fact, if APOBEC and ADAR causes more synomynous changes than normal mutation processes in the viral RNA, we should have seen a change in the codon preference between RaTG13 and SARS-CoV-2--the ratios of codon usage should have changed since an elevated level of synomynous changes over non-synomynous changes will bias the codon preference of SARS-CoV-2 significantly from "the extreme CpG deficiency of SARS-CoV-2" when compared to RaTG13.
However, as per the analysis done by Yuri Deigin
https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748
The codon usage ratio of SARS-CoV-2 is nearly identical to RaTG13. There were no bias as would be expected from an eleveted level of synomynous modification when compared to non-synomynous changes.
It turned out, APOBEC and ADAR recognized specific motifs on the RNA-- they does not recognize reading frames and the changes are just as equally likely on any of the 3 possible reading frames on any pieces of RNA. Motifs are not "Codons" because they are two times as likely to be out-of-frame than being in-frame in the context of a specific gene, which occupies only 1 of all the 3 possible reading frames.
Because APOBEC and ADAR changes equally likely on all the 3 possible locations on a single codon, the changes they make will not prefer synomynous changes over non-synomynous changes outside of specific cellular mRNAs that co-evolved with these enzymes.
Therefore, by comparing the Codon usage ratio of RaTG13 to SARS-CoV-2, we can safely assume that the RNA modification systems did not contribute to any higher syn/nonsyn ratio than normal mutation processes did.

Nerd has power
5/10/2020 06:25:36 pm

Can't agree more. Wonderful discussion and analysis. Thank you!

Exid
5/10/2020 08:11:15 pm

It also appeared that the CpG deficnency in RaTG13 and SARS-CoV-2 was ancestral-- it likely happened BEFORE the two viruses branched away on the evolutionary tree. So Whatever kind of modification system at work in the ancestor have stopped working when RaTG13 and SARS-CoV-2 came to place.

Also, SARS-CoV-2 showed a higher level of CpG deficnency than RaTG13--Confirming the previous argument that any processes that prefers synomynous over non-synomynous substitutions in this kind of genomic change will cause a significant bias on the codon preference of RaTG13/Sars-CoV-2. which, as said before, were nearly identical.

Even assuming that the editing process have reached their endpoints (which is likely not since SARS-CoV-2 is clearly more CpG deficient than RaTG13), RaTG13 and SARS-CoV-2 had vary different hosts whose codon preference are very different(Bat vs human). (see the Codon preference part on https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748 for all the different viruses, their codon preference and their hosts)

Should the introduced mutations are largely synomynous (which was not), this kind of drift and host change should have caused the Codon prefernce to diverge significantly from each other, both from normal genetic drift during editing and adaptation to different hosts. They should never be near-identical for the number of mutations we see here. Especially the Lysine codon who have AAA or AAG ratio that are identical in both strains. The A/G on the wobble position is a substrate of ADAR. which interconverts them. Should this be biased toward Silent vs sence, it should have caused the AAA/AAG ratio to drift away from each other. Quite significantly.

In fact, the only way for the codon prefernece to staty constant with significant mutations taking place is for the Sense/Silent ratio on the majority of the sequences to be normal. as any bias toward the wobble position invariably lead to a significant change in the codon preference (Wobble positions are what determines the Codon preference, therefore mutations biased toward there translates to an equal bias toward changing the Codon usage ratio significantly, especially the A+C and T+G, which was not under any selection pressure under the context for a drive toward lower CpG), Either way around. Such a change was not observed between RaTG3/SARS-CoV-2.

In fact, the near-identical codon preference of RaTG13 to CoV2 (which stand out in their own separate class, previously undetected in any other betacoronaviruses) point toward the sequences being fabricated in a way that the codon preference was closely matched toward SARS-CoV-2. This is usually done by changing the wobble position on the non-synomynous locations (as well as introducing additional synomynous mutations, keeping the total Syn/nonsyn ratio to 5:1).
Also, the large CpG deficnency also point toward optimization for quick replication as ApT replicate faster than CpG. Which were likely overdone to make the bioweapon as lethal as it can. This kind of CpG ratio was never detected in any other betacoronaviruses other than RaTG13 and SARS-CoV-2, including the alleged pangolin strains. pointing out an artificial origin of RaTG13/Sars-CoV-2. This have also excluded the idea that either strains had been evolved in pangolins for any amount of time. (which had a different codon preference and an elevated CpG ratio, instead of being CpG deficient).

Nerd has power
5/12/2020 02:18:42 pm

Thank you, Exid. Great finding! Unless they argue that SARS-CoV-2 directly crossed over from bats, they would not be able to answer why the codon bias is the same between SARS-CoV-2 and RaTG13.

The unique deficiency of CpG in both SARS-CoV-2 and RaTG13 speaks for the possibility that both might be unnatural -- SARS-CoV-2 optimized for enhanced replication/infection and yet RaTG13 sequence fabricated by following the same codon bias of SARS-CoV-2. Excellent work!

Bryan
5/14/2020 10:33:56 am

Argin,
You bring up some good points. The fact that APOBEC causes transitions, not transversions wouldn't, by itself' address the variation in syn/nonsyn rates across the genome, but only the overall syn/nonsyn rate. Maybe identifying APOBEC motifs could address the different rates in different regions, but I'm not sure how to do that analysis. It would also be interesting to look at just the transitions/posible transition rates for syn and nonsyn. Maybe there are so many transitions between RaTG13 and SARS-CoV2 because there a disproportionate amount of syn mutations, not because of APOBEC editing. That would be an interesting finding, and could blow up the APOBEC hypothesis.

I think we agree that APOBEC/ADAR edits would be equally likely in all reading frames. That's not why these edits might skew the overall rate toward more synonymous mutations. It's because of the type of mutations (transitions). For neutral mutations, there are 3 possible mutations at each nucleotide. Almost any mutation at position 1 or 2 will be a nonsynonymous mutation. Generally, the two transversions at position 3 will also be nonsynonymous where as the 1 transition at position 3 will be nonsynonymous. Thus for unbiased mutations, a mutation in a codon will by synonymous 1 time out of every 9 mutations, and will be nonsynonymous 8 times. So 11% synonymous (it's actually a bit higher than that since a few transversions in position 3 and a few mutations in positions 1 and 2 can be silent too). Whereas, if you only have transitions happening then you have 33% synonymous since a transition in position 1 or 2 are nonsynonymous, transition in position 3 is synonymous. Thus APOBEC/ADAR mutations are 3 times more likely to cause synonymous mutations than random mechanisms.

Another detail that seems to be being glossed over is this 5:1 expected ratio. There isn't one overall ratio like this. It really depends on the type of selection pressures an organism experiences. A ratio of 5 syn to 1 nonsyn would indicate fairly high purifying selection. A completely neutral ratio would have multiple nonsynonymous for every synonymous since most SNPs cause an amino acid change. If a virus jumps species, I would think that there would be high positive selection in at least some regions of the genome (e.g. the RBD), which would throw off the syn/nonsyn ration in those regions.

Nerd has power
5/18/2020 09:18:30 am

Hi Bryan, I would like to comment on the last point you raised. I agree that the 5:1 ratio is very raw. It's a general trend due to the purifying pressure that most proteins experience in evolution. You are also correct that different protein/sequences, when facing different selection pressures, would exhibit different dS/dN ratios.

For S2, however, there is high purifying pressure as this part of the protein has to maintain the trimer formation. Failure to do that would severely affect viral infectivity. So there seems to be a reason that 5:1 ratio is kept here.

More importantly, I did the analysis by comparing the natural group (ZC45 vs. ZXC21) and the suspicious group (RaTG13 and SARS-CoV-2). Basically, the ratio exhibited by the natural group would very likely be held constant by other coronaviruses even if they are jumping to other hosts -- S2 is not involved in making known interactions with host proteins. Again, its job is to mediate trimerizaiton. For S2, a dS/dN ratio of 44:1 is too far off to be coming out of nature.

Bryan
5/19/2020 03:51:34 pm

Hey Nerd Has Power,
I think the comparison of SARS-CoV2 and RaTG13 may be more typical than you realize if the only comparison you made was to ZXC21 vs ZC45. I've looked at a dozen or so S-protein comparisons like you did (that SNAP tool is pretty handy). Other natural CoVs show as big or bigger changes in syn to nonsyn mutation ratios at about the same spot in the S protein as SARS-CoV2 and RaTG13.
Higher purifying selection in the S2 portion of ORF2 is a trend in these viruses. Throughout the S-protein there is a constant synonymous mutation rate. However, the nonsynonymous rate is almost always many times higher in the S1 portion than in the S2 portion. When you are looking at really similar proteins like the S proteins from ZXC21 and ZC45 it's easy to miss this trend since there are so few mutations there’s poor signal to noise. You pointed out that the syn/nonsyn rate for the part of S2 that you chose was 44:1. That's right, and it's equivalent to the ps/pn=104 (adjusting for probabilities). Whereas the S1 portion of the protein up through the RBD, you have a ps/pn of 31. Less than a third of the S2, that’s a pretty big difference, right? Actually, it seems pretty consistent for these CoV pairs. I’ll ignore the two genomes you are suspicious about (RaTG13 and the pangolin MP789) and go slightly more distant to compare the bat CoVZC45 to the pangolin CoV GX-P5E. If you compare the S1 portions of these genomes you see the ps/pn is 3.4, whereas the S2 portion’s ps/pn=18. If the the SARS-CoV2 vs RaTG13 3X higher ps/pn ratio in S2 was big, this 5X change in the natural pair is huge. S2 is just under much higher purifying selection that S1. Like you said, “S2 is not involved in making known interactions with host proteins. Again, its job is to mediate trimerization”, thus it’s not going to be under positive selection, it’s going to be under high purifying selection. That’s exactly what we see when comparing various pairs of CoVs, including SARS CoV2 and RaTG13.

A different issue is that the overall ps/pn ratio is just higher between SARS-CoV2 and RaTG13. This issue could be partly explained by APOBEC editing, but I think it may also suggest that these viruses have been under more purifying selection than many of the other viruses. I haven’t looked at the other ORFs between SARS-CoV2 and RaTG13, do you know if they have similarly high ps/pn values? Is this a trend throughout the whole genome?

Note: I ignored the RBD (aa# 435-684) between RaTG13 and SARS-CoV2 because it’s obviously a mess. Something happened there. It could be recombination with a pangolin virus like some have suggested, or hyper selection pressure (I suspect to a new host). You see some similar RBD scrambling in other pairs of closely related CoV viruses like KY417150 and KY417146.

Argin
5/20/2020 06:21:11 am

See Farvous's comment on the next door. the ORF1ab dS/dN is a normal 6.22:1. not a sign of the extreme kind of purifying selection or an APOBEC/ADAR transition jockey here. also, ORF1ab is more purifying for viruses that have different host than S since the main mutations are on the S to mediate host switching. While in the same host the ORF1ab is less purifying than the S since the S will be more conserved to maintain host adaptation.
In fact, even with a 9:1 dS/dNe on the S2 for RaTG13/CoV2(making an unreasonably high overestimation), it will still spit out a possibility of 1 in 899 for that 44:1 S2 mutation ratio to be the result of "natural mutation".
We should see a S2 dS/dNe at most similar, most likely way lass than ORF1ab for RaTG13/CoV2-- the S is under strong CHANGING selection pressure as the host and all the conditions was entirely different. Even the S2. (may react to changing environmental conditions, but will definitely be requiring more than just 2 AA mutations across the entire stretch of 90 synomynous nucleotide changes that what it currently is. And the expected dS/dN should be definitely lower than that of ZC45/ZXC21).
The bottom line is that the 44:1 dS/dN on the S2 is completely unnatural nomatter what kind of "natural reason" you may attempt to use to explain it.

Nerd has power
5/23/2020 12:32:14 pm

Thank you, Argin, for bringing up Farvous' analysis.

Bryan, your opinion is important here in these discussions as you hold a slightly different view on this issue. I would love to hear more of you analyses and reasonings. Could you try to reconcile this discrepancy between S2 and Orf1ab in terms of the syn/non-syn ratio when comparing RaTG13 and SARS-CoV-2? Could you imagine a scenario where one could be 44:1 and the other 6.22:1?

Two other things I want to briefly mention. The overall sequence identity between ZC45 and ZXC21 is 97%, which is very much comparable to that between RaTG13 and SARS-CoV-2 (96%). Therefore, the differences in each pair are results of random mutations. If you choose to compare viral sequences that are much farther apart, other evolutionary events might be involved and responsible, which would be less appropriate in my opinion.

Finally, if it's me, I would avoid the recently published pangolin coronaviruses all together, not just MP789. If my suspicion happens to be the reality, then any comparison made with these pangolin coronaviruses would be risky. If you could, try to avoid other coronaviruses published by Zhengli Shi as well (this could be challenging since she has published so many).

John F. Signus
6/18/2020 07:29:46 am

https://drive.google.com/file/d/1Xp1zBf2d-hu32XSKkyJ7UBOg9ZHuAURS/view?usp=sharing
This is a recent collation of known SARS-CoV-2 S mutations in transfer. up to 25 mutations are already found on the S2. this is clearly NOT under purifying selection.

Bryan
7/5/2020 08:11:48 pm

Hey Nerd, sorry it's taken me a while to respond.I don’t think it’s a reasonable assumption to have uniform mutation frequencies or syn/nonsyn ratios throughout a viral genome. That might be an alright first null hypothesis, but if one actually want’s to make a case that a genome isn’t natural (either that it’s engineered or that it’s made up) you really need to look to nature, not a naïve assumption of uniform mutation rates. Is the relationship between SARS-CoV2 genome and RaTG13 different than the relationship between other related coronaviruses?
What is the expected difference in syn/nonsyn ratios between S2 and ORF1ab and is the 5-fold difference in syn/nonsyn between the two really a discrepancy? This is not an abstract a priori question that is answered with ‘everything should be 5:1’, instead this is an empirical question. How do these ratios normally differ in related coronaviruses? I’m sure a broader survey of coronaviruses could give a better range of expected values, but to pick one example that would be maybe the best apples-to-apples comparison (while avoiding any genomes from WIV or Shi Zhengli) is the original SARS and its closest bat relative (BtRs-BetaCoV/YN2018B).
SARS-hCoV and its close bat relative mirrors the relationship of SARS CoV2 and RaTG3. Both pairs of viruses show more purifying selection in the S2 protein than ORF1ab. Both also show a higher ratio of nonsynonymous mutations in S1 with a very sharp transition point near the transition to S2 with only a few nonsynonymous mutations in all of S2. The ps/pn (ratio of probabilities of synonymous to nonsynonymous mutations) jumps 7 times higher in the first SARS/bat and 5 times higher in SARS2/bat. They are not exactly the same ratios (it would be weird if they were). The SARS ORF1ab ps/pn ratio is about half-way between the S1 and S2 ratios, while the SARS-CoV2 ORF1ab ps/pn ratio is a lot closer to the S1 ratio. But overall, the trends look similar and nothing in the SARS2 RaTG13 comparison looks very far from what is typical in nature.
It doesn’t make sense to say something is not natural unless you take the time to figure out what natural really looks like. Natural does not look like some simplified model of uniform syn/nonsyn mutation rate across the genome or across the S protein. Comparing ZC45 and ZXC21 was a good start, but I think a broader survey of coronaviruses is needed to understand what natural is—especially looking at viruses from different species. The relationship between SARS CoV2 and RaTG13 does look different than that between ZC45 and ZXC21, but it looks a lot like the relationships seen between SARS hCoV and YN2018B or numerous other pairs of coronaviruses.

Exidos B freeman
5/7/2020 09:13:23 pm

https://zenodo.org/record/3786451#.XrTZjCV6uEc
See this. There is actually more evidence than just the AA sequence that point out that RaTG13 was fabricated. The Gene coding for the E protein, a 225bp sequence, is only 2nt different than ZC45/ZXC21. Which is more than 99%identical on the nucleotide level. While the entire genome is at most 89% similar to each other.
The most conservative estimate for this to be kept with the divergence between ZC45/ZXC21 and RaTG13, was estimated to be 1 in 19241.66 again when compared against the chance that the E gene being the result of artificial mainpulation.

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Bryan
5/9/2020 08:20:31 pm

I'll look at that link, is it a similar argument to that posted here?
The E-protein similarity doesn't look like a strong point though. If you look at related proteins, the E protein seems to by highly conserved. Of the sequences below from bats & pangolins, all of them have 100% amino acid identity in the E protein and over 96% nucleotide identity despite much less identity in other parts of the genome. If you look at even more distant clusters you see the same pattern too. This just looks more like a highly conserved protein than any anomaly with these particular couple sequences.
MN996532
MT040333.1
MT040334.1
MT072864.1
MG772933.1
KY417146
MT040335.1
MT084071.1
MG772934.1
MT072865.1
MT040336.1

Exidos B freeman
5/9/2020 08:52:31 pm

Well, My analysis is done on the nucleotide level-- Even assuming a highly conserved protein, the underlying nucleotides should have seen synomynous changes since it shouldn't affect the Amino Acid sequences, therefore it shouldn't affect anything at all. The point is that The newer "Evidence" (e.g. RaTG13, pangolins, etc.) Had too little synomynous substitution rate when compared to the rate that is seen in the rest of the genomic sequence. Which isextremely unusual, and seems to point out a common E sequence that were used for the fabrication of the "evidences", which were all obtained from short sequence metagenome sequencing data.

Nerd has power
5/10/2020 06:42:35 pm

Great finding on the severe discrepancies of the nucleotide sequence identity on E vs. whole genome! It seems that the more different ways we compare RaTG13 to other viruses, the more evidence we could find supporting its fabrication. Thanks, Exidos!

Bryan, I would recommend that we use these Pangolin or bat coronavirus sequences with caution. I listed my reasons in the article.

Farvous
5/11/2020 12:01:15 am

Did a BLAST on the ORF1ab polyprotein on RaTG13 to SARS2-- the Syn/nonsyn ratio is about 6.22:1.

AA(1ab)=QHR63299.1 -> 103(mut)
nt(1ab)=NC_045512.2 -> 744(diff)

which is likely overestimated since ORF1ab is more conserved when compared to S.

Using this ratio, the chance that the 44:1 Syn/nonsyn ratio being the result of natural mutation (S2 is not considered to be recombined with anything) is less than
(1-(103/744))^(90-(2*(744-103)))=1.28*10^-5. less than 1 in 77530.

It also proved that there were no elevated level of Syn/nonsyn substitutions between RaTG13/CoV2 any higher than ZC45/ZXC21. Therefore, using ZC45/ZXC21 for the mutation rate analysis is perfectly valid.

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Nerd has power
5/12/2020 02:31:17 pm

Thank you for the work! Again, they must have manipulated the sequence of Spike more than anything else --- they know people would scrutinize the RBD of S1 more than anything. However, they apparently spent too much time "editing" RaTG13's RBD that they had to be stingy about non-synonymous mutations when "editing" the sequence of S2. It is really nice to confirm that the syn/non-syn ratio is largely normal when other parts of the genome are being compared between RaTG13 and SARS-CoV-2. Thanks again!

Jogn F. Signus
6/18/2020 07:31:17 am

Also, may want to try SARS/YNLF31C.
https://drive.google.com/file/d/1Xp1zBf2d-hu32XSKkyJ7UBOg9ZHuAURS/view?usp=sharing
This is a chart of current mutations on the S2 in SARS-CoV-2. It proved that the S2 is not under purifying selection.

Exid
5/16/2020 06:52:58 pm

However,
Farvous on the next floor Did a BLAST on the ORF1ab polyprotein on RaTG13 to SARS2-- the Syn/nonsyn ratio is about 6.22:1.
No sign of a transition jockey here anywhere. the ratio is kept around 5:1 which point out that there were no APOBEC/ADAR editing to skew the ratio of dS/dN.

AA(1ab)=QHR63299.1 -> 103(mut)
nt(1ab)=NC_045512.2 -> 744(diff)

which is likely overestimated since ORF1ab is more conserved when compared to S.

Using this ratio, the chance that the 44:1 Syn/nonsyn ratio being the result of natural mutation (S2 is not considered to be recombined with anything) is less than
(1-(103/744))^(90-(2*(744-103)))=1.28*10^-5. less than 1 in 77530.

It also proved that there were no elevated level of Syn/nonsyn substitutions between RaTG13/CoV2 any higher than ZC45/ZXC21. Therefore, using ZC45/ZXC21 for the mutation rate analysis is perfectly valid.
Also, you mention that the 5:1 ratio on the S2 itself is already under purifying selection because ZC45/ZXC21 used the same host.
Therefore for RaTG13/CoV2 which had different hosts, there will be no purifying selection and the "natural" dS/dN on the S2 should be significantly lower than 5:1.
Therefore, nomatter how your hypothesis claimed, it should give an overal skewed dS/dN to >9:1 . but this ratio is not observed for the overall genome of RaTG13/SARS-CoV-2. Therefore, the APOBEC/ADAR hypothesis is still invalid on a mutation rate standpoint.

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Exid
5/20/2020 06:00:53 am

Therefore, nomatter how your hypothesis claimed, it should *Not* give an overal skewed dS/dN to >9:1 . but this ratio is not observed for the overall genome of RaTG13/SARS-CoV-2. Therefore, the APOBEC/ADAR hypothesis is still invalid on a mutation rate standpoint.
Sorry for the single word error.

Jon1717
5/7/2020 02:22:27 pm

Ok silly question, can you help me reproduce the sense / nonsene graph? I'm not getting the same results. I don't get the flatline for nonsyn mutations.

I performed the alignment with this:

https://www.hiv.lanl.gov/content/sequence/CodonAlign/codonalign.html

and I downloaded alignment in clustal forman and pasted it into SNAP

https://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html

My understanding is the spike protein for SAR-COV-2 is from 21563 to 25384

https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2?report=fasta&from=21563&to=25384

And for RaTG13 it's from 21545 to 25354

https://www.ncbi.nlm.nih.gov/nuccore/MN996532?from=21545&to=25354&report=fasta

I don't get the same flat line for nonsyn mutations. Any help?

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Exip
5/7/2020 06:00:37 pm

Maybe you accidentally made the alignment out-of-frame. Look at the assembly and locate the CDS for the “S” gene. Make sure you start with “AUG”. Your sequence may be misaligned.

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Bryan
5/14/2020 09:20:36 pm

Those are the right sequences. I think you are missing the alignment step. The input to SNAP needs to be aligned on a codon basis. I use a program called seaview that allows me to view the DNA sequences as proteins, align them (Clustal or Muscle), and then switch back to the DNA view. Then put the DNA seqs into SNAP. If you put unaligned sequences into SNAP, It will all be full of random syn and nonsyn mutations as soon as you hit any INDELs. In this case, once you hit amino acid 681 (SARS-CoV2's polybasic cleavage site) it'll all be noise.

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Mitch
5/7/2020 03:29:24 pm

While I appreciate your efforts, I agree with those who have pointed out that blog publishing is probably not the first choice of those who would like to have their information fully vetted by experts in the field.

I'm sure there are ways that your information could be submitted anonymously to journals by yourself or perhaps some of the commenters here.

Presumably, the journals' peer review process would then expose any flaws in your arguments, so that those who are not experts could have more confidence in your conclusions, or see that other experts lacked such confidence.

It would be great to know either that you have submitted your allegations to journals, or to have some explanation of why you don't think that would be appropriate.

Thank you.

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Nerd has power
5/10/2020 06:58:27 pm

Thanks for the suggestion. I don't mind having the article judged by anybody, including reviewers/experts. Apparently, the comments/discussions we got here are excellent. I have tried to reply to each one before, but I started to feel a bit overwhelmed and may not be able to keep up -- some comments are made by people who are real experts (I am an half expert) and there are too many of them now. You don't get this in a peer-review process. Reviewers' comments are typically not seen by public -- they can be unreasonable (more frequently so than you might expect) and yet the public wouldn't know. Here, on the open platform, you see everything. I personally believe that this article is being judged harsher than a manuscript undergoing peer review. If I were you, I will feel safer about the contents as they are being constantly validated.

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Alesh
7/5/2020 11:06:28 pm

Good point

Suresh K Narayanan link
5/7/2020 05:03:44 pm

I ran your analysis of SARS Cov 2 with RaTG13 by downloading a FASTA alignment file MN996532.1 x NC_045512.2.aln from NCBI virus and loading it on SNAP. I got a slightly different curve. I was wondering if I could send it to you?

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nah link
5/7/2020 05:18:37 pm

This pseudo "scientific evidence" have no value. Or maybe for reddit or conspiracy theory fans.

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Fight Against CCP
5/7/2020 05:35:31 pm

CCP dog has finally arrived. Why cannot you just disprove it? Barking around only shows your weakness!

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Kathy
5/7/2020 10:36:23 pm

Articles that clearly state an artificial origin for SARS-CoV-2 do not have a chance to be published. There is a huge censorship on the topic. What else we have that to make preprints and meet on these platforms? I am grateful that they exist.

David link
5/7/2020 07:51:55 pm

I am no native English speaker.An alternative view on it.Very impressive article and comments.I tought I was intelligent but you guys put me in my place at least in biology.I was suspicious about the way they said it was from nature in the first place because it break out where the only lab is of mainland china and where they do research with this kind of virus.Also patient zero not found on market which plays role in nature speculation.I think there were lots of infections on market because a lot of people come together there, just that. If objective mind the researchers should lower the odds of naturel accuring already a bit on that.But I often see that people have bias against a possible truth they dont like.Also scientist. When there is social research with differnt gender, race for example and they dont like the result they come with very long explanation of the least controversial possible explanation they can find.When it is 99,999 % chance from lab and you bring evidance which makes it 99,99999% from lab they would hold on to nature version.I saw some bias against bio-weapen in myself some time ago. When I realised humans are capable of nuclear weapons I changed my mind and not longer saw accident as 99% the couse.

Some think it is bad if it comes out it is from the lab.But if it does not come out I think is way worse.Then you cant learn and it is waiting for the next danger it will bring.

Also i read somewhere that when a virus is very wide spread a vaccin will lead to fast survival of the fittest of the virus and it makes kind of sense to me just as that when a virus is too strong it will be selected against the opposite is also true.If this happens also with flu it would be much slower because there are much less people infected so less mutations.But some say it is noticable for flu already. When a vaccin gives 100% protection it is safe but when it is relativelty poor effective like flu vaccin and very wide spread virus it is dangerous I think.Also it takes a lot of time to vaccin all people which gives virus more time for strongest versions to survive.

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Nonameneeded
5/7/2020 11:51:44 pm

Can someone do an in silico analysis of the role of the proline in the PRRA insert. What happens when this is removed or substituted with alanine? Asking because this may kink the secondary structure and increase solvent and protease exposure.

Also regarding the HKU9 RaTG13 discussion, apart from the geographic concerns my understanding is that HKU9 is from a large fruit bat while RaTG13 is supposedly from a horseshoe bat, so very different niches and encounter/recombination is unlikely.

Also, can someone reflect on CpG representation in this virus compared to SARS and other coronaviruses? The more suppressed CpG is, the less innate immune sensing by ZAP there is, which could account for the very prolonged incubation period and asymptomatic carriers. Any modulations in RNA motifs pertinent to the innate immune system between SARS-CoV-2 and SARS/MERS should be exhaustively examined because this is the crucial aspect that has allowed the spreading - the evasion of interferons and other intracellular mechanisms of resistance.

Finally, any data on bat cells or in vivo bat models of the virus? I have to wonder if the virus can even sustainably infect bats, or causes a very harsh disease that would obviously be incompatible with this being the natural host. This is worth looking into.

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Kathy
5/8/2020 11:38:48 am

....If the virus can even sustainably infect bats, or causes a very harsh disease that would obviously be incompatible with this being the natural host. This is worth looking into.

It is really worth looking into:

https://www.swinehealth.org/wp-content/uploads/2020/04/SHIC-106-FLI-SARS-CoV-2-Report-4-7-2020.pdf

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nonameneeded
5/8/2020 12:33:15 pm

Not sure why they did it in the fruit bats rather than intermediate horseshoe bats (and pangolins), which are the hosts that are bandied about.

Also, it occurred to me that the P of PRRA may create O-glycosylation sites nearby. Would be interesting to see if those drop out on the prediction tools when proline is taken out.

Nerd has power
5/10/2020 07:09:22 pm

Agree that some lab really should look at whether RaTG13 can infect its alleged natural bat host. I thought about this too -- what if it can't??? Maybe Zhengli Shi will blame it on the incompetency of the ones who did the test. I'm sure that, if she does the test in her lab, the infection will be "successful" and yet the bat will die very quickly -- leaving just the remains which contains the RaTG13 virus in the proper organs.

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Malcolm
5/8/2020 01:03:01 am

Re: RATG13 IS FAKE
The RaTG13 S2 subunit has a section with 84 synonymous nucleotide differences with SARS-CoV-2 without a single non-synonymous difference (over a span of 541 amino acids). The probability of this occurring by chance in related genomes (based on the 5:1 ratio) is one in five million. This is strong evidence that RaTG13 only exists on paper.

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Malcolm
5/8/2020 05:30:56 am

Calculations if anyone is interested.

The 1623 nucleotides downstream from the RaTG13 furin cleavage insert contain 84 synchronous differences with SARS-CoV-2 and zero non-synchronous differences.

Based on the expected synchronous to non-synchronous ratio of 5:1, the probability of any difference being a synchronous difference is 100/6 x 5 = 0.83333 or 83.33%

So the probability of all 84 differences all being synchronous differences is 0,83333 to the power of 84 (i.e.0.83333 x 0.83333 x 0.83333 etc etc.. )
= 2.2324 to the power of -7
= 0.00000022324
Which equals approximately one in five million chance.

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Kathy
5/8/2020 05:48:53 am

Can you exclude that RaTG13 is real but "just" edited? There is a possible record of if named BtCoV4991

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Malcolm
5/8/2020 07:05:33 am

BtCoV-4991 is a very small fragment (370 nucleotides or 123 amino acids) of the ORF1a gene, collected at the same time and place that RaTG13 was claimed to be found. A blast search gives a 100% match with RaTG13 and 98.92% with SARS-CoV-2, which is only a three nucleotide difference.

My belief is that the RaTG13 genome exists but it is actually SARS-CoV-2. Its spike protein sequence has been edited to optimize the receptor-binding-domain for human transmission, RaTG13 has 17 amino acid differences in the critically important amino acid sequence that allows SARS-CoV-2 to bind to human ACE2. It is telling that so many differences in this critical domain are non-synchronous; and that is because they were needed to make the jump from bats to humans with high affinity for human ACE2. The addition of the furin cleavage insert gives it enhanced cell fusion and pathogenity.

Prior to the belated release of the "RaTG13" genome in January this year, deliberate on paper changes were needed in order to disguise its true identity and create a plausible natural evolutionary link to SARS-CoV-2.

Mitch
5/8/2020 06:53:58 am

I understand that some of those coming here are used to an environment of strict censorship. But unless you are ready to declare that the journals covering virology have peer-review committees which are all corrupt, it seems contradictory to point to evidence of scientific fraud which is (1) obvious and (2) incapable of being published in a peer-reviewed journal.

At the very least, it would be helpful to the lay observer to be able to see the explanations (reasonable or unreasonable) that an article submitting "obvious" facts would not be published.

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Kathy
5/8/2020 08:37:05 am

I am aware that BtCoV4991 is a short sequence, but as you say, it was collected the same year, by the same group and in the same place as RaTG13 is supposed to. Coincidence? Perhaps. I am a fan of the pangolin theory, with the switch of the RBD of RaTG13 (edited) with the one from the pangolin CoV plus a furin cleavage site from another bat living in the same cave (I am still hunting this). An evolutionary study with the potential to develop a global corona-vaccine gone wrong. Interesting that these two articles were sponsored bathe same grant (Baric+Shi).

https://www.futuremedicine.com/doi/full/10.2217/fvl-2017-0143

https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC7097006&blobtype=pdf

This last review from Shi is very informative. It seems to me to present the plan to build SARS-CoV-2 and in Fig. 4 YN could be RaTG13. It is close to that strain from Bulgaria, as BtCoV4991 was here

https://link.springer.com/article/10.1007/s12250-016-3713-9

(Fig. 2).

We have different theories, but in common believe that SARS-CoV-2 is artificial. We need to find the final proof for it. Speculations can be rejected and censored, the final proof not. Then, it is good to work together.

John F Signus
5/9/2020 04:13:42 am

The paper is actually a repost of a February 17 preprint under the same title. Parts that concludes that the pangolin coronavirus have no furin cleavage site were redacted. As well as the full S gene that is very distant.
It was the same article where MP789 was first published. figures are exactly the same. Even the submission date suggest that that article was the same as the first article of ”pangolin Cov”, the MP789 article.

As for their “computational analysis”? They skewed their model so much that they conclude that a Bat-Cov had higher infectivity toward humans than SARS-CoV, at the same level as SARS-CoV-2. This is the opposite of reality since that should already caused an infection in 2013. Nothing happened.

And even after all this manipulation, they still could not bump the pangolin infectivity of either SARS-COV-2 or “ pangolin Cov” to levels above the human infectivity. Especially the “ pangolin Cov” that showed less pACE2 affinity than even SARs-CoV-2. Something that already had much poorer pangolin affinity than human affinity and were supposed to have evolved in humans. There is absolutely no chance that something that evolved in pangolins would bind to pACE2 less than something that evolved in humans, whose ACE2 was very different.

If anything that article was useful in, it actually proved that due to the dramatic difference in 4 of the 10 contact residues in hACE2 to pACE2 (H34S,L79I,M82N,G354H,especially L79I and G354H which was bulkier in pACE2 than hACE2, causes clashes that will likely destroy the binding interaction), it would be extremely unlikely that such a perfectly human-adapted RBD would bind to pACE2 efficient enough to constitute In-Vivo infection. (Which seems to be the case since this particular RBD was not isolated as a live virus, exist as a fragmented, incomplete metagenome data, and is probably introduced due to contamination)

Actually, if you look more closely, you can see a pair of new Restriction sites on the ends of the RBD of SARS-CoV-2, but not in RaTG13 or pangolin-Cov. The newly introduced restriction sites are indicative of cloning, and the underlying nucleotides on the RBD looks nothing like the alleged pangolin sequence.

https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

This is an indication that the RBD of SARS-CoV-2 was probably synthesized from the published AA sequence and swapped into the backbone via classic restriction digestion and cloning.

As the furin site is never found in any CoV spike protein that had more than 40% Sequence homology to SARS-COV-2, it lacked a natural origin (see my takedown on the alleged natural theory in my prior comments), and will need to be artificially inserted.

I agree that this may be a GOF study on the “new” pangolin RBD AA sequence (available to the WIV since October 2019) that have gone wrong, possibly due to the insertion of a furin site after the researcher was dissatisfied of it’s infectious capacity.
Probably after serial passage in VERO E6 as there were indication that the removal of the furin site causes it to infect VERO E6 more than anything else, by a high margin.

https://www.sciencedirect.com/science/article/pii/S0092867420302622

In fact, the high VERO E6 affinity prior to PRRA insert is already an indication that it have been passed extensively in the lab prior to the artificial insertion of the PRRA furin site. The Intermediate host is a cell line that exist nowhere except in biolabs.

Also, the Cryo-EM study done by the same article have disproved the idea that an O-linked glycan exist around the Furin site. the S1-S2 junction was NOT glycosylated.

John F Signus
5/11/2020 02:49:01 am

Read the Nature article more thoroughly.

They clain that their "Serological test" had an inter-test variation of 15% (which mean that there is a 15% chance for a false positive and a 15% chance for a false negative). and they conducted their test only once.

For reference, commercial COVID-19 serological test kits, even at April 15, was expected to have a specifity <90%. e.g. They will have a false positive rate >10%. And that is a modern antibody test. We can not expect their much earlier tests conducted before 16 Feburary (the date of submission) to have a specifity any higher than the figures we have today. Most likely they will have a specifity much poorer than that, since the first three FDA approved antibody tests have a specifity ranging from 83 to 96 percent--giving an average of 10.1% of all tests being false positives.
https://www.discovermagazine.com/health/covid-19-antibody-testing-tougher-than-true-false

https://www.healthleadersmedia.com/clinical-care/antibody-tests-coronavirus-can-miss-mark

As there were no FDA approved test kits for SARS-CoV-2 before 31 March 2020, We can reasonably put a lowest false positive rate on serological tests being >17% (since that if a test managed to have a higher specifity before, it should have already been approved by the FDA) . Therefore, we can consider the author's claim of a 15% inter-test variation rate to be the absolute minimum of the false-positive rate of their serological assay. (Their ELISA kit does not actually state anything about their specifity, other than that "it had no cross-reactivity to SARS-CoV, common and avial influenza viruses, mycoplasma, and chlamydia." Obviously there were far more than just these 5 pathogens that may cause cross-reactivity and false positives.)

Using a false positive rate of 15%, we can expect to see one false positive sample out of at most every 7 samples tested using the kit (an average of 1 in (1/0.15)=6.67 samples)

they only had 1 out of all 8 plasma samples that were tested positive, out of the 3 PCR-positive samples for their "pangolin coronavirus", which itself is only 93.21% complete even until today. (It was the same sequence as MP789 (possibly an earlier, more incomplete version) as indicated by their collection data, the date of analysis, the identical figures and the small fragment of sequence they put out after heavy censorship. See https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748 for details about the specific event and collection data. the entire article was the exact same preprint at 17/02/2020 and have been extensively criticised for the poor data curation and lack of independent verification).

Which mean that out of every 8 samples (12.5%) it's completely expected to see a false positive given a false positive rate of at least 1 in 7-- It could be hACE2 contamination from the handling of the samples since their tests used the RBD of SARS-CoV-2, which binds hACE2 very well. It may also be cell fragments from human ACE2+CD147 cells contaminating their samples during handling (which will bind both the S1 and the RBD), as they have their sample frozen for at least 4 months before testing (as indicated from their collection data), and they tested only plasma samples instead of actual serum samples that are free of cell debris. Also, the fact that this is not human antibodies will also significantly impact the specifity of such a test in a negative manner, as we can not be certain that antibodies from a non-human animal will react to the test materials in the same way the antibodies from a human will.

This mean that their alleged "Serological test" evidences are well within the false positive rates of their early, experimental and therefore must less accurate tests, and are not really evidence at all.

The date of the testing (well after the COVID-19 outbreak) may also indicate contamination from fragments of SARS-CoV-2 that were being tested inside their lab. Which was supported by the fact that none of the pangolin Cov submissions dated before 08-FEB-2020. A time where many of the chinese labs have already begun to perform studies on SARS-CoV-2 itself.
Contamination may explain the "identical RBDs", alongside with straight data fabrication. As by the time they have begun testing this in their labs they already have samples of SARS-CoV-2 within the same lab.
RT-PCR is a very sensitive technique, and could pick up even the slightiest traces of RNA contamination. This is especially problematic since you are already looking for sequencces that resemble SARS-CoV-2 as closely as possible. You may pick up actual SARS-CoV-2 fragments contaminating your samples and think it is a part of your expected virus (like the E protein gene), especially when dealing with metagenome data from samples which can contain mixed genetic information from multiple sources, that may or may not agree with each other on the ends (which were used for "

John F Signus
5/11/2020 06:24:50 am

Which was used for "Assembling" the genome. It's very easy for the researchers to mistake a contamination from SARS-CoV-2 itself, or other "more similar sequences" that they pulled off "The raw reads from public databases" as something that is more close to SARS-CoV-2, and mistakenly assemble it into the resulting genome.
Their Methods section have also pointed out that "The raw reads from public databases" were used for the assembly of their alleged "pangolin CoV" as well as "some in-house metagenomic datasets" which noone else had nor independently sequenced.
The short sequences were then "trimmed to remove adaptor and low-quality sequences". and "mapped to the SARS-CoV-2 reference sequence (MN908947) using BWA-MEM (v0.7.17) with >30% matches". It looke like that they actually discarded any dissatisfying reads and pulled data that is not from their alleged samples, in order to assemble a genome that is as closely related to SARS-CoV-2 as possible. It looked more like straight up data fabrication than any actual, serious science.
There was a reason why their "assembled full-length genome" wasn't available on GenBank apart from the incomplete MP789 sequence. Even though the data was available since at least the end of march.
And even the MP789 sequence, originally published on their preprint indicating that this Exact experiment as done on the alleged samples using the exact same method have in fact failed to produce a complete genome, was likely either heavily contaminated or was straight out fabricated using their skewed sequencing/assembly methods.

Troy T Dow
5/8/2020 11:59:34 am

I wish John F Signus would comment again. I'm worried his real name is Bing Liu, murdered in Pittsburgh on May 2nd.

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J17
5/8/2020 07:23:23 pm

I talked to my lab guy and he agrees with the Yuri article. He wonders how often furin sites are so perfectly added to close relatives without any extra junk. So I'm going to be doing analysis of closely related RNA viruses w/ and w/o furin to see how often that happens.

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Anonymous
5/9/2020 12:01:24 am

If the argument is that the COVID-19 E protein mutated over time and that E proteins mutate at a constant rate within bat populations, why then are ZC45 and ZXC21 identical? Or am I missing part of the logic?

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John F Signus
5/9/2020 03:27:53 am

ZC45 and ZXC21 are very closely related—while RaTG13 or CoV2 are very distantly related to ZC45/ZXC21.

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Anonymous
5/9/2020 04:11:50 am

Sorry, John F. Signus, but I still do not understand the logic of your argument. Although they were collected 1.5 years apart, because ZXC21 and ZC45 are "related," they should have not undergone any mutation and, therefore, are identical. RaTG13 is not related to them, but has an identical E protein. How does the short term mutation of COVID-19 fit into that argument? That is, would not any E protein mutation also apply to ZXC21 and ZC45, which would then show a sequence difference? Thank you in advance for a clarification.

John F Signus
5/9/2020 11:26:51 am

ZC45/ZXC21 was 98% similar. They are more similar to each other than RaTG13 to SARS-CoV-2. Also collected at the same site. The evolutionary distance is very short between ZC45/ZXC21. Much shorter than ZC45/ZXC21->RaTG13/CoV2. See the RaTG13 article to see how short their distance was, Evolutionarily. The reason that E was suspicious, was, due to the fact that there were closer Bat-CoVs and SARS-CoV, with a different E protein and genetic similarity higher than RaTG13->ZC45/ZXC21.

FreeFreedomain
7/2/2020 07:14:03 pm

John, thanks for the detailed explanation. I'm trying to understand it. If ZXC21 and ZC45 are 97% similar and CoV2 is 95% similar to both ZXC21/ZC45. Why having identical E protein when similarity is 97% is ok and natural, but when similarity is 95%, then it's nearly impossible to have identical E? There is only 2% difference between these two cases.

John F Signus
5/10/2020 02:51:09 am

https://zenodo.org/record/3786451#.XrFN4YijeUk
See this. The real problem is that even assuming perfect conservation of the E protein, the underlying nucleotide sequence still show too little synomynous substitutions than it should given the large distance between ZC45/ZXC21 and RaTG13/CoV2.
E is a protein coding gene. as long as the protein itself remain unchanged mutations on the underlying nucleotides should not cause any difference.
ZC45 and ZXC21 was 98% similar. It was expected that the small amount of mutations between them have not landed anywhere on the E gene.
However, with RaTG13/ZC45 it was a 89% identity on a pair of sequences where only 72% that can mapped to each other ("Coverage").
By comparing the ORF1ab polyprotein with a very distantly related coronavirus that is not on the same lineage, SARS, we can estimate that the total number of positions within the Lineage B coronavirus genome that can tolerate mutation being

29855*(3315+3*(7073-3315))=20526. or 68% of total.

As the part of two genomes that can not be aligned are typically more different than the part that can be aligned, We could use a conservative estimate for the total number of nucleotide substitutions between ZC45 and RaTG13:

29855-(19227/21597)*29855=3276.21.

Using the figure of ORF1ab, the range of which these 3276.21 substitutions could land on becomes

29855*(14589/21219)=20526.63 nucleotides.

Assuming that the E proteins of Bat coronaviruses of lineage ZC45/ZXC21-RaTG13 was perfectly conserved (e.g. no amino acid substitutions are tolerated), since there was no Tryptophan(W) within the E proteins in neither proteins, and
the Start codon must be ATG for Methionine, This
gives a total number of places where a mutation can be accepted within the E gene being 75-1=74 nucleotides.
Getting the first two mutations to land within the E gene will require an average of 2*(20526.63/74)=544.77 Substitutions, which leaves the other

3276.21-544.77=2731.44

nucleotide substitutions to land on the places other than the E gene.
The chance of which all the other 2731.44 nucleotide substitutions did not land on the E gene is
((20526.63-74)/20526.63)^2731.44=5.197056e-5, or 1 in 19241.6.

Therefore, the phenomenon that the underlying nucleotide sequence of the E gene being only 2nt different between ZC45/ZXC21 and RaTG13 being the result of "Natural evolution" was less than 1 in 19241.6.

The GX-CoVs had more than 9 synomynous substitutions in the E gene--well inside the "natural" range that would be estimated from Synomynous substitution rate analysis data alone. Therefore their E proteins does not have such a problem, which was reflected by the fact that the GX-CoVs were complete genomes.

MP789 had only 1nt different from RaTG13 but the total genomic distance was only 88.9% similarity to RaTG13, 90.1% similarity to CoV2 with 2 mutations on the E gene.
While the sequence itself is only 93% complete with 1872 unsequenced nucleotides marked as "N".
Applying analysis using similar techniques, the chance of this difference being the result of natural evolution (assuming the E was conserved), was only about 1 in 1846.
The MP789 sequence was incomplete, and was not isolated as a live virus.
The analysis above seems to point out that the particular nt sequence of the E for MP789, which was "obtained" from short sequence data just like every other "pangolin Covs" and RaTG13, seems to be an archived filler that was used to "fill in" the blanks on the E gene during the "reconstruction" (e.g. fabrication) of the genome, with synmonymous mutations thrown in to make it look different. BLAST on the E protein actually revealed that much of the incomplete Coronavirus sequences published by the CCP used the exact same nucleotide sequence for the E protein, despite the large distances otherwise.
Notably, the only E protein that is shown in the "identical protein group" with the SARS-CoV-2 E protein was RaTG13, ZC45 and ZXC21. No other E protein sequences were listed, including the alleged "pangolin Covs", which seems to point out that the RNA sequence was likely added to the other "identical E CoVs" as a filler during metagenome assembly. Indicating that the other "identical E proteins" from the nt sequence was not real.
This is a sign that this particular sequence was being used as the filler/placeholder for the E protein during short sequence assembly, as no way in nature that across all these distantly related coronaviruses that the E sequence was perfectly conserved, without even a single synomynous substitution.
This may also indicate that the Particular RNA sequence for the E protein was a sequence that was used extensively in Coronavirus engineering. Therefore, whenever a new coronavirus partial genome was assembled, particularly when using SARS-CoV-2 as a template, the sequence was used as filler for the E protein.
And whenever a chimeric/synthetic coronavirus was engineered, the particular E protein sequence was used for it's c

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John F Signus
5/10/2020 03:00:28 am

And whenever a chimeric/synthetic coronavirus was engineered, the particular E protein sequence was used for it's construction. It may then acquire the (unnaturally few) synomynous mutations after brief passage in cell culture.
Alternatively, they may throw in one or two synomynous substitutions to make the E protein gene more unique for strains that were used for Bioweapons--an 100% identical E gene down to the nucleotide level is a very clear sign of engineering.
As well as for fabricating sequences that were used as "critical pieces of evidence". Again, an 100% identical nucleotide sequence for any genes of an RNA virus will be a very clear sign of fabrication.

Unfortunately, they seems to have underestimated the mutability of the E protein gene. so whenever a complete, proven genome comes out, the E shows much more synomynous substitutions than the incomplete ones that were used as "evidence", bringing the complete genomes well into what was expected for the genomic distance by synomynous substitution rates.

John F Signua
5/10/2020 03:11:01 am

"By comparing the ORF1ab polyprotein with a very distantly related coronavirus that is not on the same lineage, SARS, "
should be
"By comparing the ORF1ab polyprotein between two very distantly related coronavirus that is not on the same lineage, SARS and MERS,". Sorry for the writing error.

Nerd has power
5/10/2020 07:36:56 pm

Thank you, John. Fantastic analysis and reasoning as always!

Physics Guy
5/11/2020 02:44:00 pm

Hi John F Signus, Nerd has Power and others. As my name suggests I am not a biologist, but I follow this discussion with interest. As an outsider I can in any case see that a moratorium was tried on research that creates viruses with pandemic potential

https://www.sciencemag.org/news/2014/11/moratorium-risky-virology-studies-leaves-work-14-institutions-limbo?fbclid=IwAR2o3fIfA1mvCppqg41u3ZbFl_oFST0IKepXFDII0qD47F7r9Ua2ItvtqUs

and with good reason

https://www.nytimes.com/2014/06/30/opinion/anthrax-thats-not-the-real-worry.html?_r=0&fbclid=IwAR0jvHFvnmN-0DRmTLdiP-bUpOAX89gz4rgSDcPWYpeMtW1NUCNzfHXFzCo

That the epidemic started in Wuhan of all places to me suggest that without any other information the lab-leak scenario is about 100 times more probable than the natural scenario. I have no idea which percentage of the viruses that are handled regularly at such a lab is natural and which percentage is non-natural.

Anyway, my reason for commenting is that I noticed a small mathematical error in your calculation. Where you calculate the probability that at most 2 mutations fall in the E-protein part, I think you should use a binomial probability distribution with p=74/20527, n=3276 and number of succes x<=2. This gives a probability of ~1 in 1655.

I wonder if you also took into account the "look-elsewhere-effect"? Do you calculate the probability for the E-protein because the is an a priori reason why this part of the gen is special in this regard or is it picked merely because it looks the most improbable? In the latter case you should multiply by something of the order of 20527/74=277 to account for the other 277 same length sequences that you could also have selected because they could have looked improbable. I honestly don't know whether this is an issue in this case or not. If so you get a probability of ~1 in 6.

John F signus
5/11/2020 05:01:38 pm

The actual problem it that the E protein is a protein coding gene—it is Significant in the sense that it is Both a distinct part of Coronavirus biology and Coronavirus reverse genetic systems. It is not a “random sequence”. Other sequences are non-significant, therefore they Can not be compared to a plausible synthetic origin By engineering.
However, for the E itself, it DO have significance in engineering (It’s a structural protein of the coronavirus virion, also the first things they consider when looking at coronavirus genomes) and therefore The chance for natural origin is compared against the chance for engineering (it would be what you expect for bioweapons engineering, take an existing archived sequence and throw in one or two synomynous substitutions to make it more unique.) . That’s why such a comparison is valid.
From your reasoning, for that sequence to be exactly that of a gene that is of the same length (the E), it would have to land exactly on the location the E was located. Then the chance becomes 1/(29855*6)=1/179130.

Physics Guy
5/12/2020 01:02:48 am

Great. I don't follow your last bit 1/(29855*6)=1/179130. My estimate for the "look elsewhere effect" was just a maximum. How many of these locations like the E-protein one are there, for which you could a priori expect something "fishy" if the virus is not natural? Roughly the "look elsewhere effect" says that if there are for example 4 of such a priori locations and you find something fishy for 1 of those 4, then you divide your final probability by 4.

John F Signus
5/13/2020 12:01:56 am

Well, there is only ONE E gene in SARS-CoV-2. the only other suspicious protein is the first part of S2. using the "look elsewhere" effect, for the specific sequence (btw it was an 225bp sequence out of 29855, not a 75bp sequence), the maximum should be 1 in 6*(225/29855)=796.16, or based on the only 2 identical proteins of significance, 1 in (1655/2)=827.5.
Here you have. the maximum chance for a natural origin of the E gene nucleotide sequence, assuming the maximum of "look elsewhere" effect, being either 1 in 796.16, or 1 in 827.5. more than 99.85 chance that it was the result of deliberate genetc manipulation.

K. Evangeline Luk
5/9/2020 02:37:34 am

I'm not sure if @Raoul89573207 that tweeted me this blog was you, but you need to come onto our Riot server.

What we've found might inform you on the science front, and maybe you'd know something in the Chinese researcher circle or history of biological sciences there that can help us with the investigation as well.

The most I can write here is that the whole thing started decades ago, and at least three more countries are involved (none publicly funding the labs I think), and about a dozen of Chinese virologists/immunologists' lives are hanging by the thread right now (cue: Bing Liu).

I'll see you at https://matrix.to/#/!fseEHHdfaSBvFYbopt:matrix.org?via=matrix.org&via=junta.pl

EV

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Eva Celia
5/9/2020 11:23:36 pm

Have you read about the odd pneumonia outbreak in Virginia back in summer 2019 https://www.nbcwashington.com/news/local/health-officials-to-give-update-after-respiratory-illness-sickens-dozens-at-virginia-retirement-community/135890/ and also this from France in Nov 2019 https://www.scmp.com/news/china/society/article/3083599/france-had-covid-19-november-hospital-says-after-analysis-chest

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Kathy
5/9/2020 11:28:55 pm

COVID RIDDLE Fears coronavirus arrived in Europe in OCTOBER ‘when French athletes at World Military Games in Wuhan brought it home

https://www.thesun.co.uk/news/11565077/fears-coronavirus-europe-october-french-athletes-military-games-wuhan/

https://www.sundayguardianlive.com/news/corona-scare-india-part-wuhan-games

https://www.web24.news/u/2020/05/military-world-games-in-wuhan-in-focus-we-are-all-sick.html

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Fight Against CCP
5/9/2020 11:41:06 pm

Well done, Kathy! :) Such a large scale virus couldn't have originated from anywhere else and hidden for so many months until the first largest outbreak in Wuhan. That's why COVID-19 = CCP Originated Virus Infectious Disease-19. As you can see, CCP propaganda and dogs are everywhere. They would not know how evil CCP are until they themselves become CCP's victims.

Fight Against CCP
5/9/2020 11:48:54 pm

Here are more to add to your list:

https://twitter.com/jenniferatntd/status/1229921498464505856

http://bbs.creaders.net/life/bbsviewer.php?btrd_id=5367211&btrd_trd_id=1451321

Nerd has power
5/10/2020 07:46:59 pm

Agree. Given how easily this virus spreads and how little people know back then about having to use masks to stop it, there is no chance that France (or any other place that the CCP wants to mis-label as the "true origin") could have avoided community spread back then.

to go deeper
5/10/2020 12:21:12 am

But summer comes before fall

See message from Eva Celia

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CCP is NAZI
5/10/2020 09:58:57 am

Have you actually compared its sequence with CCP Virus' sequence side by side? If so, why don't you bring its sequence here to let virus specialists verify? Even look at it from a non scientific perspective view, with such a high infectious rate that CCP virus has, why nothing happened in U.S. afterwards until after Wuhan lock down? Interestingly, CCP tried their very best to help this deadly CCP virus spread as fast and wide as possible all over the world by silencing 8 doctors like Li Wenliang , by destroying all virus samples, by stressing it's preventable and controllable, by denying the fact about human to human transmission, by letting people out from Wuhan to everywhere in the world but not within China? Were CCP able to prevent and control it? Exactly, what's CCP's hidden agenda? Well, aren't these https://www.newsmax.com/navrozov/china-biological-russia/2009/09/17/id/335042/ and https://www.theepochtimes.com/did-chinas-plan-to-destroy-the-united-states-backfire_3223117.html enough to tell CCP's world domination goal?

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Jane
5/18/2020 05:37:49 am

Did you read this?

https://www.globaltimes.cn/content/1181292.shtml

Flu, vaping or novel coronavirus: experts suspect the US might have failed to identify causes of deaths

CCP is NAZI
5/10/2020 11:16:38 am

Where is the bat woman and the virus queen - Shi Zhengli? Why don't you invite her here to see what she has to say? Where is the Patient Zero, Huang Yanlin? Is she already dead or hidden? Why did Wuhan P4 lab take her picture down while still leaving her name there? Look at her with her lab mates on 2018 New Year Day: https://www.youtube.com/watch?v=NVzne_ZQ0Vo I'm sure that CCP really has hard time to find someone who not only looks like her, but also has a virologist's knowledge. Further, U.S. has the best CCP virus tester to test her to see if she was infected with or is currently infected with CCP Virus.

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detailfreak
5/10/2020 02:07:13 am

Two tracks to follow now that we know that RaTG13 is BtCoV4991:

https://twitter.com/schnufi666/status/1259035090811846661

1) check the Pairwise identities of nucleotides and amino acids % (nt/aa) of RaTG13 in comparison with the CoVs in the supplementary data here

https://europepmc.org/article/med/26920708

Search everywhere a publication (maybe in Chinese?) with the spike of BtCoV4991

PCR screening of coronaviruses and sequencing One-step RT-PCR (Invitrogen, San Diego, USA) was used for the amplification of a 440-bp fragment target-ing the RNA-dependent RNA polymerase gene (RdRp)of all known alpha- and betacoronaviruses (de SouzaLuna et al., 2007). Amplification of an 816-bp fragment extending the 440 bp was performed using published methods (Drexler et al., 2010). Spike (S) genes wereamplified using degenerate primers designed based on the alignment of known coronaviruses (sequences provided upon request).

The cherry on the cake: Remdesivir targets RdRp

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Lincolin
5/10/2020 06:31:43 am

Did a BLAST on all of them.
https://www.uniprot.org/uniprot/A0A126K9A8 -> Bat coronavirus 1B
https://www.uniprot.org/uniprot/A0A126KBA2 -> Bat coronavirus 1B
https://www.uniprot.org/uniprot/A0A126KBA0 -> Bat coronavirus 1B
https://www.uniprot.org/uniprot/A0A126K9U6 -> BtMf-AlphaCoV/AH2011
https://www.uniprot.org/uniprot/A0A126K9I9 -> Bat coronavirus 1B
https://www.uniprot.org/uniprot/A0A126K9J8 -> BtMf-AlphaCoV/AH2011
https://www.uniprot.org/uniprot/A0A126KB98 -> Bat coronavirus 1B
https://www.uniprot.org/uniprot/A0A126KBA9 -> Bat coronavirus 1B

Bat coronavirus 1B-> https://www.uniprot.org/taxonomy/393768
Is an Alphacoronavirus.

BtMf-AlphaCoV/AH2011-> https://www.uniprot.org/taxonomy/1503278
Is an alphacoronavirus.

NO betacoronavirus Spike proteins were sequenced on https://europepmc.org/article/med/26920708

There were still NO confirmation of RaTG13 Spike protein anytime before 7 Mar 2020.

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Lincolin
5/10/2020 07:00:55 am

There were a total of 8 Spike proteins Listed there.
KU343205==A0A126K9I9 -> MfulBtCoV/3759-1 = AMB43195
KU343206==A0A126KBA9 -> MsBtCoV/4001-1 = AMB43196
KU343202==A0A126K9U6 -> MsBtCoV/3710 = AMB43192
KU343203==A0A126KB98 -> RsBtCoV/3716 = AMB43193
KU343201==A0A126KBA0 -> MfulBtCoV/3709 = AMB43191
KU343208==A0A126K9A8 -> MsBtCoV/4068 = AMB43198
KU343207==A0A126K9J8 -> MsBtCoV/4056 = AMB43197
KU343204==A0A126KBA2 -> MfulBtCoV/3736-1 = AMB43194

All of them were Alphacoronaviruses, and obviously none of them was the 4991 Spike gene.

Record on 4991:
AMB42835
Rhinolophus bat coronavirus BtCoV/4991 partial RNA-dependent RNA polymerase

KP876546
Rhinolophus bat coronavirus BtCoV/4991 RNA-dependent RNA polymerase (RdRp) gene, partial cds.

A0A126KB67 A0A126KB67_9NIDO RNA-dependent RNA polymerase RdRp Rhinolophus bat coronavirus BtCoV/4991

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Lincolin
5/10/2020 07:46:28 am

Therfore, there were still no Spike gene dequence data, or any pieces of data other than the tiny 370bp BtCoV/4991 Partial RdRp, of RaTG13 that were published, or confermed, before the pandemic.

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Dartagnan
5/12/2020 03:00:04 pm

Wow... so the gene fragment that codes for the RdRp of the virus is conveniently put out there 3-4 years ago. Just enough time for someone to develop an effective RdRp Inhibitor drug (like remdesivir or likely something even more effective given in pill form which is not public yet) that can be given prophylactically to high level officials or others you want to favor. These people will be protected from infection or treated quickly once the pandemic hits, while the rest of the world is physically and economically paralyzed. Gilead's tenofovir, which is the adenine analog chain terminator cousin of remdesivir, is already used this way as a reversetranscriptase inhibitor prophylactic for HIV. Then folks like Peter Daszak can go on 60 Minutes and say that their dangerous GOF research helped lead to life saving breakthroughs like remdesivir...

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CCP is NAZI
5/12/2020 03:35:08 pm

No surprise. CCP's evilness is way more than what you can imagine. CCP's Nazi conduct are doomed to be severely punished. I wish more people can wake up to knock down CCP with whom the world would not have any peace.

Javier
5/10/2020 07:46:23 am

Hello. Excellent work here in this blog. Thanks.

Following, is one recent paper that claims to have proven natural mutation origin of the FCS (furin cleavege site) in CoV-19
title:"A mutation model explaining acquisition of the furin cleavage site in the SARS-CoV-2 genome". Shan Gao (Nankin University 03-2020)
(https://www.researchgate.net/publication/340091740_A_mutation_model_explaining_acquisition_of_the_furin_cleavage_site_in_the_SARS-CoV-2_genome)
Extracted from this paper (still non peer reviewed):
"In a preliminary study, we also unexpectedly found that some avian influenza viruses acquired FCSs through mutation, indicating that
acquisition of the FCS in the SRS-CoV-2 genome through natural mutation is probable".
"Based on the above findings, we proposed an original theory that the accumulation of non-neutral mitochondrial mutations (CNV of STR as the predominant type) may constitute one of the most important causes for ageing and cancer [6]. In the present study, we found similar findings in the mutation pattern of SARS-CoV-2 within one host (human).
Based on the mutation data of animal mitochondrion and viruses, we propose the non-neutral theory of molecular evolution, and have used the theory to interpret acquisition of the FCS in the SARS-CoV-2 genome. This leads to revealing the natural origin of SARS-CoV-2 at the molecular level".

Can someone put more light about these arguments.

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John F Signus
5/10/2020 07:51:03 am

The article claimed that the Furin site arose in humans-- However, if that was the case, why the very first sample of SARS-CoV-2 already had the exact perfect PRRA furin cleavage site? Why there were never a detection of a "Progenitor virus" in humans that lacked this site? If it arose in humans, it should have already been detected. no intermediate CoV2 W/O PRRA is a clear indication that id did not arise that way.

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John F Signus
5/10/2020 08:03:41 am

From phylogenetic data and the comment made by Firox TD Jackinson, We already knew that a furin site will not survive nor persist in wild animals for Spike gene that is more than 40% homologous to SARS-CoV-2. So it can't arise in an animal. And there were no detection of a "intermediate virus" in humans that is near-identical to SARS-CoV-2 while laching the PRRA. Therefore, it can't arise in animals and did not arise in humans, leaving the only possible source of the furin site as either deliberate insertion or lab-based passage. Cell passage may cause it to arise in the lab in that mechanism, however. (the AIV example cited by the paper is actually in-vitro passage data) Since cell culture lacked an immune system, they will not eliminate the furin-cleavable spike immediately like wild animals, but since they are not humans, the intermediate will be shielded inside a lab and therefore will not be discovered in actual, live humans. explaining the lac of an intermediate in humans.

Javier
5/10/2020 09:12:50 am

Very interesting reply John.


Then, we have no existing known progenitor virus in humans with these features.
then, any room to exist any other known virus able to jump to and mutate in humans in such a specific way?
You know, perhaps, this slow gradually "cooking" in humans since several months earlier.

John F signus
5/11/2020 05:20:05 pm

The problem is that both the FCS containing precursor and the RBD containing precursor are potent enough to cause an epidemic in humans. More so than SARS. We already knew that the FCS is not tolerated in wild animals for betacoronavirus spike protein genes that is more than 40% homologous to SARS-CoV-2. So it can’t arise in that way.
If this arose in humans, then there should already be an epidermic of a precursor virus that is exactly like this, that either lacked an RBD (outbreak of FCS-contain RaTG13) Or lacked a FCS (outbreak of FCS-lacking SARS-COV-2). Such an outbreak should have already alerted the health authorities and should have been sequenced like SARS or MERS.
We didn’t see any of such precursors causing an outbreak.
Therefore, even if your assertion was valid (which is not since the mechanism they proposed are only observed in INFLUENZA viruses, an entirely different family of viruses with a Negative sense RNA genome. coronaviruses are positive sense RNA viruses that are replicated and packaged in entirely different mechanisms and are just too distantly related to possibly share this same mechanism), it should have already caused an detectable outbreak in humans before it have reached the state as we observed like today. We did not observe such an outbreak, therefore it can only happen in a non-animal-non-human situation. Which mean that it can only occur in a lab.

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IdoNotLikeCherries
5/10/2020 09:36:32 am

Anther cherry on the cake:

https://www.nytimes.com/2020/04/21/magazine/pandemic-vaccine.html

The final list — which did contain SARS and MERS, along with seven other respiratory, hemorrhagic or otherwise-lethal viruses — also included something the W.H.O. dubbed “Disease X”: a stand-in for all the unknown pathogens, or devastating variations on existing pathogens, that had yet to emerge. Daszak describes Covid-19, the disease caused by the virus SARS-CoV-2, as exactly the kind of threat that Disease X was meant to represent: a novel, highly infectious coronavirus, with a high mortality rate, and no existing treatment or prevention. “The problem isn’t that prevention was impossible,” Daszak told me. “It was very possible. But we didn’t do it. Governments thought it was too expensive. Pharmaceutical companies operate for profit.” And the W.H.O., for the most part, had neither the funding nor the power to enforce the large-scale global collaboration necessary to combat it.

well, it looks like that at the end they got some money for it ;)

https://www.newsweek.com/dr-fauci-backed-controversial-wuhan-lab-millions-us-dollars-risky-coronavirus-research-1500741

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Nerd has power
5/10/2020 08:21:50 pm

Fantastic efforts, everybody! This place is becoming alive, with all your insights and excellent analyses. I must admit that they are much better than what I was able to offer in the original article. Please keep adding it.

Only if Zhengli Shi and Peter Daszak could visit and join our discussions, right? Maybe it will happen. We'll see.

I will become more of an observer at this point and will not try to participate in all discussions -- some of them are out of my reach actually. Also, next two weeks will be a bit packed for me. I apologize in advance for delays in responding to questions directed at me. Thank you!



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Batman link
5/11/2020 02:38:51 am

In 2013-15, Zhengli Shi and colleagues thought the Rhinolophus sinicus bats they had found in Yunnan caves had a Coronavirus capable of infecting human lungs.

In early February 2020 , Zhengli Shi and colleagues published their paper claiming the similarity of Covid-19 to RaTG13.

Earlier on 27 January 2020, they filed the complete genome of Bat coronavirus RaTG13, with GenBank: MN996532.1, after supposedly having found it seven years earlier in 2013!
https://www.ncbi.nlm.nih.gov/nuccore/MN996532

Supposedly, this genome was derived from droppings from the cave in Yunnan.

But from which of the seven Bat species they found in the cave?

In the complete genome of Bat coronavirus RaTG13, filed
by Zhengli Shi and colleagues, they claimed the source was:
“ /host=Rhinolophus affinis" and the Rhinolophus sinicus bats were not mentioned.

Really?

“Rhinolophus affinis” bats are widespread over Asia, but suddenly they appeared in winter, in Wuhan alone, with a deadly and virulent Coronavirus RaTG13?

Really?

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Marco Belladama
5/11/2020 05:37:25 am

I'm a system integrator.... need to download the genome and a little guide to remake the picture like in this post

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Sam
5/11/2020 09:52:18 am

Does the existence of BtCoV/4991 RdRp - a fragment of a viral genome that got published in 2016, 4 years before RaTG13, and seems the same as the RatG13 sequence published in Jan 2020 affect your argument (that RatG13 is a fake) at all? https://www.ncbi.nlm.nih.gov/nuccore/KP876546

Peter Daszak tweeted that: These are sequences from the same sample, 1st RdRp using sample code at that time, then full genome using 2019 sample naming system.
https://twitter.com/PeterDaszak/status/1259111768217063424?s=20

What do you make of 4991? Thanks.

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Simen
5/11/2020 08:03:47 pm

The existence of RaBtCoV/4991 actually does not prove anything. The sequence is only 370bp in length and everything elve were still not published until 27-JAN-2020 . The only other reference of RaBtCoV/4991 was a single figure in that 2019 paper, which published no sequences. Notably, if what they claim was true on the S protein, we should already seen the RBD of RaTG13 being published before. We didn't.
Therefore, we can not rule out fabrication/manipulation for the rest of RaTG13/RaBtCoV/4991. Without being able to rule out such fabrication/manipulation, The argument that RaTG13 is invalid as "evidence" for a "natural" origin of SARS-CoV-2 is still as valid as it currently is.

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Nerd has power
5/12/2020 03:22:37 pm

Thank you, Simen! Agree with your analysis.

Sam, I answered your question in a later post of yours. Please scroll down to look at it. Again, I sent Peter the invitation with hope that he would join the discussion here. So far that has not happened.

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Sam
5/13/2020 03:35:14 am

Thank you so much for your interesting reply and for taking the time to respond. Much appreciated.

Robespierre
5/11/2020 10:34:31 am

China knew at least on March 7, 2020, probably earlier, that RaTG13 and BtCoV/4991 were the same. Why the charade?

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Batwoman
5/11/2020 11:36:33 am

Maybe few in China knew it, but most of the others not. Are you aware that in science you can’t change the name of a sample that it has been published to avoid to lose the track of previous works? Only who has something to hide would do it. Are you a friend of Peter Daszak?

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Sam
5/11/2020 12:45:40 pm

Not at all. I think he has very little credibility and has clearly lied about a number of things, not least his claim to “have no conflicts of interest”! But I am interested in what Nerd makes of 4991.

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Nerd has power
5/12/2020 03:20:10 pm

Hi Sam, here is my take on this 4991.

They did publish it in the 2016 paper:

https://www.ncbi.nlm.nih.gov/pubmed/26920708

In there, it was named RaBtCoV/4991. According to the paper, the sample was collected in 2013.

First, why do they change the name in the 2020 publication? They didn't have to. Also, why didn't they cite this 2016 publication in their 2020 Nature paper? For something this important, they should do all they can to explain its origin in the paper. Yet the reality is the opposite: they breezed through this part light-heartedly. In addition, they changed its name and they failed to cite their own 2016 paper when 4991 was first published.

Second, if they have sequenced the RBD of 4992 back then, why didn't they publish anything about it? That RBD sequence is so bright that Shi's eyes would hurt just by looking at it. Could she curb her enthusiasm so well and then go out strenuously to hunt for other bat viruses? Many of the ones Shi did publish in between 2013 and 2020 are far less attractive in comparison to the 4991/RaTG13 in terms of sequence.

If they could not sequence the RBD back then (they must have tried because it is THE most interesting piece in their eyes), how come they could sequence it now? The sample did not improve. If anything, RNA tends to decay easily.

I have said this in an earlier post: I think the reason that Shi did not mention this 4991 in the 2020 Nature paper is because she would struggle to explain the above points.

BTW, I actually invited Peter under that tweet to come over and join our discussion here. I don't think he posted any comments yet (maybe he did???)

Dusha
5/11/2020 11:51:09 am

Many thanks for your interesting article! I have one question. You say, "As stated in the [Shi's] paper, RaTG13 was discovered from Yunnan province, China, in 2013." What is the reference for the 2013 date please, as I can't find it anywhere in Shi's 2020 paper here:
https://www.nature.com/articles/s41586-020-2012-7
Is there some other evidence to show that this was a 2013 isolate?

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Batwoman
5/11/2020 11:59:32 am

The article where BtCoV4991 is cited is here:

https://www.ncbi.nlm.nih.gov/pubmed/26920708

They did not cite it in the article in Nature. They just said that they magically found the RdRp sequence of RaTG13, that is 100% identical to the nucleotide sequence of BtCoV4991. We forced them now to admit that it is the same strain.

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Nerd has power
5/12/2020 03:32:39 pm

Yes, the publication listed by batwoman is the original one that reported about how this 4991 was discovered back in 2013. Please see my answer right above for my take on this 4991.

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guesswho
5/13/2020 02:34:18 pm

Something more on BtCoV4991. Here https://www.ncbi.nlm.nih.gov/pubmed/26920708

BtCoV4991 clusters with GU190215 from Bulgaria.

https://www.ncbi.nlm.nih.gov/pubmed/20686038

The RBD of GU190215 showed high similarity with that of SARS-CoV. Can they really claim that the spike of BtCoV4991 was not sequenced before 2020, after that they have seen that phylogenetic tree? We should ask Drosten. He does the same dirty experiments in Germany and he was one of the supporters in Lancet of Shi, present also at the WHO meeting in Ginevra where all started.

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Aydin
5/11/2020 03:27:28 pm

Interesting to read Peter Daszak@PeterDaszak's tweet:

"The answer is already in the papers & obvious to people working in virology: These are sequences from the same sample, 1st RdRp using sample code at that time, then full genome using 2019 sample naming system. Oh yes, & by the way this is NOT the same virus as SARS-CoV-2"


What is this "2019 sample naming system"?

Recently saw someone interprets RaTG13 as "Total Genomic 2013"' . A bit strange.

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Dalstra
5/11/2020 05:25:59 pm

Also add in the fact they have never produced a physical copy of RaTG13 nor let anyone else resequence it’s genome. And the fact that it was not submitted nor published anywhere (Except for the 370bp RaBtCoV/4991) until January 27, well after the outbreak.

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Tongguanzhen
5/12/2020 08:56:58 pm

https://twitter.com/paleovanguard/status/1257416405835792388

Thread on Twitter seemed to be on a right track regarding naming: TG being Tongguanzhen

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cippi
5/13/2020 02:17:17 pm

TG sounds to me more Total Genome

Aydin
5/11/2020 03:44:01 pm

Per "The Bats Behind the Pandemic" Wall Street Journal by Matt Ridley (4/9/2020)

"RaTG13 is the name, rank and serial number of an individual horseshoe bat of the species Rhinolophus affinis, or rather of a sample of its feces collected in 2013 in a cave in Yunnan, China. The sample was collected by hazmat-clad scientists from the Institute of Virology in Wuhan that year. Stored away and forgotten until January this year, the sample from the horseshoe bat contains the virus ..."

https://www.wsj.com/articles/the-bats-behind-the-pandemic-11586440959

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John kelleher
5/11/2020 03:46:53 pm

https://connect.biorxiv.org/relate/content/181
Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag
Prashant Pradhan, Ashutosh Kumar Pandey, Akhilesh Mishra, Parul Gupta, Praveen Kumar Tripathi, Manoj Balakrishnan Menon, James Gomes, Perumal Vivekanandan, Bishwajit Kundu
doi: https://doi.org/10.1101/2020.01.30.927871
This article is a preprint and has not been certified by peer review [what does this mean?].
AbstractFull TextInfo/HistoryMetrics Preview PDF
Abstract
We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this

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Nerd has power
5/12/2020 03:35:30 pm

I have commented on this article a few times, I believe. Sorry that I won't repeat myself here again, except to say that I'm not completely bought by their evidence and conclusions (although I agree that the virus is of non-natural origin).

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Jane
5/18/2020 06:00:28 am

Can we exclude that Daszak & co. figured out that the spike of BtCoV4991 modified with the RBD of the pangolin CoV and furin site had HIV properties and for this reason they inserted in the BtCoV4991 backbone to develop a super vaccine/drug?

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Nerd has power
5/18/2020 09:39:07 am

Sorry that I don't think I fully understand your question. But I would simply say that 1) I personally have not been convinced that there are HIV sequences inserted into the genome of SARS-CoV-2; 2) no one would try to create a vaccine this way --> making it resemble SARS and then, on top of that, adding a Furin-cleavage site to make it more infectious. The manipulations are intended for the opposite.

Hans Fields
5/11/2020 07:49:46 pm

I ran a sequence alignment on RaTG13 vs Sars-CoV-2 and it only turns up 4 nonsyn mutations on the N protein (419 len). It seems a bit low given that there are 18 nonsyn mutations comparing ZC45 to Sars-Cov-2.

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BreakingBad
5/12/2020 05:06:10 am

China’s own scientists have provided evidence that COVID-19 is man-made

https://www.wionews.com/opinions-blogs/heres-what-chinas-scientists-have-to-say-about-the-origin-of-covid-19-298213

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yano
5/12/2020 06:39:07 am

What is the probability that the SARS-CoV-2 E protein become 100% similar to ZC45 and ZXC21 naturally?

Why do ZC45 and ZXC21 have the same 100% similar E protein?

Why are scientist ignoring this?

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Nerd has power
5/12/2020 03:43:18 pm

Please scroll back to see the conversation between Anonymous and John F. Signus on this topic. John explained it very well.

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yano
5/12/2020 06:51:11 am

The Feb 11 2020 E protein for SARS-CoV-2 is 100% similar to ZC45 and ZXC21. It is thought that the virus went into humans in Sept-Oct 2019.
Why was there no mutations in the E protein between Oct 2019 and Feb 2020?

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Nerd has power
5/12/2020 03:46:57 pm

You need the size of infected populations to be big enough to see these mutations showing up in sequencing. Please note that not all samples are sequenced. There might be mutations in E from earlier patients, but those may not be selected for sequencing.

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Claire Robinson
5/12/2020 06:53:46 am

An article has been published about Nerd Has Power's research here:
https://www.gmwatch.org/en/news/latest-news/19396

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Nerd has power
5/12/2020 04:05:04 pm

Thank you, Claire and your team, for publishing the article. You have successfully identified me being a male. One error in your article is that you mentioned (twice, I believe) that ZC45 and ZXC21 were discovered by Zhengli Shi. It's not correct. The discovery was made by another lab in China that has a military background. Here is the publication:

Hu D, Zhu C, Ai L, He T, Wang Y, Ye F, et al. Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats. Emerg Microbes Infect. 2018;7(1):154.

I hope you get to update things in there. But either way, great article and summary! Thanks again!

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Claire Robinson
5/13/2020 02:14:06 am

Corrected the article--many thanks!

Claire Robinson
5/12/2020 06:55:31 am

An article has been published about Nerd Has Power's research here:
https://www.gmwatch.org/en/news/latest-news/19396

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ioderESTEl
5/12/2020 05:41:15 pm

As if on queue, now that questions about the cleavage site have been raised more widely, Shi has produced another 'natural' bat coronavirus with the requisite cleavage site.

https://www.cell.com/current-biology/pdf/S0960-9822(20)30662-X.pdf?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS096098222030662X%3Fshowall%3Dtrue

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nonameneeded
5/12/2020 08:14:25 pm

Chinese propaganda in overdrive. I hope no one buys into this nonsense about a new lineage B betacoronavirus with a furin cleavage site that was reportedly collected in May-October 2019 that they sat on until April 2020.

I looked through the methods. It seems to me that the NGS has poor coverage. The sequences were reportedly found in only a single pool (1 out of 55). They make no mention of the read depth at the S1/S2 hinge, so for all we know there was a handful of reads off of which this is based. Then the Sanger sequencing supposedly isolated the RmY02 in only a single bat feces sample which was collected in June 2019.

Also, I can't understand why it took them until April 2020 to put together this paper, not least because the lead author of this paper (Professor Weifeng Shi in Shanghai) was also one of the senior authors of a high profile Lancet paper in January, which had very similar types of figures, including sequence identity, phylogenetic analysis, amino acid comparison, and Swiss-Model protein modeling). The Lancet paper was submitted 20 days after publication of the SARS-CoV-2 sequence, so why it took 95 days to do the comparison of SARS-CoV-2 and RmY02 is not clear to me, well except for the fact that this is a clear attempt to undercut one of the major arguments that this virus was lab-manipulated.

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30251-8/fulltext#seccestitle190

Unsurprisingly, one of the co-authors of this newest paper is none other than Edward Holmes, who was the co-author of the fraudulent "Proximal origin" paper:

https://www.nature.com/articles/s41591-020-0820-9

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Lima
5/13/2020 12:19:46 am

It was actually the very old paper on RmYN02. you will need to look at the sequence/alignment more closely.
Count the number of amino acids from Y674 to V687 on their alignment for RmYN02. Based on how you intepret the grey dashes (it was supposed to represent codons that were only partially known), it may either be ?NSPAAR? or just NSPAAR.
Which is either a completely normal sequence length for S1-S2 in Lineage B, or two deletions. It is most likely a completely normal length sequence with 2 unknown AAs. as the S1-S2 junction will need at least an RS/RN (R followed by a polar residue) to be properly cleaved during the process of cell-cell fusion. RV is not one of the possible S1-S2 cleavage sites for coronaviruses, and it should have completely deactivated the virus assuming the deletion scenario.
Therefore, the only possible scenario was the identical-length ?NSPAAR? sequence for Y674-V687 of RmYN02. with a length of 8 amino acids just like every other normal Lineage B betacoronaviruses that exists.

yano
5/13/2020 10:10:30 pm

What kind of crazy system allows un-verified and possibly fake virus data to be manufactured and uploaded without verification.

No one will know if these viruses are real or a fake unless they have a sample.

I'm waiting for the angry mobs of pitch forks and torches.

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Henri
5/18/2020 03:31:46 pm

Has it not occurred to you that sending live, potentially pathogenic viruses through the post to another lab would be a serious safety risk?

Henri
5/18/2020 01:08:19 am

Are you suggesting that RmYN02 is also a 'fake' sequence?

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Lima
6/18/2020 07:53:32 am

https://drive.google.com/file/d/1gGX4EobrnP3EKYLQDsl9Xa6tKoiwmI8O/view?usp=sharing
Counting the AAs there--NO inserts at all.

yano
5/12/2020 07:42:07 pm

Is this new work with the likely copy and paste 100% E protein and the mysteriously appearing furin cleavage site causing virologists to accept the lab creation theory?

If not, how can this information get elevated to the right people? This just seems crazy that we have top ignorant scientist pushing the natural virus propaganda.


Just for curiosity, what is the probability that SARS-CoV-2 is a natural (not man made) virus? 0%, 5%, 10%????

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nonameneeded
5/12/2020 08:16:13 pm

It is not ignorance. It is a conspiracy of silence by virologists who know much, much better.

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nonameneeded
5/12/2020 08:45:25 pm

Edward Holmes is also on the Malayan pangolin coronavirus paper that appeared in Nature.

https://www.nature.com/articles/s41586-020-2169-0

Seems like there's a circle of very fishy researchers and administrators who were deeply involved in this, including Shi Zhengli, Ralph Baric, Edward Holmes, Anthony Fauci, and Peter Daszak.

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John F Signus
5/13/2020 12:22:09 am

See my takedown on it at 5/9/2020 04:13:42 am.

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Dex
5/19/2020 09:54:46 pm

Edward Holmes lab work was outsourced to the PLA.

"The Daily Telegraph can reveal that the “sequencing” and “virus isolation” on which the study relied was done by laboratories run by the PLA in China.

https://www.dailytelegraph.com.au/coronavirus/the-covid-files-australianfunded-coronavirus-paper-used-in-chinese-military-facility/news-story/7241a6b112816f3951495e0fa52ed2aa

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Even Yahalom
5/13/2020 11:26:20 am

Who are you?
Email me.
I am a virologist on this situation.
Thanks,
Even

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Lilian
5/13/2020 02:44:51 pm

I have a question abt the RaTg13 spike protein:

it was updated https://www.ncbi.nlm.nih.gov/protein/QHR63300.1

and in the old version there is an extra coding sequence (MFLLTTKRT) that is actually present in both RaTG13 and SARS-CoV-2 but it has not been annotated. It overlaps with the end of ORF1b in SARS-CoV-2. Is that normal?

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Lilian
5/13/2020 02:55:26 pm

I just noted that it has 100 % match with SARS Urbani, just after Ratg13

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Lilian
5/13/2020 03:04:09 pm

but missing there the starting ATG...mmh...

Nerd has power
5/23/2020 01:08:10 pm

It may be a conserved sequence at the end of Orf1b. Orf1b and Spike are located right next to each other in the genome, so the end of Orf1b should be right in front of the beginning of the spike.

John F. signus
5/30/2020 08:49:03 am

However, this leader sequence were not found in other SL-CoVs as this in-frame insert. All the other SL-CoVs appeared to have 1 less Nucleotide on the junction meaning that they won’t translate to such.
Highly likely that they removed just that nt to get to an extra Start codon to serve as an in-vitro protein tag(but will not affect the virus In-Vivo.)to potentially purify the Spike for use in the lab.
Also. The genome assembly Coverage on TG13 is really, really bad. Not much from the “sample” even cover the S gene. Not on the beginning of the S, either. The Tg13 sample is mostly garbage. And had 4% primates and 21% rodents in it. And the MtDNA readings contain oddly-spaced “N”s that seems to be duplicated on a sequence-specific manner. This is not the kind of “N”s you may see in real ILLUMINA runs as these came from mechanical errors (like bubbles) and are co-located with the Position index of a nt in a read. Instead it imply In-Silico work being used to fabricate most of the dataset to give it a “bat” origin.
It in stead imply an origin in cell culture or lab rats. This is the incomplete GOF backbone that were used to make CoV2 by genetic engineering.

Physics Guy
5/14/2020 02:23:24 am

Has someone checked the non-synonymous to synonymous ratio of the S2 part of the spike for the mutations that are now naturally occurring in COVID-19? Is there evidence/non-evidence for natural selection in favour of synonymous changes there?

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Libres
5/14/2020 03:20:23 am

Well, the dS/dN on the ORF1ab between RaTG13 and SARS-COV-2 was 6.22:1. Ruling out the possibility that selection in this lineage favoring Syn over Nonsyn.
The dS/dN between the S2 of ZC45 and ZCX21, two viruses that used the same host, is 5:1. This rules lou selection favoring Syn over Nonsyn on the S2 as well.
Since neither ORF1ab nor ZC45/ZXC21 S2 suggest an elevated dS/dN during selection, the 44:1 dS/dN on the S2 of RaTG13/SARS-COV-2 was a very clear sign of either one of them being unnatural.

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Physics Guy
5/14/2020 03:36:38 am

I know, but further evidence as I suggest could further prove/disprove the hypothesis.

nonameneeded
5/14/2020 09:09:16 am

Can you check the dS/dN between specifically GD pangolin vs SARS-CoV-2?for

1) RBD of S
2) S1
3) S2
4) Entire S protein
5) other structural and nonstructural proteins, particularly E and N

This paper suggests that despite the very high amino acid identity, the underlying nucleotides evince a high dS/dN in the spike protein (compared to the rest of the virus) pointing to positive selection.

https://academic.oup.com/nsr/advance-article/doi/10.1093/nsr/nwaa036/5775463

I am not very handy with these alignments or calculations, but I think if the RBD is demonstrated to be a near replica of GD pangolin RBD when it comes to protein identity, yet the nucleotide analysis reflects significant drift and positive selection pressure, we can infer that either any recombination event occurred implausibly long ago given the clinical and epidemiologic characteristics of the SARS-CoV-2 epidemic, OR that the selection was artificial and accelerated, pointing to laboratory manipulation.

I am sure this can be put on a surer footing mathematically, I just don't have the skills or expertise to do so, and would be curious if others have looked at this.

John F Signus
5/14/2020 03:20:50 am

https://www.sciencedirect.com/science/article/pii/S0092867420302622
There is a smoking gun indication that the virus have been passaged through VERO E6 BEFORE the insertion of a polybasic furin cleavage site.
These scientists, in order to assay the binding affinity of the S1 RBD portion of SARs-COV-2, constructed pseudoviruses with the SARS-COV-2 with the furin site removed. Termed S fur/mut. What they found is that such a spike enters VERO E6 cells nearly one and a half times more efficiently than BHK cells that expresses the hACE2 receptor. The SARS-COV-2 S with the furin site removed also enters VERO E6 More than one and a half times efficiently than the original SARS-COV Spike.
This mean that the S RBD of SARS-COV-2 binds the ACE2 of VERO E6 more than 1.5 times more efficiently than hACE2, 1.5 times more efficiently than the SARS-COV RBD could on the same cells.
This is way higher than even the heavily skewed calculation of the RBD’s affinity to the pACE2 receptor in that Nature article (which, despite being skewed so much that they even conclude that RaTG13 had more infectivity in humans than SARS, still showed a higher hACE2 affinity to pACE2. Their “pangolin-1” showed even lower pACE2 affinity than SARS-COV-2 in their own models—not what you expect for a virus that was supposed to have been evolved in pangolins)
Normally, an ~1.5 time higher infectivity in an organism than any other host cells is a clear indication that this organism was the intermediate host— but in this time, Physical assay revealed that the Intermediate host was VERO E6.
VERO E6 is a cancerous cell line mutated from the VERO 76 cell lines with attenuated contact inhibition, the VERO 76 cell line itself was mutated from the original VERO cell line in 1968, distinguished by a lower saturation density. Then the VERO Cell line was a cancerous derivative from a monkey cell line isolated in 1962. There is absolutely no way that VERO E6 can even be turned back into the original organism, let lone be found in wild nature in any way. The only place cells like VERO E6 exists and can exist is within a lab.
VERO E6 is used for the cultivation and isolation of viruses from all kinds of sources.
This Spike had a history in VERO E6 before the PRRA furin cleavage site was added in. Which could only mean one thing. SARS-COV-2 have been in a lab before it gets the polybasic furin cleavage site and went into humans.

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yano
5/14/2020 07:30:52 am

Great! I am so happy people are digging out this information. The world is waiting for justice. Keep up the good work!

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Javier
5/16/2020 09:07:07 am

Congratulations, good work. I'm trying to understand your reasonings, but I have one doubt.
You say:
“This Spike (SARS-CoV-2 ) had a history in VERO E6 before the PRRA furin cleavage site was added in. Which could only mean one thing. SARS-COV-2 have been in a lab before it gets the polybasic furin cleavage site and went into humans”
“Normally, an ~1.5 time higher infectivity in an organism than any other host cells is a clear indication that this organism was the intermediate host— but in this time, Physical assay revealed that the Intermediate host was VERO E6.”

In the paper you mentioned, by making assays in two different cell culture types (VERO E6 and BHK cells, this latter being transiently transfected with hACE2) the capacity or affinity of coronavirus spike (S) glycoproteins to enter into cells was tested.
In this paper, next findings are reported (according to the figures 1A and 1B):

- SARS-COV-2 S-MLV (9000 RLU) and SARS-CoV S-MLV (8500 RLU) ,both pseudoviruses entered VERO E6 cells equally well.
- SARS-COV-2 S-MLV fur/mut (fcs removed) present 1.5 times higher affinity (13500 RLU) to VERO E6 than SARS-COV-2 S-MLV (9000 RLU) and SARS-CoV S (8500 RLU).
- SARS-COV-2 S-MLV presents higher affinity (13000 RLU) to BHK cells (hACE2 transfected) than SARS-COV-2 S-MLV fur/mut (9000 RLU)
-¿¿¿ SARS-COV-2 S-MLV presents NULL affinity to BHK cells (NOT hACE2 transfected)??? (What does it mean?)

Author's conclusion: “The observed transduction efficiency of VeroE6 cells was higher for SARSCoV- 2 Sfur/mut-MLV than for SARS-CoV-2 S-MLV (Figure 1A), whereas the opposite trend was observed for transduction of hACE2-expressing BHK cells (Figure 1B). These results suggest that S1/S2 cleavage during S biosynthesis was not necessary for S-mediated entry in the conditions of our experiments (Figures 1C and 1D. We speculate that the detection of a polybasic cleavage site in the fusion glycoprotein of SARS-CoV-2 could putatively expand its tropism and/or enhance its transmissibility, compared with SARS-CoV and SARSr-CoV isolates, due to the near-ubiquitous distribution of furin-like proteases and their reported effects on other viruses”.
Speculation says (in my opinion successfully) that the polybasic cleavage site enhance its transmissibility.
Now, my doubts about your second reasoning: I don’t see clear enough since (see above) SARS-COV-2 S-MLV presents higher affinity (13000 RLU) to BHK cells (hACE2 transfected) and SARS-CoV S-MLV (8500 RLU) to VERO E6 cells.
So, it seems that really in both cultivation cells, not only in VERO E6, SARS-COV-2 S-MLV has high ACE2 affinity.

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John F Signus
5/20/2020 05:55:31 am

Well, the hACE2 transfected BHK cells had less affinity to FCS removed S than the FCS on S. BHK itself does not express any ACE2 and is used to model human cells by transfecting it with hACE2 and overexpressing it on the cell surface. the real point is that SARS-CoV-2 fur/mut had the highest affinity to VERO E6 (13500 RLU) than any other assays done. including the SARS-CoV-2 S on hACE2/BHK (13000 RLU).
The NULL affinity on non-transfected cell is just to be used as a control--to prove that there were no other interference and the hACE2 receptor assay data is valid.
technically, the hACE2 transfection/overexpression on BHK should make it even MORE suspectible than actual human cells in-vivo--most human cells does not overexpress ACE2 like that.
yet it still had less affinity to even the intact S than the fur/mut S to non-transfected (ACE2 expression level normal) VERO E6.
The real comparison is here. S(fur/mut) display no pre-cleavage on the S1-S2 junction making the S1-S2 and S2 machinery a homologue of the SARS-CoV S1-S2 and S2. therefore the comparison is between these 3 pairs:
SARS-CoV RBD->VERO E6 ACE2 (normal expression)
SARS-CoV-2 RBD->VERO E6 ACE2 (normal expression)
SARS-CoV-2 RBD->hACE2 (overexpressed in BHK)
the RBD had way higher affinity to the VERO E6 ACE2 than hACE2, and even the BHK overexpression did not knock it down in comparison. this strongly suggest that the SARS-CoV-2 RBD is far more adapted to the VERO E6 ACE2 receptor than the hACE2 receptor.
As a Control, SARS-CoV-RBD (known to NOT be native to VERO E6) displayed an affinity to the VERO E6 ACE2 that is similar to the SARS-CoV-2 RBD to hACE2. Implying that the SARS-CoV-2 RBD is not native to the hACE2 receptor.
comparison is between these 3 pairs:
SARS-CoV RBD->VERO E6 ACE2 :Non-native(known)
SARS-CoV-2 RBD->VERO E6 ACE2 :Native. far higher affinity than the other two interactions.
SARS-CoV-2 RBD->hACE2 :Non-native. same level as SARS-CoV RBD to VERO E6 ACE2.
Compiling the two pieces of data, we can deduce that the actual native host receptor of the SARS-CoV-2 RBD/S1 is the VERO E6 ACE2. implying that the S1 and RBD was evolved there.

Nerd has power
5/23/2020 01:33:01 pm

Thank you so much, John. Great finding and analysis!

Bugsy
5/14/2020 06:45:46 am

A two-step approach, first receptor binding, then the addition of a furin site was implied in a study by Ralph Baric's group "SARS-like WIV1-CoV poised for human emergence," replacing the spikes of SARS Urbani and WIV1.
"Whereas the receptor binding domain had garnered the
most interest, changes in the remaining portion of S1 as well as the
S2 portion of spike may also play a critical role in facilitating CoV
infection, transmission, and/or pathogenesis (20). Differences in
these regions of spike may yield increased protease targeting,
enhanced spike cleavage, and/or expanded tropism leading to
more robust infection for the epidemic SARS strains."

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nonameneeded
5/14/2020 08:39:45 am

For those who are interested, this is the 2014 patent for the chimeric virus construction and serial passaging by Ralph Baric at UNC. The patent was assigned to NIH in 2017.

https://patents.google.com/patent/US20170096455

Methods and compositions for chimeric coronavirus spike proteins

"1. A chimeric coronavirus spike protein comprising, in orientation from amino to carboxy terminus:
a) a first region comprising a portion of a coronavirus spike protein ectodomain that precedes a coronavirus spike protein receptor binding domain (RBD) as located in a nonchimeric coronavirus spike protein, of a first coronavirus;
b) a second region comprising a coronavirus spike protein receptor binding domain (RBD) of a second coronavirus that is different from said first coronavirus;
c) a third region comprising a portion of a coronavirus spike protein S1 domain as located in a nonchimeric coronavirus spike protein immediately downstream of the RBD, contiguous with a portion of a coronavirus spike protein S2 domain as located immediately upstream of a fusion protein domain in a nonchimeric coronavirus spike protein, wherein said third region is of said first coronavirus; and
d) a fourth region comprising a portion of a coronavirus spike protein from the start of the fusion protein domain through the carboxy terminal end as located in a nonchimeric coronavirus spike protein of a third coronavirus that is different from said first coronavirus and said second coronavirus."

Special attention should be given to the figures in the pdf, where clear reference is made to serial passaging in mice to enhance pathogenicity (see figure 16).

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Andrew M
5/14/2020 01:01:17 pm

Isn't the differential ratio of synonoymous to non-synonymous mutations simply down to the effect of purifying selection in the region concerened - exactly what you might expect?

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John F Signus
5/14/2020 05:03:38 pm

Well, ZC45 and ZXC21 uses the same host, yet their ds/dn on the S2 was 5:1 with 5 nonsyn mutations. CoV2/RaTG13 have different hosts— we should see both a bigger difference on the S2 and at least a similar dS/dN. However, In RaTG13/CoV2 it was 44:1 with just 2 nonsyn substitutions. Somehow it ended with more nucleotide substitutions than ZC45/ZXC21, yet had less Nonsyn substitutions on the S2 with ZC45/ZXC21. This is completely unnatural.
Remember that ZC45 and ZXC21 Had the same host. There can not be higher “purifying selection” in RaTG13/CoV2 than ZC45/ZXC21.

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Tony
5/15/2020 07:19:04 am

https://www.biorxiv.org/content/10.1101/2020.02.27.969006v1.full.pdf

Physics Guy
5/15/2020 08:20:23 am

As I have suggested above I think it would be good to also look at syn/non-sym ratio of the spike regions in the current mutations of COVID-19. As an outsider to this field to me it seems that this could either further prove the the hypothesis or disprove it.

Andrew M
5/16/2020 01:37:29 am

Sorry, I can't follow this. You just seem to be asserting that different ratios in different places are "unnatural", when in fact it could just reflect stronger selection in certain region(s). The article linked by Tony below seems to this.

Tony
5/16/2020 07:53:45 am

Why there can not be higher “purifying selection” in RaTG13/CoV2 than ZC45/ZXC21?
Such statements can only be made if you count over the evolutinary history of the organism.
Standard methods rely on the assumption, that the base composition is at the equilibrium.
Moreover, the assumption that the ratio of these mutations (N/S) must remain constant over the entire gene is not correct. It has also been shown for RNA viruses that that synonymous mutations can have a wide range of fitness effects and that they can contribute to adaptation the same way as non-synonymous ones. This is also immediately obvious when looking at the RNAs secondary structures of SARS-CoV2. One can be sure that changes here have an impact on the virus and altered RNA structures are particularly important when dealing with polycistronic transcripts.

John F Signus
5/20/2020 05:38:17 am

ZC45/ZXC21 never left the same cave and had the exact same host. There can't be a more stable, purifying selection than this.
RaTG13 was in YunNan. Sars2 was in Wuhan. >1000km geographical distance. RaTG13 was allegedly found in a mineshaft in bat poop while SARS2 broke out in a crowded metropolis in humans. The location, condition and host are totally different. You can't expect to see "purifying selection" holding across such a great geographical distance. Also, S2 have already started to mutate in later versions of SARS-CoV-2 during human transmission. Implying that neither the Bat host(ZC45/ZXC21) not the human host(WuHan-Hu-1/later CoV2) are capable of purifying selection for the S2 of betacoronaviruses. The RBD was located on S1. S2 is not critical for host selection as it's buried within the S (it does not interact with host receptors) and it's sole job is to facilitate membrane fusion AFTER the virus have attached to/internaized by the right kind of cell using receptors determined by the S1/RBD. From all the other viruses (fig3), it's very clear that there is a lot of flexibility in S2. other way, there can not be a higher purifying selection in RaTG13/CoV2 than ZC45/ZXC21.

John F. Signus
6/18/2020 07:57:05 am

And--unfortunately, CoV2 S2 is not the type of S2 that is under purifying selection. https://drive.google.com/file/d/1Xp1zBf2d-hu32XSKkyJ7UBOg9ZHuAURS/view?usp=sharing
up to 25 mutations on S2 have appeared during transmission. this is not "purifying" since none of these S2 mutations confers an adavantage.
https://www.biorxiv.org/content/10.1101/2020.05.21.108506v1

Zhong Hua link
5/14/2020 01:05:27 pm

https://www.blogger.com/blogger.g?blogID=1454999851131440366#editor/target=post;postID=4346645706657233226;onPublishedMenu=allposts;onClosedMenu=allposts;postNum=0;src=postname

Please help spread the truth.

Reply
John F Signus
5/14/2020 05:07:08 pm

Sorry, but it keep saying that you does not have the permission to view this page. Could you update it?
Thanks

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Andrew M
5/14/2020 03:47:27 pm

Another question having read your paper more carefully. You say recombination is rare and thus can't explain differences between the S1 portions of the various coronaviruses. But I have read elsewhere that recombinations are quite common in coronaviruses. The % difference between the genomes and typical mutation rates of such viruses suggest their evolutionary paths 2 diverged decades ago - plenty of time for such recombinations to have taken place. Any thoughts on this?

Reply
Nerd has power
5/19/2020 09:07:40 pm

Sorry for having missed this. I posted something on 5/18 in a reply to charly. You will find my answer to your question there. Please scroll down to read it. Thank you!

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John Kelleher
5/14/2020 07:45:19 pm

I think our governments have no brakes on their train. If the release was planned ,Shi could have published the RaTG13 paper before the release. Maybe ?
nonameneeded the patent you posted seems to be worded to be a vaccine . ,https://patents.google.com/patent/US20170096455
Funded by the U.S. Government.

Reply
Tony
5/16/2020 08:19:08 am

"nonameneeded the patent you posted seems to be worded to be a vaccine . ,https://patents.google.com/patent/US20170096455"

Yes, maybe a vaccine. It is the double-edged sword of "dual use", which is always part of gain-of-function research.
If you prohibit GoF, you should also prohibit loss of function, because often you do not know what will come out later.
Besides, I believe that a general ban on GoF is counterproductive with regard to protection programmes such as vaccine development.
If we want to understand the basic mechanisms of these pathogens in advance, there is no way around it.
These are of course questions that need to be discussed openly, but it is a different question whether Shi wanted to produce a biological weapon. If so, there are many many ways to make this easier.
And it is of course another question whether the virus is of natural origin or whether it is made in a laboratory.
Unfortunately a lot of things are mixed up, some of which are not based on evidence but on rumours.

Reply
Jane
5/18/2020 06:12:46 am


Do you really need GoF studies to develop vaccines? Maybe for viruses that might arise in the future, that maybe will never appear. And you take the chance to cause the next pandemic with your experiment that might go wrong. This was the case of SARS-CoV-2, I fear.

Robert Piller link
5/15/2020 02:10:17 am

A Nerd has Power.

I have to say I was intrigued by the title, I for one have never been in any doubt about this, but all too often this power can be highly destructive. So much so I've written a publication on this.

The Hapless Conservationist. Please take a read.

https://static.wixstatic.com/ugd/74da12_dde38c5de5ea45389debda0a22ab3038.pdf

If you get in touch I'll put your details onto our mailing list.

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John Kelleher
5/15/2020 12:48:49 pm

https://www.youtube.com/watch?v=GUW51MYQ_XQ
This a video from 2005. It is listed with no information. A women is presenting a vaccine to reduce religious fundamentalism in the Middle East.

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Andrew M
5/16/2020 02:11:47 am

https://www.metabunk.org/threads/debunked-funvax-pentagon-briefing-on-removing-the-god-gene-hoax.317/

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Adam
5/15/2020 08:12:31 pm

I am not a virologist so can't comment on the analysis and its accuracy however why don't you reveal your identity? Are you are based in China and forsee a threat? As you judge others of being biased, would be great if you let the readers judge your biases as well. Not accusing you of anything just making sure we know the person behind the article. I dont understand Chinese hence can't really read the Chinese version.

Regarding whether the virus was man made or not, world is fucked as we know it anyway. How will you penalise China if its proved with absolute evidence that China created the virus and it was leaked?

Reply
Fight Against CCP
5/16/2020 12:26:54 am

I'm not a virologist either. Neither am I the author of this article. However, why do everyone have to disclose their identity especially on internet? Have you ever tried to figure out why and how the world known virologist died suddenly and mysteriously back in this January? Have you ever tried to figure out Czech Senate leader Jaroslav Kubera's sudden death right before his trip to Taiwan? What about the vaccine developer, Liu Bin's being assassinated? I'm sure that you have this common sense as well, as otherwise, you would have disclosed your real full name.

Letting as many scientists as possible see through CCP's Nazi conduct is all what a scientist can do. As for how CCP will be punished, the governors of each country will have their own decision, Besides, does truth really require disclosing one's identity to be proven to be true? Don't worry, just wait to see how CCP will end up with the similar fate as NAZI. The evil will eventually be punished.

If you really want to understand what the Chinese comments are, just simply right click the mouse to select "Translate to English" in Chrome to understand everything.

Reply
Nerd has power
5/18/2020 04:38:02 pm

Thank you, my brother. We are in this fight together.

Hi Adam, I have my own reasons (part of it is fear) to not reveal my identity. If you are willing to believe your own ability to reason and judge, who I am does not matter. I can assure you that I'm no comparison to Andersen of the Nature Medicine paper, which argued for a natural origin of the virus. So, if names are important for you in judging this, I would admit failure right away. If you don't feel safe believing it, please help invite real experts over to join the discussion. The comment section here offers even more than what's in the articles. More critical insights, the closer we will be to the truth.

True Crime
5/16/2020 06:56:39 am

Is Shi trying to say now (new paper) that it was not Rhinolophus affinis from Yunnan, but may be Rhinolophus sinicus from... Hubei?

https://www.biorxiv.org/content/10.1101/2020.05.13.093658v1

R. Ebright seems to have noticed it and replied accordingly.

https://twitter.com/R_H_Ebright/status/1261649950078836736

Reply
True Crime
5/16/2020 07:17:54 am

All out:

https://twitter.com/R_H_Ebright/status/1261660326082367489

Richard H. Ebright
@R_H_Ebright
En respuesta a
@DrEricDing
WIV constructed a series of novel chimeric viruses encoding different receptor binding domains--with different receptor binding affinities--in an otherwise constant genomic context. And did so using "seamless ligation" procedures that leave no signatures of human manipulation.

Reply
CCP is NAZI link
5/16/2020 12:40:37 pm

EXCLUSIVE: Virus researchers uncover new evidence implying COVID-19 was created in a lab:


In a LifeSite exclusive, reporter Matthew Cullinan Hoffman breaks the story about the preliminary results of a new study that strongly suggests that the novel coronavirus was produced in a laboratory, presumably a lab of the Wuhan Institute of Virology.

The study, which was carried out by a team of Australian scientists, produces evidence that the virus that causes COVID-19 is specifically optimized for attacking human cells, indicating that it did not arise in an animal host.

Hoffman has been researching this issue for weeks, and this is his first major piece, with more to come in the days ahead.While preliminary, this new study raises serious questions about the narrative on the virus from China, the World Health Organization, the global health establishment, and social media censorship.

https://www.lifesitenews.com/news/exclusive-virus-researchers-uncover-evidence-implying-covid-19-was-created-in-a-lab

Reply
Lori
5/16/2020 04:11:07 pm

I've been following your work along with reading the comments and just wanted you to know you are not the only one who thinks this virus was manipulated to make it transmissible to humans.

The Coronavirus Is Man Made According to Luc Montagnier the Man Who Discovered HIV

https://webcache.googleusercontent.com/search?q=cache:oDzY1m720UYJ:

https://www.gilmorehealth.com/chinese-coronavirus-is-a-man-made-virus-according-to-luc-montagnier-the-man-who-discovered-hiv/+&cd=2&hl=en&ct=clnk&gl=us


EXCLUSIVE: Virus researchers uncover new evidence implying COVID-19 was created in a lab
https://www.lifesitenews.com/news/exclusive-virus-researchers-uncover-evidence-implying-covid-19-was-created-in-a-lab?fbclid=IwAR0OhZqkRBeysQCrx5rEcsDH7VWOhnjqjq4_0o5axzVQi3iPYalJSt2Q3Po

BTW China now claims they destroyed the original samples.

China admits to destroying early virus samples sought by Pompeo
https://www.washingtonexaminer.com/policy/defense-national-security/china-admits-to-destroying-early-virus-samples-sought-by-pompeo

Why would China destroy the virus samples?

What is China hiding were they altering the virus to use as a bio weapon?

Ex-Chinese Official Details Plan for World Domination
https://www.newsmax.com/navrozov/china-biological-russia/2009/09/17/id/335042/

Reply
Ape link
5/16/2020 04:39:02 pm

Instinctively skeptical of useful novel organisms. This article seems compelling, (and great illustrations). https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

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nonameneeded
5/16/2020 06:51:02 pm

Sad that it's the dailymail that reports on this and not the more "prestigious" mainstream media a la NYT, BBC, Guardian, WaPo. I doubt this will go into a high profile journal, although quite excellent.

They compared SARS-CoV and SARS-CoV-2 evolution and show that the dN/dS is flat in the latter and in SARS-CoV at the end of the epidemic, whereas it was quite steep for early SARS-CoV. They suggest the high dN/dS early on in SARS-CoV suggests a process of animal->adaptation which SARS-CoV-2 never demonstrated, suggesting very high adaptation to human infection from the very beginning. And they finally do allude repeatedly to the lab possibility being very real.

They also put the stupid wet market theory to death.

Preprint below:

https://www.biorxiv.org/content/10.1101/2020.05.01.073262v1

Reply
Nerd has power
5/18/2020 01:42:49 pm

Thank you for sharing it. Great article! Reading in between the lines, I can tell that the authors are as convinced as many of us here, and yet they just can't say it in a submitted manuscript. They are so brave to bring their work to the public in this manner. Doesn't matter whether it makes to a top journal, we should spread it.

The statistical aspect of this study is the real force. Given their data, one has to agree that there has been no sign of adaptation of SARS-CoV-2 in humans, which is in great contrast to how SARS worked its way (clear adaptations) in the early stages to eventually settle down in the human population.

Reply
Lori
5/16/2020 11:31:11 pm


Database Of Wuhan's 'Batwoman' Altered 48 Hours Before COVID-19 Samples Ordered Destroyed

https://nypost.com/2020/05/06/what-is-china-covering-up-about-the-coronavirus-devine/

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Henri
5/18/2020 01:34:14 am

No, it was not 'ordered destroyed' according to the news article. 'The article says 'made substantial changes' which, upon further reading, turns out to be 'remove the terms 'wild life' and 'wild animal'.

So, you have exaggerated the report.

Reply
Lilian
5/18/2020 01:41:44 am

You need to practise reading Henri:
Lori writes database altered, he is right

Days before the Wuhan wet market was bleached, whistleblowers were punished and virus samples were destroyed, someone at the high-security Wuhan Institute of Virology censored its virus database in an apparent attempt to disassociate the laboratory from a novel-coronavirus outbreak that would become a global pandemic.

Henri
5/18/2020 02:46:58 am

Read the article more carefully, Lilian. Read ALL OF IT. That 'censoring' was not destroying the data base at all. It was changing some words or deleting some words viz: wild animals. Your other points, even if true (which I doubt) does not involve the database.

Lilian
5/18/2020 04:38:49 am

Who is using the word destroying in relation to the database? The article says that the samples were destroyed, not the database.
Please read carefully what Lori writes. He writes the same.

Why do you doubt that SARS-CoV-2 is Disease X?

Ridan
6/18/2020 07:58:36 am

Unfortunately, that 64.3MB database is now gone. completely.

Reply
Ape link
5/17/2020 08:47:47 am

When asked by Zhong Nanshan if her lab is responsible for creating and losing the virus, Shi says, "She said that's totally ridiculous, she had never been doing anything like that," said Zhong, who called Shi a "good friend."
"She said based on their equipment and facilities and manpower... they were unable to do anything, any kind of artificial virus at this time." Was her lab technically "unable" to manipulate/modify viruses? - https://www.cnn.com/2020/05/16/asia/zhong-nanshan-coronavirus-intl-hnk/index.html

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CCP is NAZI
5/17/2020 10:05:03 am

How would you expect a CCP criminal to admit their crime? They know exactly what kind of consequence admitting their crime would lead to. None of those CCP's mouths has any credibility including CNN which is short for ChiNiese News who serves as CCP's propaganda. Sadly, none of the sources you use have any professional ethics.

Reply
Henri
5/18/2020 01:10:53 am

What are these 'reliable sources' that the Wuhan lab does not have a hard copy of RaTG13?

Reply
Lilian
5/18/2020 01:18:18 am

Is it not enough that the name of the sample was changed from BtCoV4991 to RaTG13? What do you need more than this demonstration of scientific fraud?

Reply
Henri
5/18/2020 01:41:18 am

This does not answer my question

charly
5/18/2020 03:34:28 am

You can see there https://www.ncbi.nlm.nih.gov/nuccore/MN996532 that they published in 2020 a sequence from a sample collected in 2013. While this does not constitute any fraud, and their claims support it, it is unusual.

Reply
Lilian
5/18/2020 04:31:16 am

It is a fraud to change the name of a sample. In 2016 BtCoV4991, in 2020 RaTG13.

Nerd has power
5/18/2020 02:02:20 pm

This is what I was told by people I trust (hence my reliable sources):

Upon the publication of Shi's 2020 Nature paper, many people in the field wondered whether RaTG13 could infect humans directly. As I said in the article, anyone with adequate knowledge in coronaviruses would immediately think of such a possibility if they simply peek at the sequence of RBD. Some people even intended to obtain the gene of RaTG13 and try to prove or disapprove the theory of such a virus jumping directly from bats to humans. As you could imagine, many of them know Shi personally and asked her, either for an answer or for a copy of gene. The message from Shi was consistent: she does not have a physical copy of the virus and she only has the sequence info.

You know who the best source is? Zhengli Shi herself. If you got the time, you could email her directly and see what her answer would be. It would be even better if you could manage to get her here. I would love to see her comments in this section.

Reply
Henri
5/18/2020 02:49:30 pm

Do, your 'reliable source' is hearsay snd nothing else. You email Zhengli Shi and get a response before you make unfounded allegations

Henro
5/18/2020 03:34:55 pm

So, your 'reliable sources' turns out to be hearsay.

YOU are the one making the allegation that RaTG13 is a made up sequence. So, I suggest YOU email Zhengli and at least give a chance of a response. So, find her email address and get on the ball. Fair enough?

Nerd has power
5/18/2020 04:16:00 pm

You are right. I am the one making the allegation that RaTG13 is fabricated. I wrote a whole article about it. However, this "reliable sources" thing is just a small part of my reasoning. Don't forget there are other more substantial evidence in there. Calling the allegation unfounded is, in my opinion, unfounded.

BTW, is there any evidence that Shi has shared any copy of this RaTG13 virus with anybody? Many did request. Her close collaborator Peter Daszak confirmed on tweeter that Shi only obtained the sequence info. I actually invited Peter under that tweet to come over and comment, but I don't think he did. I doubt Shi would be responding any more positively. A request sent by a neutral person might be treated differently though.

Henri
5/18/2020 06:06:58 pm

In other words you want someone else to do your dirty work because you din't have the courtesy or courage to do it yourself

Nhele
5/18/2020 09:02:11 pm

How curious Henri that you make such a statement about 'dirty work' and a lack of courtesy and courage. It appears the author of this article has, in a very reasonable way, indicated that he has invited these experts to share their point of view here. Your response sounds childish and repudiates your attempts to present a serious alternative to some very in depth analysis appearing here in the commentary.

Henri
5/18/2020 10:09:00 pm

Nhele,

Nerdhaspower has made serious allegations against Zhengli Shi.NerHasPower suggested I should email her to clarify issues. My reply was that since he / she has made the allegation, he / she should email her. His / her response was that someone else should email her on his / her behalf to get a better response.

I find that a cop out and graphically said as such. Zhengli Shi's email address is available. I feel very strongly that NerdHasPower should contact her directly and invite a response.

If you think my suggestion is childish, so be it. I still feel NerdHasPower should be the email and not have the cheek to request someone else to do it on his / her behalf.

Maybe you should email Zhengli Shi on NerdHasPower's behalf. Will you do it?

Leo
5/18/2020 11:38:55 pm

I bet that RaTG13 is not functional. Otherwise someone would have already synthetized it to make a proof of existence using this method:

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

https://www.nature.com/articles/s41586-020-2294-9

I hope that a group not involved in this story will do it soon and show how RaTG13 behave.

@Henri: I am totally at Nerd’s side. Are you aware of all people involved with this story that have been killed?

Justice Defender
5/19/2020 12:03:51 am

The author wrote down his or her analysis, hence s/he is already done with his/her job. Now that you are the one making "unfounded allegations" statement, why cannot you first prove how it is unfounded? If you can show your so called "unfounded"
evidence, I'd be happy to email her directly once you provide her email(I cannot find it.). This is absolutely NO big deal! Further, are you sure that she will come to comment on this site in spite of the CCP's strict censorship? Hasn't Wang YanYi wechated everyone i the lab not to discuss about the virus? If I email her without any response, how will my time be compensated just because you are barking around here?

ll11ll11
5/19/2020 10:19:53 am

I wouldn't argue with this "henri", he apparently just a stupid troll, just ignore him.

Lilian
5/18/2020 01:21:29 am

Synergistic China–US Ecological Research is Essential for Global Emerging Infectious Disease Preparedness

https://link.springer.com/article/10.1007/s10393-020-01471-2

EcoHealth volume 17, pages160–173(2020)

Do you need more evidence?

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Lilian
5/18/2020 01:24:32 am

Here you are:

How Scientists Could Stop the Next Pandemic Before It Starts

On a cold morning in February 2018, a group of 30 microbiologists, zoologists and public-health experts from around the world met at the headquarters of the World Health Organization in Geneva.

https://www.nytimes.com/2020/04/21/magazine/pandemic-vaccine.html

Disease X = SARS-CoV-2

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charly
5/18/2020 03:44:53 am

Some red flags triggered during my reading:
- 'When comparing sequences, one can compare either gene sequences or protein sequences. For viruses, however, this makes almost no difference as the whole genome of a virus is practically translated into proteins'
I dont agree, mutations occur at the nucleotide level, and many mutations will not translate into a different amino acid when isinde a gene (aka silent mutations). However, such mutations must be accounted for when you try to compare the genetic distance between two individuals.
So the whole amino acid interpretation is biased in my opinion.

- 'This is extremely rare because natural evolution typically takes place when changes (mutations) occur randomly across the whole genome. You would then expect the rate of mutation being more or less the same for all parts of the genome. '
I dont agree. A species will likely accumulate mutations not killing itself. So there are genes which will accumulate very few mutations because most of them would be deadly for the virus. In other genes however, they may only alter the host the virus can infect, which is no issue for the survival of the species.

- 'In evolution, recombination events happen much less frequently than random mutations.'
I don't have the frequencies in mind. But I know for sure recombination still occur during each meiosis in drosophilia, which lead morgan to push genetics forward. Also, it seems recombination is known to occur in coronavirus since a few years now, with the implications on human health.
Also, some recombination mechanism has been more or less conserved during evolution, so this is probably non negligible. On the other hand, multiple mechanisms to correct random single point mutations exists across various species.

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Nerd has power
5/18/2020 03:27:46 pm

Thank you for the comments. I was just about to call the day for blogging and move on to other things. Your red flags gave me a reason to linger here a bit longer. I will address each of the points you raised.

First, would the sequence comparison based on amino acid sequences be appropriate or biased? Honestly, a big concern that I had was whether or not they have manipulated the sequence of the Wuhan coronavirus when “creating” it. If a person is making a bioweapon by basing on a template, he/she wouldn't dare to change the amino acid sequence much because it would more likely affect the function. In contrast, he/she has more room to play with nucleotide sequences. As you pointed out, there could be silent mutations, right? The benefit there is clear – the template and the product would look further apart. Out of this fear, I felt that it is safer to compare amino acid sequences.

Importantly, doing the comparison using AA sequences does not mean that we completely ignore silent mutations in nucleotide sequences. In fact, if you read the newer article “RaTG13 is fake”, you will see that I did a thorough analysis on the synonymous (silent) and non-synonymous mutations for the spike gene. That analysis actually indicated further, and very firmly, that RaTG13 was fabricated.

Second, certain genes may accumulate more mutations than others. You are correct on this. In fact, I mentioned the same thing in my article:

“Although the spike proteins of different coronaviruses are more likely to differ, greater discrepancy in S1 may only be expected if two viruses have been long separated during evolution and have adapted, through random mutation, to their respective hosts for a long, long time.”

I’m not hiding this fact. Can random mutations convert an average bat RBD to an ACE2-binding, SARS-CoV-2-like RBD? It actually can! That would have to come from convergent evolution. The problem is that convergent evolution takes enormous amount of mutations and therefore a long, long, long time. Well, could such a convergent evolution leave the E protein intact (remember E is 100% identical between ZC45 and early samples of SARS-CoV-2)? I say – NO WAY. Not to mention all the other proteins also remain highly identical between ZC45/ZXC21 and SARS-CoV-2. In fact, I will challenge you to find an example where convergent evolution (not recombination) has been shown to be responsible for 69% identity in one protein and 95-100% identity in other proteins.

Finally, how often does coronaviruses see recombination? Pretty often. I did not state that in the article, but I did say so multiple times in the comments. However, just because recombination could happen in coronaviruses does not mean any pattern of the genome can be achieved through natural evolution. There have been some great discussions here, which clearly dismissed the possibility of Furin-cleavage site being acquired through recombination. I also listed the reason why the RBD region (more precisely the RBM, receptor-binding motif) of SARS-CoV-2 is next to impossible to be obtained through such a precise recombination. Importantly, both of these two recombination events have to occur to get to SARS-CoV-2. I did the best I can to show that this is not possible, but I certainly did not expect to convince everybody.

Also, drosophilia may not be the best comparison here. If ZC45 is only recombing with itself, it can never get to SARS-CoV-2. What it would take is for ZC45 and another beta coronavirus (viruses too distant from ZC45 most likely would not work because recombination needs high sequence identity) to be in the same cell of the same bat (it’s horrible, but this bat should not die right away, otherwise the recombined virus don’t get to be passed around). This other beta coronavirus has to somehow steal all the good tricks from SARS RBD and yet drop a bunch of non-essential elements (it actually would be less suspicious if the RBD here simply resembles SARS RBD). And then, the recombination has to occur in a very specific manner – just the RBD being swapped between ZC45 and this other SARS-resembling/non-identical coronavirus. I mean, this last step alone is just so so so unlikely to happen. The most common form of recombination in coronaviruses is template switching, which is not going to give you what is needed here. Alright, putting all these requirements together, how likely is it going to happen in nature? When you get a number for it, don’t forget to multiply it with the probability of recombination to acquire Furin-cleavage site too.

Alright, I think this would be the last time I try to re-explain why recombination is impossible here – at least in my eyes.

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John
5/19/2020 07:04:06 pm

People win the lottery sometime too, Covid likely a gain of function experiment but not impossible that is came from wet market. On the other hand, how many doctors get reprimanded for using WeChat for simply noticing a SARs like infection in the hospital and sharing it with other Docs in Wuhan. No real reason to "kill the messenger" over a natural spillover event unless they are covering something up.

Nerd has power
5/23/2020 02:08:11 pm

Yes, you are right in that they can always argue that, even if the chance is one out of one million years, it is still a possibility. That's the beauty of recombination.

But, then again, what is the need to publish a fake RaTG13 virus and fabricate its sequence?

As you said, the weirdest thing is actually not about scientific evidence. It is how the CCP government censored discussions on the topic, strictly screened scientific manuscripts looking into the origin of the virus, and reacted furiously to foreign requests of investigating the Wuhan institute of virology. Shouldn't the CCP government be the most interested in finding out where it came from and how the pandemic initiated?

Shannon Entropy
5/20/2020 07:44:36 pm

I tried to do my own mathematical analysis of the Wuhan virus genome compared to the other known beta corona viruses. I was able to download all the genome data from www.viprbrc.org.
Since I am not a biologist, the only biological information I am using is the genetic code. I excluded all other 2020 data.

The only extremal property of the Wuhan virus (and of RaTG13) among all these corona viruses which I was able to find was the codon usage percentages for certain codons (I used the ORF1ab-polyprotein). This is implicitly also oberved, in the paper https://www.biorxiv.org/content/10.1101/2020.02.15.950568v3.full and probably best explained by the CpG Deficiency
https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa094/5819559

I am interested in the genome of RmyN02 to repeat the analysis done by Nerd has Power. This genome is not in the Genbank as it seems and only available via https://www.gisaid.org/
as EPI_ISL_412977.

But this is secret "science" since gisaid says when I like to access the data:

"You may not distribute GISAID data outside the GISAID community, such as by releasing genetic sequences obtained in GISAID in any publication, transferring the data to colleagues that are not registered users, or offering GISAID data on a server accessible by others who are not duly registered with GISAID".

Could someone with access please make the sequence somehow available? This should be in the public interest since it is said that it proves the natural origin of the Wuhan virus.

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Verner Von Rovan
5/21/2020 06:42:22 am

RmYN02 actually had two deletions. one at T678 and the other at S686. the nt sequence appears to possess multiple short indels scattered across the S1-S2 junction when compared against ZC45. a hallmark of an low-quality sequence being used for assembly and resuled in a misplaced contig at S1-S2.
the deletion of S686 will also deactivate S1-S2 junction cleavage-- either an R!S, R!N or R!G is needed at mimimum for TMPRSS2/Cathepsin L/Trypsin recognition and proper cleavage of the S1-S2 junction during entry.There is no furin site at the S1-S2 location--which prevents cleavage of S1-S2 during exit.
the result is a S protein that can't be cleaved. and therefore a deactivated virus that will not be able to infect anything In-Vivo. RmYN02 is either very poorly curated, or is straight up fabricated.
either way, RmYN02 is not an example of "insertions" happening in lineage B in the wild, nor is it an example of a furin site.

The real problem is the completely normal 6.22:1 dS/dN on RaTG13/CoV2 orf1ab and the completely impossible 44:1 dS/dN on the S2. nomatter which kind of model you propose the maximum dS/dN possible in the natural origin model is <9:1. still leave a nearly impossible 1 in 899 chance for natural mutations to cause a dS/dN like this.
The S2 still make it crystal clear that either RaTG13 was fake, or SARS-CoV-2 was engineered out of it.

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Henri
5/21/2020 09:41:29 pm

If what you say is correct, then why not send a letter to the Editor of Cell and see if you get a response? They should pass you letter on to the authors and allow them to reply followed by publication of both letters at the same time.

yano
5/22/2020 07:54:46 am

https://www.biorxiv.org/content/10.1101/2020.05.01.073262v1.full.pdf

" In a side-by-side comparison of evolutionary dynamics between the 2019/2020 SARSCoV-2 and the 2003 SARS-CoV, we were surprised to find that SARS-CoV-2 resembles SARSCoV in the late phase of the 2003 epidemic after SARS-CoV had developed several
advantageous adaptations for human transmission."

More scientist are starting to see the evidence.

Maybe someone could comment on this interesting issue of a stable S protein from the beginning.

A pre-adapted to human transmission virus implies one of two things. It was manipulated in the lab to do this. Or it circulated in humans for months/years without getting anyone sick.

The former seems more possible

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Andrew M
5/22/2020 11:26:06 am

You appear to be reading something in this article which isn't in it. Search this discussion thread for "Bill Gallagher". Read that. Go to his site and read the rest of his comments. There is nothing about the S protein which implies a man-made creation.

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yano
5/22/2020 12:14:41 pm

I understand I am reading something into this article. This is why I am asking for input.

Is this not suspicious that the S protein was already tailor made to infect humans? The SARS CoV-1 came from animals and the S mutated significantly over time to better "FIT" humans.

SARS-CoV-2 was "FIT" for humans from the beginning. This seem very suspicious.

What are possible explanations for this? 1) It was created this way by humans in the lab, or 2) it was mutating inside humans getting no one sick for months or years?

I'm suggesting this is another piece of information that points to a lab created virus.

Tony
5/23/2020 10:25:28 am

You mean Bill Gallaher(?):
http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384

Indeed that is a very good and different view. It's the first time I've read that the construction of the insert is actually not in frame. If you take a closer look at the nucleotide sequences, you wonder why somebody should make it so complicated.
Of course you can, but that's extremely weird.
Also the palindrome sequence in connection with a copy-choice_recombination is impressive. Overall, the analysis just confirms that RaTG13 is simply not the ancestor of CoV2.

Nerd has power
5/23/2020 02:35:49 pm

Thank you, Yano, for bringing up this preprint article again (someone did it in an earlier comment too). It is THE best work so far, in my opinion. Very powerful analysis. The pattern is not just seen in the S protein. It's the overall feature of adaptation: SARS-CoV-2 is well adapted to humans from the very beginning, while SARS clearly worked diligently in the earlier stages to gradually settle down in the human population. In other words, SARS did look like a virus jumping from an intermediate host to humans. SARS-CoV-2 did not.

To other commenters here, yes, comparing what is laid out here with what Professor Bill Gallaher has been saying must be very interesting. I don't think anything is not in frame for the furin-cleavage site, but it's been too long since I read his post and I forgot his actual argument. I tried to leave a comment under his blog, but somehow wasn't able to. Anyways, I'm glad people are looking both ways. It is the best way to stay unbiased.

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Med
5/22/2020 11:29:53 am

Hi,
The epoch times documentary said there are 3 gp 120 hiv and 1 hiv gag insertions in the sars-cov-2.
The only other known viruses to have a similar sequence is the:
- Hiv virus
- a bat coronavirus that zenghli shi found.
Isn't this bat coronavirus ^ the RaTG13 ?
Thanks.

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Andrew M
5/22/2020 12:33:34 pm

The Epoch Times is Falun Gong propaganda. They have less interest in science and more in discrediting the CCP. Their motives may well be good ones - I make no comment on that. But the HIV insertion story is nonsense - it has been thoroughly debunked and is not worth pursuing. It detracts from Nerd's theory, which is at least worth parsing,

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Shakti
5/22/2020 12:52:21 pm

You have presented a lot of very interesting information here but I don't understand one thing - why is everyone referring to Dr. Shi Zhengli as Dr. Zhengli Shi? I'm pretty sure that Shi is her first name.

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mz1-2-3-4
5/22/2020 03:38:50 pm

You are simply clueless in this very topic (the name). Her name: Last = Shi; First = Zhengli. In Chinese: 石正丽

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Shakti
5/22/2020 03:51:46 pm

My sincere apologies for the mistake. I only read articles in my native language, English, where she is always referred to as Dr. Shi Zhengli - and in English, the name that comes first is the first name.
Example: https://www.scientificamerican.com/article/how-chinas-bat-woman-hunted-down-viruses-from-sars-to-the-new-coronavirus1/

Keith link
5/22/2020 01:46:09 pm

I believe you will find this blog review of your paper useful

https://www.randombio.com/ratg13.html

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Andrew2
5/22/2020 02:44:37 pm

I think This is HIV insertion paper...Please discuss it after reading not before reading!
https://osf.io/d9e5g/

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Andrew M
5/22/2020 03:41:31 pm

I can't say I understand this paper - particularly when I got to the part about the golden ratio numbers in DNA. The 2 authors really do not check out very well. Montagnier, true is a Nobel prize winner who made excellent discoveries in the past but is now an 87 year old embracing some quack homeopathy "science" theory and the other guy - well - read his Twitter.

I don't think this should really be taken as support for Nerd's theory, as it detracts rather than adds to the plausibility.

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Annette
5/23/2020 01:13:51 am

I found Montagnier paper very informative and robust. My understanding is he was able to detect electromagnetic signals at high dilutions and is quoted as saying "High dilutions of something are not nothing. They are water structures which mimic the original molecules.” A Homeopathy uses high dilutions the proof that remedies have an effect when prescribed professionally should not be debunked but further understood. It is a poor scientists who closes his mind to research as it will limit his/her ability to see things outside of the expected.

Regarding the discussion, does anyone have information to say if a lab created synthetic virus in plant/medicine will challenge the immune system in the same way as the natural virus and leave a person with long term immunity or will they become chronic with an auto immune disease?

Andrew2
5/22/2020 04:15:54 pm

I am sure there is something there as original sars was also had similarity with HIV...also some of the signs of this disease are similar to aids but no one compare those! I am especially worried for long term chronic form of disease with some kind of immunodeficiency for life ..also a paper shows the same genetic risk factor for HIV, could influence your resistance to covid, please see PMID= 23361009 ...I really hope I am wrong!

this is also jean Claude Perez twitter...May be he engaged more in real discussion...he is a little younger!

https://mobile.twitter.com/jcperezcodexlang=en

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Andrew M
5/22/2020 04:39:30 pm

There isn't "similarity" - they are completely different viruses. They have different proteins doing different things (with some overlap, admittedly). This paper seems to find repeated short sequences, after searching through all HIV genomes available (there are quite a few!). Before we even get to considering why viruses and their proteins might have common short sequences for natural reasons, where is the statistical analysis to demonstrate this is anything other than random?

Can't get your Perez link to work, but try mine:
https://twitter.com/JCPEREZCODEX?ref_src=twsrc%5Egoogle%7Ctwcamp%5Eserp%7Ctwgr%5Eauthor

"Golden ratio"! looks like a nutcase (he's 72).

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Andrew2
5/22/2020 04:52:51 pm

I know but those sequences are unique to this virus...don't forget francis Boyle that for many years claim all SARS are man made!

Andrew2
5/22/2020 04:48:48 pm

oops...that was older paper...The pmid=32348495. In that paper please check ref10.

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Andrew2
5/22/2020 07:36:32 pm

Ref8

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Greg
5/22/2020 11:58:41 pm

Hello, I found your blog post very interesting but I have one question regarding the dn/ds ratio that you use as evidence to show that either ratg13 or sars-cov-2 is not natural. First, isn't it a more parsimonious explanation that the skewed dn/ds ratio in certain portions of the S protein gene is due to the fact that cov-2 contains sequences from the pangolin derived coronavirus, specifically in the RBD region of the S protein gene and therefore in those regions, the dn/ds ratio would be different compared to the rest of the genome? Shouldn't one compare the dn/ds ratio in the RBD region of cov-2 to the RBD region of the pangolin derived virus? Also, even if they are different, a skeptic could say so what. Although this may not occur often, it could occur just due to random chance. Is there any way to test whether the differences are statistically significant using a Chi square test or something like that?

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Nerd has power
5/23/2020 02:19:28 pm

Thanks for the comment. The RBD is in S1, not S2. So the comparison of dn/ds for S2 is not affected by a possible RBD-specific recombination.

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Greg
5/24/2020 06:51:50 pm

Thanks for your response. But what about the fact that positive or negative selection can also skew the dn/ds ratio? Couldn't a skeptic just say that the S2 region underwent strong positive selection, accounting for a high dn/ds ratio, while the rest of the genome is under negative or purifying selection which produces a low dn/ds ratio? This alternative explanation could account for the genetic peculiarities of Cov-2, but it seems like it would raise more questions, specifically it seems like cov-2 is well adapted to humans, its genetic polymorphism resembling the original SARS virus late into the epidemic after human adaptation. The question then becomes in what host this adaptation (positive and negative selection) occurred. It wasn't pangolins and it wasn't bats since the binding affinity of the S protein to human ACE-2 receptors is greater than to the bat/pangolin ACE-2 receptor. There's also no evidence of early adaptation of Cov-2 to a human population. Isn't the most likely explanation that Cov-2 adapted to human cell culture lines?

John F Signus
5/25/2020 10:40:53 am

Even the pangolin strain had 8 sense mutations on the S2. The virus itself is about 10% different and the mutations on the S2 appears to have been saturated. However, with the ZC45/ZXC21 bearing 5 dN at a level of similarity being ~97% and pangolin/CoV2 bearing 8dN at a level of similarity being ~90%, the ~96% similar RaTG13/CoV2 can never have just 2dN. The expected dN should be between 5 and 8– and the pangolin strain tells that the dN on RaTG13/CoV2 is far from saturated. The 44-to-1 ratio should never happen at all.

Further examination revealed all the original samples of the pangolin-CoV were heavily contaminated.
Using the NCBI GenBank Trace tool (called Krona, Accessed via the “analysis” tool of the SRA dataset), The pangolin-09 on Nature is 40% human, Lung07 is 3% human, Lung08 is 2% human (7% hominid RNA), Lung09 is 65% human and Lung11 is 1% human. These corresponds to up to 1.4mg out of an 100mg sample sent to sequencing in Lung11(which have only 2Kb reads) , 4.2mg out of 100mg in Lung08, 5.2mg from the 100mg Lung07, up to 86% of Lung09 and up to 44.9% of the Nature article sample (pangolin-09 being the only sample with mapped reads for “Sarbecovirus” and “WuHan seafood pneumonia virus” That were “identified as SARS-COV-2 related” by their methods section).
The samples were supposed to be lung tissue. As we know, Lung tissue does not directly contact Any outside surfaces within the animal since the lung is located deep inside the body cavity. You may get bacteria there but Human RNA can only enter via one route: direct contact and contamination of the sample after the sample have been taken from the original animal/organ/tissue.
The Human RNA fraction is very high when comparing the fraction of reads from human against the fraction of reads from pangolin. Up to a significant fraction of the total weight of the sample. Even things like improper handling of the samples without globes won’t give such a high fraction.
The only logical explanation? The Samples were artificially spiked with material from virus cultures in human-derived cells. The contamination fraction in Lung07, Lung08 and Lung11 were in fact consistent with someone putting a swab or a pipette tip worth of Human cell culture material into the sample before sequencing was conducted.
We knew that in order to design a very high binding affinity RBD, extensive cell passage must first happen in order to artificially select for an RBD that best binds to human cells. The RBD design would have taken during September to November. The same timeframe where the “sequencing” of the samples allegedly happened. (During the initial sequencing of the samples, they did not assemble a genome and have never produced a S or an RBD. That part may not even be from their datasets as they claimed to obtain it using “specific PCR assays” of which the data was never available to anyone. Even the PLOS Pathogens article recovered a very poor coverage on the Spike, leaving 5 gaps in their published Spike gene.)
The most logical reason for the alleged “detection” of a similar S RBD will simply be the result of contamination from one of the experiments being done in the labs. The Lung09 data imply a significant mix-up of sample as it’s mostly human.
The coverage on the Spike in both the PLOS and the Nature article were very poor—Less than 144 reads out of a total of more than one hundred million reads from all 4 samples. Statistically insignificant.
judging by the high level of contamination in the samples, it may have came from a researcher that have previously handled virus cultures before, or from contaminated instrumentation that were previously used for the analysis of the Gain-Of-Function project on the RBD. RNA sequencing is a very sensitive technique, and The Spike gene reads was simply too sparse to have been significant from contamination in any way.
In addition, the Nature sample was sequenced well after the First outbreak of SARS-COV-2. Their sample, which is 44.9% human, is best explained by deliberate mixing of Human cell-derived virus culture material Into the sample (note: the Nature article claimed to use VERO E6, but they are not human cells and should not leave behind a definitive Human (homo Sapiens) DNA signature which accounts for 26% of total human+ pangolin reads.)
This may actually be the proof of GOF being conducted there— either playing with an RBD extracted from these samples during their Pathogens gathering and testing project (the RBD nucleotides have a very high TMRCA of >19.8 years Ruling out a recent recombination, High RBD infectivity to hACE2 rule out early recombination (should have triggered an outbreak) but is consistent with synthetic constructs where large Amount of dS Being introduced either via the Backtranslation process from the AA sequence, or for optimizing the construct during the cDNA design and s

John F Signus
5/25/2020 10:42:33 am


This may actually be the proof of GOF being conducted there— either playing with an RBD extracted from these samples during their Pathogens gathering and testing project (the RBD nucleotides have a very high TMRCA of >19.8 years Ruling out a recent recombination, High RBD infectivity to hACE2 rule out early recombination (should have triggered an outbreak) but is consistent with synthetic constructs where large Amount of dS Being introduced either via the Backtranslation process from the AA sequence, or for optimizing the construct during the cDNA design and synthesis process.), Splicing in a PRRA which neither the bat nor the pangolin had, Which ended up with a leak of the engineered virus;
Or it was the result of a virus culture bearing the RBD being inadvertently mixed into the samples from a GOF experiment conducted in the same lab (all the sequencing were conducted in Chinese military labs, from the Nature article.), resulting in a misread of the RBD. Which is supported by the fact that all samples positive of Coronavirus reads showed an unacceptably high level of contamination by human-derived material.

John F Signus
5/25/2020 04:13:59 pm

The tens of million refers to all sequencing reads.

Greg
5/25/2020 10:22:38 pm

John F Signus, that’s quite an explosive claim. You are saying that the pangolin lung tissue has up to 40 percent human DNA? It would be easy to test whether this came from a cell culture or whether it was a contaminant, if it was a contaminant it should’ve been from human rna from skin epithelial cells, ie basal cells in the skin or something like that. If it’s a cell culture, there should be a particular rna that’s more likely to be expressed in human cell lines

John F. Sugnus
5/26/2020 08:52:08 am

Exact RNA type may not be known or important--there are hundreds of different cell culture lines, many of hich were used to culture viruses within. also, we do not know how the samples were contaminated, other than the fact that it can only happen after the sample have been taken and that it formed significant mass fractions in all samples with reads positive for the "pangolin coronavirus". with samples that tested negative showing no such contamination.
Three routes are possible--two of which may not leave a specific RNA signature other than that it was human RNA.
First route: deliberate "spiking" of the samples with GOF research material in order to "prove" the existence of a "natural" origin of the RBD. This is likely how the Nature sample gets contaminated during sequencing.
Second route: by inadvertant mixing of GOF research material from a researcher, or from within the lab. If this is the case, the material will be mixed with Human Epithelial cells from the hands of the researchers who have previously handled GOF material. the RNA signature may be washed out, or even become useless.
Third route: Contamination of the sequencing instruments by GOF research material sequenced prior to the sequencing of the sample. If this is the case, contamination will be from multiple cell lines and the RNA signature will too be messed up.
However, we DO know that all such samples with positive mapped reads were contaminated, and the negative samples were not. and we also know that lung tissue does not make contact with human skin or tissue materials until they were sampled, as the lungs, especially the inside of the lungs, were located deep inside the chest cavity and the mechanisms in a living animal excludes solid and RND-bearing contaminants very efficiently.
Graphical summary and proof pulled from the GenBank SRA database:
https://drive.google.com/file/d/1020E8X4iQF5-XF98Tnd0GS5epaqQ4yDW/view?usp=sharing

John F. Signus
5/26/2020 08:57:11 am

Lung09 is nearly 65% hominid DNA in total-- and judging by mass of the sample (Human DNA/pangolin DNA), the actual mass fraction directly attributed to humans is more than 69% the total weight of the sample--and they do say that some of the S reads were mapped there......Very likely thatthe RBD was one of the previous GOF experimental cultures being mixed up with the actual sample.

Nerd has power
5/26/2020 09:14:13 pm

That is fantastic, John. Thank you for sharing it with us. Have you seen this preprint article?

https://www.biorxiv.org/content/10.1101/2020.05.07.077016v1

He is saying pretty much the same things. Did you analyzed pangolin viruses from all three reports (ref 1-3 in that preprint) or a subset of them? Either way, this is SOLID proof!

John F. Signus
5/27/2020 05:46:54 am

On the Dataset SRR10168374, or Lung12. This dataset was claimed by the third article to contain Coronavirus-related reads. however Genomic analysis on the NCBI database failed to identify any.
It contained 26% reads from "Catarrhini"--the order which apes and humans belong to. It imply a large mass-fraction of Primate-derived material in the sample. However, this does not solidly home to the Human genome.
https://en.wikipedia.org/wiki/Catarrhini
In order to plot the fraction of Human DNA within that contamination, we chose to analyze the concentration of MtDNA from the two species of interest: The Human MtDNA reference genome NC_012920.1 and The pangolin MtDNA reference genome NC_026781.1. This represent only a very small fraction of the transcriptome of both species, but the MtDNA concentration serves a good proxy of how much of each and every part of a sample was composed of when the NCBI analysis tool did not give a detailed enough classification.
BLAST analysis on the Mitochondrial DNA revealed 229 150bp reads from the Human mitochondrial genome and 10217 150bp reads from the pangolin Mitochondrial genome. which gives a total fraction of human-derived sample mass at a minimum of 2.19% out of all Human+pangolin derived material (bulk mass of the sample). This MtDNA fraction is well supported by the 26%"Catarrhini" reads indicating at least 20% of the read "Catarrhini" reads were from human. (the Human MtDNA tends to form a smaller subset of the total cell transcriptome when compared to other animals for these samples. Likely due to cell culture conditions reducing the total mitochondria content of cultured cells when compared against animal cells In Vivo. The other samples with Definitive Homo Sapiens readings appears to underrepresent Human MtDNA. (though readings of the Human MtDNA were found at a significant number in all the other contaminated samples.) the other 4 SRR databases with BLAST results for Coronaviruses, as claimed by that article, were the Lung07(SRR10168378), Lung08(SRR10168377), Lung09(SRR10168376) and Lung11(SRR10168375). which were all contaminated by Human DNA and explained in detail by my last comment.
There is another dataset, deposited at 15-Feb-2020, was an abridged dataset containing only virus reads: "SRX7732094: RNA-Seq of GD Pangolin coronavirus: Pangolin 2". However, examining the only other Complete SRR dataset under the same name in NCBI SRA, revealed no traces of Nidovirales nor Coronaviruses in general.
Metadata of that dataset says: "Design: This dataset contains coronavirus-like sequence reads, based on BLAST search." Indicating it's likely a pooled-up dataset of the other "coronavirus-related sequences" pulled from "published databases and some in-house metagenomic datasets" mentioned by the Nature article.

The other SRR experiments, experiments with a more complete Sequencing dataset, under the same BioProject, are all datasets for the GX-Pangolin-CoVs which lacked the RBD.
Tracing back to the Biosample revealed no source tissue/organ recorded, unlike the GX samples which specified lung, blood, intestines or cell culture material(the P2V dataset).
Interestingly, this dataset contained definitive Reads from SARS-CoV-2 sequences that are distinct from the "Pangolin Coronavirus" they talked about earlier. (they may be Ultra-conserved sequences, but is not certain.) and the sequencing date was After the Outbreak have begun. This definitive reads fraction of Sarbecoviruses and "WuHan Seafood Market Pneumonia Virus", which were both at 2~3Kb in total, indicate that this dataset is likely the "Blast search results" dataset mentioned by the Nature article, which likely included the "pangolin 9" dataset that had the same level of SARS-CoV-2 and "Bat SARS-like Coronavirus" contamination in their analysis result.
Further examination of the reads across all 5 datasets there revealed identical "Pangolin Coronavirus" sequences with the earlier data reads, with only 2 SNP positions being different for 5 records at highly redundant positions in Lung08, likely due to sequencing error in the Lung08 dataset. This indicate that all the different raw sequence datasets contained only one strain/sequence of GD-Pangolin-CoV, with the only SNPs across all the datasets being 3 locations across 7 reads. easily attributed to RNA sequencing/amplification error.
The databases (the other database was of the Nature sample, one likely duplicated into the abridged one at SRX7732094 as an "in-house metagenomic dataset") with definitive reads mapped to SARS-CoV-2/WuHan Seafood Marked Pneumonia Virus and Bat SARS-Like coronaviruses were all sequenced well after the SARS-CoV-2 outbreak--most likely source was contamination by COVID-19 test material from the same lab as the methods section in the Nature article with the unabridged dataset claimed to have "serological test" capability. and the only unabridged sample with the same two phylogenetic analysis hits, the Nature article "pangolin 9", was 1

Nerd has power
5/27/2020 06:17:52 am

Thank you so much, John. I have to read it a few more times to better understand it as I'm no expert in such research. But from what I can understand so far, it's already rock solid. Amazing work!

Nerd has power
5/27/2020 06:22:17 am

Judging from the quality of the figure, I can see a publication coming out of this, which would be amazing. Thanks again for revealing this truth! I look forward to the published article!

John F. Signus
5/27/2020 06:34:18 am

17% human by sequence and 26.1% human by total sample mass--Indicating a heavy contamination by SARS-CoV-2 containing COVID-19 test material. The strange dataset was heavily redacted and contained no other non-coronavirus-related reads making analysis of contamination difficult. However, the sequencing date was after the Outbreak and are very close to the Nature article sequencing date, meaning that it was likely a workbench version of the "pooled reads" from the method section of the early Nature article, which have sequenced a contaminated sample that returned the same two signals with a total mass fraction being 26.1% human, so far remaining the only unabridged dataset that had these two hits.
Deep analysis of sequence sizes revealed that the contaminating sequences we seen in this dataset match in numbers with the Nature sample--indicative of an abridged preprint version of the "pooled dataset" in the Nature article.
It also appeared to be far more incomplete and evenly fragmented when compared to the other non-abridged datasets. Coverage analysis also revealed a lack of coverage of the RBM in this paticular dataset.
As the methods section explictly says that they searched public databases and "some in-house metagenomic datasets" for matches toward SARS-CoV-2, it appeared that SARS-CoV-2-related reads of the other contaminated sample was included here as well.
In addition, both datasets with identified SARS-CoV-2-related contamination were sequenced well after the outbreak and happens in a time where the labs have already been processing COVID-19 patient genomes--therefore the existence of such sequence in the abridged SRX7732094 dataset have the same origin as the "pangolin 9" genome sequenced at a very close date with the Nature article on the exact same set of samples--Contamination from COVID-19 testing ongoing in the lab.
Therefore, the detection of SARS-CoV-2 related sequences ("related", with only 11 identical reads that may also be from highly conserved areas, typical of an in-lab contamination since this is after COVID-19) in this Post-COVID-19 Dataset is an solid indication that even that dataset was contaminated--and they used the same contaminated sample-- Not just by a in-house Gain-Of-Function experiment, but also by SARS-CoV-2 itself currently being tested in the lab (from clinical samples being tested or sequenced before using the same instrumentation. This is during the time where all major Chinese labs have already begin testing, sequencing and processing COVID-19 clinical samples. also a proof that lab contamination of samples are very commonplace).

Rubber Ducky
5/23/2020 04:14:21 am

Synonymous mutations and the molecular evolution of SARS-Cov-2 origins https://doi.org/10.1101/2020.04.20.052019

"We perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human SARS-CoV-2 and related strains. We show that the synonymous divergence between the bat derived viruses and SARS-CoV-2 is larger than between GD410721 and SARS-CoV-2 in the RBD, providing strong additional support for the recombination hypothesis. However, the synonymous divergence between pangolin strain and SARS-CoV-2 is also relatively high, which is not consistent with a recent recombination between them, instead it suggests a recombination into RaTG13."

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Johnny M
5/23/2020 05:33:02 pm

1. CoVID-19 symptoms relates to Nipah Virus - it has massive cell-to-cell fusion and primarily relates to endothelial cells.
https://pubmed.ncbi.nlm.nih.gov/21054904/

2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807285/ 181-182

3. https://pubmed.ncbi.nlm.nih.gov/9484801/

4. https://www.sciencedirect.com/science/article/pii/S2214750020302924

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yano
5/25/2020 02:27:48 pm

https://arxiv.org/abs/2005.06199

The devastating impact of the COVID19 pandemic caused by SARS coronavirus 2 (SARSCoV2) has raised important questions on the origins of this virus, the mechanisms of any zoonotic transfer from exotic animals to humans, whether companion animals or those used for commercial purposes can act as reservoirs for infection, and the reasons for the large variations in susceptibilities across animal species. Traditional lab-based methods will ultimately answer many of these questions but take considerable time. In silico modeling methods provide the opportunity to rapidly generate information on newly emerged pathogens to aid countermeasure development and also to predict potential future behaviors. We used a structural homology modeling approach to characterize the SARSCoV2 spike protein and predict its binding strength to the human ACE2 receptor. We then explored the possible transmission path by which SARSCoV2 might have crossed to humans by constructing models of ACE2 receptors of relevant species, and calculating the binding energy of SARSCoV2 spike protein to each. Notably, SARSCoV2 spike protein had the highest overall binding energy for human ACE2, greater than all the other tested species including bat, the postulated source of the virus. This indicates that SARSCoV2 is a highly adapted human pathogen. Of the species studied, the next highest binding affinity after human was pangolin, which is most likely explained by a process of convergent evolution. Binding of SARSCoV2 for dog and cat ACE2 was similar to affinity for bat ACE2, all being lower than for human ACE2, and is consistent with only occasional observations of infections of these domestic animals.


Overall, the data indicates that SARSCoV2 is uniquely adapted to infect humans, raising questions as to whether it arose in nature by a rare chance event or whether its origins lie elsewhere.

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CCP is NAZI
5/25/2020 04:52:02 pm

Australian scholars reveal that CCP virus is carefully lab engineered and optimized to infect human:

https://twitter.com/i/status/1264876787881791488

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Alesh Aras
5/25/2020 05:22:34 pm

How will the below change your conclusions?

1. Can you do a similar cumulative syn, nonsyn, and indels analysis between pangolin and ZC45 (MG772933) and ZXC21 (MG772934) coroviruses?
2. How will the recent suggestion (https://www.futuremedicine.com/doi/full/10.2217/fvl-2020-0066) that most of the mutations between RaTG13 and SARS CoV2 are due to base editing by APOBEC proteins, not the more typical copying errors, affect your mutation rate analysis?

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Mr. Tuesday
5/25/2020 10:23:37 pm

https://www.biorxiv.org/content/10.1101/2020.02.15.950568v1.full

SARS-CoV-2 was first identified in human, but its codon usage was very different from other four types of Betacoronavirus that infecting human (Supplementary Figure S5A). In fact, the codon usage at both the amino acid level and synonymous level denoted that the orf1ab gene in SARS-CoV-2 had closest relationship to bat-origin SARSr-CoV, especially RaTG13. The CoVZX45 and CoVZXC21 had similar amino acid usage but relatively different synonymous codon usage to SARS-CoV-2 (Figure 3). This result in orf1ab was in accordance with the full-genome phylogenetic analysis (Supplementary Figure S7), showing a close relationship between SARS-CoV-2 and RaTG13 by the overall backbone of the genome.

The S protein are responsible for receptor binding which is important for viral entry. The genetic variability is extreme in spike gene26, and this highly mutable gene may possess valuable information in recent evolution history. In our results, the synonymous codon usage of SARS-CoV-2 in spike gene was distinct from those of RaTG13 and other phylogenetic relatives (Figure 3A), which was not observed in orf1ab or nucleocapsid gene. Although the codon usage in spike of SARS-CoV-2 and RaTG13 were similar at amino acid level, the difference at synonymous codon usage level indicated that they are unlikely to share a recent common ancestor. It is more likely that SARS-CoV-2 and RaTG13 might have undergone different evolution pathways for a certain period of time. The amino acid usage of SARS-CoV-2 in membrane was clustered with bat SARSr-CoV, however the synonymous codon usage of SARS-CoV-2 was still distinct to these bat coronaviruses. Our result supported different evolutionary background or currently unknown host adaption history in SARS-CoV-2. The codon usage of SARS-CoV-2 in nucleocapsid gene was similar to bat SARSr-CoV both at amino acid level and synonymous level, suggesting that there was no highly significant mutation happened in this gene.

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John F Signus
5/26/2020 08:38:29 am

Did you just say "unique synomynous codon usage" on the S protein? it would be exactly what is expected for a synthetically constructed protein being back-translated to a nucleotide sequence, with a bunch of synomynous mutations thrown in to make it more "distinct" from known nucleotide libraries. they failed to account for the codon usage, though.

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Nerd has power
5/26/2020 09:27:43 pm

I know it's hard for any author in a submitted manuscript to call it out, but really our claim here works perfectly in interpreting their finding that "SARS-CoV-2 and RaTG13 might have undergone different evolution pathways". It is this way because at least one of them is not natural. I will have to study their paper more, but can't help comment on it first.

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Mother Jones
5/26/2020 10:40:00 am

https://link.springer.com/article/10.1186/s12985-017-0766-9

In 2017 Xing-Yi Ge and Zheng-Li Shi report a RRAR furin polybasic cleavage site in a rat coronavirus. In Feb 2020 Xing-Yi Ge would report the RRAR in SARS-CoV-2. Also in Feb 2020, Xing-Yi Ge and Zheng-Li Shi would report on RaTG13. What a coincidence.

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Nerd has power
5/26/2020 09:47:03 pm

Thanks for sharing. Yuri first found out about this and posted it on Twitter. It's an alpha coronavirus that is far away from SARS-CoV-2 or beta coronavirus in general in terms of sequence identity. Therefore, RRAR in SARS-CoV-2 could not be acquired through recombination.between a precursor beta coronavirus and this alpha Coronavirus.

On the other hand, Zhengli Shi apparently knew, at least since 2017, that a RRAR sequence is a functional Furin-cleavage site. Talking about the knowledge and tools Shi has gathered in understanding coronavirus biology (and for the creative work too). She could have used them to help the world......

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Henri
5/27/2020 02:58:26 am

I have asked you this before and got no reply. I'll ask again. Why don't you write up your hypothesis that RaTG13 is fake, based on your analyses presented in this blog, and submit a letter to the Editor of Nature Medicine? The Editor should pass the letter to Zhengli Shi and allow a reply. Then, the two letters might be published together in a future issue of nature Medicine.

Nerd has power
5/27/2020 05:38:19 am

Thanks, Henri. I appreciate your suggestion. Challenging Shi and RaTG13 in a reputable journal would be a powerful approach. However, these journals may not be as accommodating for "conspiracy theorists" as people wish. If you looked back on some earlier comments, you will see how Nature has refused to publish an article written by a reputable scientist, arguing for a lab origin of SARS-CoV-2. My attempt would not turn out any better. On top of that, I prefer to not reveal my identity as many do here. Things might change, who knows. If you can't wait, please check out the link below. Some people have already published preprint manuscripts challenging the authenticity of RaTG13:

google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwiF5bC6mc3pAhWmneAKHXLTCi4QFjACegQIBBAB&url=https%3A%2F%2Fwww.preprints.org%2Fmanuscript%2F202005.0322%2Fdownload%2Ffinal_file&usg=AOvVaw1jpIVRdCYIwaBLBRuLcrrE

Henri
5/27/2020 07:42:31 pm


The link you gave me does not work. Please give me the title and author (s) of the pre-print so I can Google it myself.

The only other pre-print I can find alluding to this 'RaTG13 is fake' idea is something by Dean Brengston who claims that all publications using RaTG13 should be withdrawn on the basis that the origin of the virus is in question. He notes a partial sequence published in 2016 is identical to a sequence in RaTG13. For some reason, he seems to think that this proves that the RaTG13 sequence is fake. I do not understand his reasoning. Perhaps you can enlighten me, in simple terms.

I note that you are unwilling to submit a letter to Nature Medicine since you wish to remain anonymous and you doubt that they will publish something so strongly attacking someone of such a high research profile. I therefore have another proposal. I am not afraid of identifying myself and I wish to get to the bottom of this. Thus, I will write the letter to Nature Medicine, drawing attention to your blog and summarising the most important points. I will NOT attempt to take credit for your work. Bioinformatics is not my field so I politely request that you read my letter first and check that I have got the information and ideas correct.

Although I am now retired, coincidentally I am affiliated with a research laboratory in Wuhan. Thus, there can be no allegations that my letter is 'political aimed at stirring up trouble'.

This would not be the first time that potential fraud has been brought up in the science world. See, for example, the claim that HIV was present in a 1959 blood sample and the whole Wakefield situation. I still have faith in Zhengli Shi and her research group. I do not believe that the whole group could be knowingly involved in such a fraud, if, indeed it is fraud. However, I remain open minded.

Give me 1 or 2 weeks to compose the letter. I will get back to you.

Nerd has power
5/29/2020 04:14:09 am

Thank you very much, Henri. That's a wonderful proposal. It would be great if you could send a letter to Nature Medicine, drawing their attention to my blog. I would be more than happy to edit your letter. When you post the letter, please use a fresh comment as it gets hard to roll back and check old comments.

I have always considered Zhengli Shi a victim as well, a victim of the system. She is no doubt a great scientist and could totally be on the other side of the aisle in this situation. Well, I probably should not say too much about it just yet. We should let the truth tell the story.

Accessing the link above would lead to a direct download of the PDF file. So, check your download folder. You might have it there already.

Uncle Weatherby
5/26/2020 10:01:07 pm

China Rules Out Animal Market and Lab as Coronavirus Origin

https://www.wsj.com/articles/china-rules-out-animal-market-and-lab-as-coronavirus-origin-11590517508  

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CCP is Nazi
5/26/2020 10:11:07 pm

Who gives it a damn shit on what China has to say? What did they say that is not a lie? Isn't WSJ bought by CCP as CCP's mouthpiece? Which NAZI criminal would admit their NAZI conduct?

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Alesh Aras
5/26/2020 11:01:13 pm

I would rule out wet market as well: China in a hurry to reopen wet markets is prima facie evidence that the CCP doesn’t think that the virus came from there.

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CCP is NAZI
5/26/2020 11:12:54 pm

But CCP rules out their P4 lab as the CCP virus origin as well. It's like saying this shit is not from their ass but from someone else'. Once I see the subject in the URL, I would not even waste a single second to read on.

Alesh Aras
5/26/2020 10:59:20 pm

Bat woman Shi Zhengli has resurfaced.

https://youtu.be/8opSfaCD5iE

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Nerd has power
5/27/2020 05:55:49 am

The head of the Wuhan Institute of Virology also gave an interview:

https://www.youtube.com/watch?v=_P0kH5wVj4s

Don't worry about her speaking Chinese. The CCP's propaganda machine CGTN has been so sweet that they made English subtitles for readers like you! There is no real meat in almost all her answers, except this:

Zhengli Shi does not have the real RaTG13 virus. She only has its sequence info.

It happens that some commenters here recently challenged this same notion that I made in the article. I can't believe the head of WIV would come out and help me clarify. My "reliable sources" turned out well in the end. I'm so happy for them.

One thing I have to warn you about is that the way they interacted in the interview may seem unfamiliar to you ---- the questions are so soft and comforting. Indeed, the two of them are just acting together, not interacting :).

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Tony Soprano
5/27/2020 09:12:04 pm

The Wuhan Seafood Market theory of the origin of SARS-CoV-2 is out and the "multiple origins" theory is in.

https://weather.com/en-IN/india/news/news/2020-05-27-coronavirus-originate-wuhan-multiple-origins-claims-china-covid-19

It is based on this scientific article.

https://www.nature.com/articles/s41586-020-2355-0

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CCP is NAZI
5/27/2020 10:15:30 pm

Who gives it a damn shit on what China has to say? Ya, Nature with a bunch of Chinese "scientists" who only speak what their NAZI CCP want them to say. Otherwise, the lab in Shanghai would not have been ordered to shut down shortly after the lab shared the virus sequence. But are you sure Nature is still science centric and not bought by CCP if CCP have already bought bigger organization like WHO, WTO and many other world organizations? I am so glad that President Trump stopped funding WHO. Let's see how many lies WHO continue to tell the world without the world's biggest donar.

Multiple origins? Lol, sure, thanks to CCP's NAZI conduct, their lab made virus has been spread to multiple so called "origins" simultaneously:
https://www.news18.com/news/sports/new-revelations-from-world-military-games-participants-hint-at-covid-19-spread-in-china-in-october-2625391.html

No matter what, if NAZI CCP really hadn't done anything wrong, why is it so hard for CCP to open their P4 lab to let world scientists investigate? The more they are trying to cover up, the more evident their NAZI crime is.

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Leo
5/27/2020 11:11:26 pm

The journal Nature is corrupted, they have so many connections with EcoHealth Alliance.

In this article

https://www.nature.com/articles/s41586-020-2355-0_reference.pdf

How is it possible to accept such a phrase: our time-resolved phylogeny analysis suggests that the earliest zoonotic spillover event might occur at late November 2019, which is in agreement with the analysis by Andersen et al.

No, it was already going around during the Wuhan Military Games.

And what about these cases?

https://flutrackers.com/forum/forum/united-states/pneumonia-and-influenza-like-illnesses-ili-ae/virginia-aa/815013-va-three-dead-others-hospitalized-in-unidentified-virus-outbreak-at-fairfax-retirement-community-rhinovirus

Rhinovirus?!

Try to do a little research on the internet. From Europe there is no access to most of the sites.

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CCP is NAZI
5/27/2020 11:26:17 pm

Have you actually compared its sequence with CCP Virus' sequence side by side? If so, why don't you bring its sequence here to let virus specialists verify? Even look at it from a non scientific perspective view, with such a high infectious rate that CCP virus has, why nothing happened in U.S. afterwards until after Wuhan lock down? Interestingly, CCP tried their very best to help this deadly CCP virus spread as fast and wide as possible all over the world by silencing 8 doctors like Li Wenliang , by destroying all virus samples, by stressing it's preventable and controllable, by denying the fact about human to human transmission, by letting people out from Wuhan to everywhere in the world but not within China? Were CCP able to prevent and control it? Exactly, what's CCP's hidden agenda? Well, aren't these https://www.newsmax.com/navrozov/china-biological-russia/2009/09/17/id/335042/ and https://www.theepochtimes.com/did-chinas-plan-to-destroy-the-united-states-backfire_3223117.html enough to tell CCP's world domination goal?

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Leo
5/28/2020 12:35:24 am

We need a forensic analysis on these early cases in USA.
Their CDC has the samples.
Why USA has now so many cases? Could it be a sum of early cases starting in summer 2019 + the cases fired back from Wuhan? Do you know about vaping illness in 2019 in USA? Are you aware of the tight collaboration between USA and China on coronavirus research?

Smiley Evans, T., Shi, Z., Boots, M. et al. Synergistic China–US Ecological Research is Essential for Global Emerging Infectious Disease Preparedness. EcoHealth 17, 160–173 (2020).

https://doi.org/10.1007/s10393-020-01471-2

Both China and USA are probably trying to cover up the whole story to avoid to pay for all the rest of the world.

CCP is NAZI
5/28/2020 06:38:24 am

I know everything including how NAZI CCP have been faking their total sum of infected cases, how Obama administration used their tax payers' money to fund HIN who then outsourced to Wuhan P4 lab, how CCP have been threatening each country and scientist who is revealing the truth and facts, how CCP have been developing biowar non stop with still tons of under developed virus in their P4 lab which are far more lethal than what was already released, the so called CCP virus, how CCP have been destroying the virus samples, how CCP have been shirking their crime, how https://nextstrain.org/ncov/global shows all CCP virus mutation could be traced back to China. Oh, too much to list.

Is's absolutely irresponsible, illogical and non scientific to compare the number from a country that has the most extensive and accurate testing with a country with totalitarian regime that has been severely under reporting their actual infected cases using testing kits with at most 30% accuracy.

No matter what, if NAZI CCP really hadn't done anything wrong, why is it so hard for them to open their P4 lab to let world scientists investigate? Don't CCP want to find the source of the virus as well?Merely shirking around never solves the problem until CCP opens their P4 lab.

Bubba Gump
5/28/2020 12:31:29 am

This article "Viral and host factors related to the clinical outcome of COVID-19" https://www.nature.com/articles/s41586-020-2355-0, published on May 20th, which is now being used by China and the media as the basis for the "multiple origins" theory for SARS-CoV-2, appears to suffer from many of the same problems as "On the origin and continuing evolution of SARS-CoV-2" https://academic.oup.com/nsr/advance-article/doi/10.1093/nsr/nwaa036/5775463, published in March 2020, as explained in a criticism of it http://virological.org/t/response-to-on-the-origin-and-continuing-evolution-of-sars-cov-2/418

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CCP is NAZI
5/28/2020 06:45:19 am

At this moment, the most important is not who has published what considering how many world organizations and journals CCP have been buying, threatening and silencing(Isn't WHO the best example to prove my point?) but when CCP can open their Wuhan P4 lab.

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Leo
5/28/2020 07:02:16 am

I am not sure if USA has the most accurate testing for covid19 cases:

https://www.nytimes.com/2020/04/18/health/cdc-coronavirus-lab-contamination-testing.html

https://edition.cnn.com/2020/04/06/health/us-coronavirus-death-count-cdc-explainer/index.html


I agree completely with you on which crimes CCP did. What USA did, comes as extra. USA and CCP did this together. Both pushed first on the natural origin of the virus and in the meantime some of them accused each other of lab release. Is it that not weird?

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Leo
5/28/2020 07:04:52 am

This article is quite good for an overview of the facts

https://asiatimes.com/2020/05/us-china-blame-game-a-lose-lose-proposition/

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Leo
5/28/2020 07:20:32 am

This is also interesting:

https://www.globaltimes.cn/content/1185291.shtml

Ancestral type of COVID-19 virus mainly found in the US: study

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CCP is NAZI
5/28/2020 07:32:53 am

I'm sorry, but I don't waste even a single second on CCP propaganda such as global times, cnn, nytimes. Trump should craft executive order on fake news just like how he crafts one for social media. I cannot wait to see how 50 cent army will suffer from this: https://www.foxnews.com/politics/trump-crafts-executive-order-on-social-media-weighs-commission-to-probe-bias-amid-twitter-fight I will be eager to test out if anti CCP words still keep disappearing on YouTube and will definitely sue them once the order comes to legal effect.

I'm also eager to see how long CCP's money can last to pay those media to speak for them now that the world is pulling their manufacture chain out of China The best time to see if those media still speak for them is when CCP is running out of money. Let's wait for a year to check those media again?

Again, no matter what, if NAZI CCP really hadn't done anything wrong, why is it so hard for them to open their P4 lab to let world scientists investigate? Don't CCP want to find the source of the virus as well?Merely shirking around never solves the problem until CCP opens their P4 lab.

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Leo
5/28/2020 07:47:12 am

It is probably hard for CCP to open their lab as it is for Fort Dietrich.

They might have worked in parallel on the same stuff

https://pubmed.ncbi.nlm.nih.gov/30849247/

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Bubba Gump
5/28/2020 07:53:02 am

Can we stick to scientific analysis here? Political diatribes contribute nothing to what we are trying to achieve.

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Leo
5/28/2020 08:02:52 am

You are right, but it is difficult to avoid to make the discussion political. If this virus is lab made, it is normal to try to find out from which lab it might come. We probably need to wait for more analysis like the one done by Forster with more data.

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charles
5/28/2020 10:31:50 am

Stop sidetrack the discussion. You are just trying to throw in multi-origin conspiracy theories to deflect the spot light on CCP - a typical CCP propaganda and a trick by the Global Times shills. The quickest and easiest way is always transparency from CCP. They are stonewalling from day 1 and their further actions to destroy evidences speak loudly of their complicity in their role in the pandemic to say the least.

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Nancy
5/28/2020 11:02:40 am

You're absolutely right, charles! 50 cent army is everywhere. Luckily, this site isn't paid or operated by CCP. Otherwise, the truth and facts discussed here would be censored or deleted just like YouTube /Twitter/Facebook. There isn't anything easier than being transparent or opening CCP's P4 lab to prove if CCP are NAZI criminals or not. Unfortunately, Xi Jinping suggested to open their lab to the world in a year, after all evidence are destroyed or everyone forgets about it. Come on, no one would ever forget this NAZI crime! If we let it go this time, we don't even know how we die tomorrow!

Bubba Gump
5/28/2020 11:26:00 am

I recommend that the administrator delete the childish political comments before they ruin the site. The children can go waste people's time ranting on Twitter or elsewhere and let the adults have a serious discussion about the science here.

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Nancy
5/28/2020 01:21:27 pm

Which adults fist started the political games here? Which adults only talked about who says what, but nothing substantial or scientific from
their own brains.

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CCP is NAZI
5/28/2020 12:20:17 pm

I also request the administrator to remove CCP's "multiple origins" conspiracy theories along with CCP propaganda to clean up the trash and lies. Just bring your own analysis, virus sequence and facts here.
We have plenty of conspiracy theories and CCP propaganda elsewhere. No need to bring those trash here to distract everyone.

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Bubba Gump
5/28/2020 08:08:35 pm

Here's the problem in a nutshell - when you have people like "CCP is Nazi," who pollute a scientific discussion with useless political comments, it ruins the site. "CCP is Nazi" either did not read or did not understand the articles about "multiple origins" upon which my post was based. The last citation is a scientific criticism of the articles supporting "multiple origins." So, my post about "multiple origins" debunks rather than supports the theory of "multiple origins." Not surprisingly, "CCP is Nazi" got it all wrong, is the one who is the distraction, adding nothing to the scientific discussion and will end up ruining it for everyone. Sadly, that type of behavior is all too common on social media sites, people with big mouths and little brains.

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CCP is NAZI
5/28/2020 09:56:47 pm

This is not the first time I name like this. I don't know why you are the only one fussing about this. Anyone can decide what name to use just like how you name yourself as Bubba Gump instead of your
real name. I have brought an article into the discussion previously just like what you have done, yet it seems that you have a bigger brain even though I'm not seeing any scientific analysis from your own brain. But isn't this "Scientific evidence and logic behind the claim that the Wuhan coronavirus is man-made" talking about how Wuhan virus is lab made? What? Releasing lab made Wuhan virus is not a NAZI conduct? Really, how many scientists on earth do not work for political purpose? I wish all scientists only work for science not politics, so that we would not even have this bio war in the first place.

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Bubba Gump
5/28/2020 10:23:05 pm

The irony is people like "CCP is Nazi" and "Nancy" practice exactly what thw communists and Nazis did, that is, the political interpretation/use of science e.g. Soviet Lysenkoism and Nazi racial science. Offer scientific analysis here and reserve your political views to a more appropriate forum.

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CCP is NAZI
5/28/2020 10:42:01 pm

While Bubba Gump is asking others not to talk about politics, he himself is just doing the same. How funny! Those who do not agree with CCP is NAZI are undoubtedly NAZI themselves.

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Rose
5/28/2020 11:21:45 pm

I suggest to use rather our energy to definitely demonstrate that SARS-CoV-2 is lab made, in a way that it is finally accepted from the rest of the people who still think is natural, because I fear they are still the majority.

I was really surprised to see that genetic manipulation of the spike protein of coronaviruses started already before the first SARS:

https://pubmed.ncbi.nlm.nih.gov/10627550/

https://pubmed.ncbi.nlm.nih.gov/11162792/

And that Daszak was hunting bat viruses also before that:

https://science.sciencemag.org/content/287/5452/443.long

But I could not find a direct record of coronaviruses in bats before the first SARS. Is anybody aware of it? Maybe some records in Chinese?

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Bubba Gump
5/29/2020 03:07:47 am

"Rose," Peter Daszak started his career in parasitology and migrated into virology. Here is a review article he published in January 2000 with some of his citations. He was into Nipah virus before coronavirus, I believe.
https://science.sciencemag.org/content/287/5452/443.abstract

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Rose
5/29/2020 03:46:44 am

Ah yes thanks, it is the same article I posted. I am just wondering if he could have seen before emerging of SARS, that bats carried CoVs, since he was already studying their viruses before 2000.

Nipah virus is another big part of his research, even more scary than CoV:

https://www.nytimes.com/2018/06/04/health/nipah-virus-india-vaccine-epidemic.html

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732787/

https://books.google.de/books?id=pymSBkVU-FsC&pg=PA148&lpg=PA148&dq=genetic+manipulation+nipah+virus&source=bl&ots=H4l0r-ulwG&sig=ACfU3U27gz3PNwtt7ajfQh-wI__d5loEyQ&hl=de&sa=X&ved=2ahUKEwjbvbrCiNfpAhVIyoUKHd95A4MQ6AEwBnoECAoQAQ#v=onepage&q=genetic%20manipulation%20nipah%20virus&f=false

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Nerd has power
5/29/2020 04:50:06 am

I appreciate everyone's contributions to this comments section. It has certainly surpassed my exceptions. Many analysis and findings that people shared here are just brilliant. We are piercing deep into the thickness. It's great.

We are all here trying to dig out the truth and protect it from being smeared or hidden. However, not everyone's approach is the same. While I think all of these actions are honorable, I agree with some of the above comments that the best use of this space is to discuss the scientific aspects of this suspicious virus. In light of this spirit, I suggest that we don't have to involve too much politics in every thread. Avoiding politics completely is of course impossible here. But we can try to keep it at a minimum and focus more on the scientific evidence and analysis. In the end, hard facts will convict these criminals. Sometimes, opposing views on a scientific topic and a fair discussion are what's needed to get to the bottom of a particular point. So, as long as it is not a clear case of the CCP's 50-cents army being involved, we don't have to blame the CCP for every opposing view. That's my two cents.

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Henri
5/30/2020 04:42:22 am

I have composed a 'diplomatic' letter for Nature Medicine. The technical bit, which I need you to check over and correct if necessary is below.

I will require a copy of the synonymous non-synonymous Figure to submit with the letter. Can you give an email address so we can communicate more efficiently?

Among others, the main 'anomalies' raised by the website's author are as follows:


A) SARS-COV2 has overall 95% sequence similarity to bat corona viruses ZC45 and ZXC21. However, this similarity is not evenly spread over the genome. It ranges from 94 -100% for the nucelocapsid, S2, membrane and E proteins, but is only 69% for the S1 portion of the spike protein. This pattern extremely rarely occurs naturally but is common in laboratory chimeric viruses.

B) The E protein has a structural role and can withstand repeated mutation without loss of function. It is highly unusual that the E protein found in the earlier identified SARS-COV2 has an identical amino acid sequence to a distantly related virus, RaTG13. Moreover, later isolates of SARS-COV2 have already shown mutation in this region in just a few months.

C) The synonymous (s) to non-synonymous (ns) mutation ratio remains at the typical 5:1 ratio when ZC45 is compared to ZXC21 I (Fig. 1a). In contrast, comparison of SARS-COV2 with RaTG13 shows an unusual pattern, including a flat region which is highly unlikely to occur in nature.

D) In an interview with the Institute's head, which can be found online (7), it is admitted that the laboratory does not have an isolate of RaTG13 and is no longer in possession of the sample taken in 2013 from which the sequence was derived. Thus, significant conclusions about the origins of SARS-COV2 is relying upon a computer-generated sequence and not on the characteristics of an established virus.

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Henri
5/30/2020 07:26:24 pm

The letter to nature is ready to be submitted. Please check the technical bit given above and make any changes you feel necessary. If I don't hear from you soon, I shall submit on the assumption I have described your main objections correctly.

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Lilian
5/29/2020 06:09:28 am

I have found some interesting information on the ORF10 in SARS-CoV-2

https://www.biorxiv.org/content/10.1101/2020.02.17.952895v1.full.pdf

https://chemrxiv.org/articles/Molecular_Insights_into_the_Contagious_Nature_of_Pandemic_Novel_Coronavirus_2019-nCoV/12118839/3

If you blast the protein in NCBI, there are only few matches, mostly in Australia and USA, and with the weird pangolin sequence.


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Jule
5/29/2020 09:47:41 am

What I do not understand is why Shi did not simply synthesize RaTG13 to avoid any suspicion.
After all, from what I read here several times, this is supposedly very simple.
With all the criminal energy she is accused of, an icredible energy which she has used in a rather cumbersome way to cover up the true origin, the obvious thought would be to do just that.

Furthermore, I don't understand why Shi's blood must have boiled when he saw the RaTG13 sequence. If I look at the Bat CoV sequences listed here (upper half), my blood would boil exactly at the ones Shi actually published.
„Importantly, RaTG13 preserves the binding motifs as much as, if not better than, any other bat coronavirus in Shi’s list. „
I do not see it like that. There is actually only one amino acid to which this applies: Position 472, all other amino acids are worse compared to the published Bat-CoVs.

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Rose
5/29/2020 10:31:21 pm

I bet that RaTG13 is not functional as designed. Let’s see if someone try to synthetize it.

And the boiling blood of Shi is expected because in 2013 they were still searching desperately for the source of SARS1. In that cave where 4991 alias RaTG13 was found, this was the only SARS like CoV. Some miners got SARS-like pneumonia and three even died. But no, at WIV it was decided to leave RaTG13 in the freezer and to take it out only 7 years after, when SARS2 popped out.

To prove that SARS2 has a natural origin.

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Tim Pearson
5/29/2020 12:51:36 pm

When the SIV of sooty mangabees crossed over into humans and became HIV, there weren't any alternative theories that some local laboratory within West Africa had been responsible for that occurring.

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Rose
5/29/2020 10:34:47 pm

You can ask Fauci, he is the HIV expert and best friend of Daszak

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ioderESTEl
5/29/2020 03:00:16 pm

So SARS-CoV-2 has another dangerous characteristic tied to ORF8. I wonder if another animal virus sample showing this particular characteristic will suddenly be published.

Have any of you scrutinized this section?

"The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I"

https://www.biorxiv.org/content/10.1101/2020.05.24.111823v1.full

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Rose
5/29/2020 10:18:32 pm

You can have a look here:

https://twitter.com/Daoyu15/status/1265499547666657282

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Reminder
6/8/2020 07:16:05 am

If the link is down, here is a mirror to that file.
https://drive.google.com/file/d/18twqD8_FOwLBdUZ375O6T3Zkr9wnC1ny/view?usp=sharing

Pete Ross
5/30/2020 05:56:29 am

To add or subtract from the mystery of SARS-2 (CoV2) origins are this series of tweets from Dr. Peter Daszak of EcoHealth Alliance, recently deprived of NIH funding just last month.
NIH funding seems to be a small fraction of the Pentagon-associated Alliance, being all at once a private charity NGO , a global megacorporations' NGO and a defense contractor, with an annual estimated annual operating budget ranging from $16 to $99 million and biosafety laboratories in at least 30 countries.
EcoHealth Alliance is a spinff of the Rockefeller's WWF - World Wildlife Fund.


Peter Daszak
@PeterDaszak
·
Oct 17, 2019
What better way to show our
@EcoHealthNYC
Board members the work we do - visiting farmer Wei’s rat farm in Guilin China where
@nycbat
is giving us a sampling demo! Watch out for the big ‘chompers’
0:00
480 views
Peter Daszak
@PeterDaszak
·
Oct 17, 2019
We’ve been working with him for over 8 years - he’s the head of a wildlife farmers trade group in China
Peter Daszak
@PeterDaszak
·
Oct 17, 2019
Never thought I’d be live tweeting from a rat farm
Peter Daszak
@PeterDaszak
·
Oct 17, 2019
And by the way these animals are bamboo rats bred for food - they’re extremely cute which makes you a bit sad but at least it means they’re not taken from the wild
Peter Daszak
@PeterDaszak
We’re testing for viruses to see if the trade is a rack for public health I. Southern China
11:20 AM · Oct 17, 2019 from Guangxi, People's Republic of China·Twitter for iPhone
1
Retweet
2
Likes
Peter Daszak
@PeterDaszak
·
Oct 17, 2019
Replying to
@PeterDaszak
And here’s me with farmer Wei, his wife and my goddaughter Luo Yuan

https://twitter.com/PeterDaszak/status/1184746055050694661

for more background material:

https://www.npr.org/sections/goatsandsoda/2020/04/29/847948272/why-the-u-s-government-stopped-funding-a-research-project-on-bats-and-coronaviru

https://globalbiodefense.com/2015/12/09/who-got-biodefense-funding-from-dtra-in-2015/

https://www.gatesfoundation.org/Media-Center/Press-Releases/2000/03/Infectious-Disease-Research-Institute

The Pandemic Century: One Hundred Years of Panic, Hysteria, ...books.google.co.il › books
For Daszak and his colleagues in the EcoHealth Alliance, the buzz generated by Disease X presented an opportunity to leverage additional funding for research ...
Mark Honigsbaum - 2019 - ‎Medical


https://sites.google.com/a/housatonicits.com/home/research/dr-peter-daszak?authuser=0

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Alexandrian LC rocks
5/30/2020 09:11:06 am

This is an important lead. Remember Shi’s previous statement? They were gathering bits and pieces from naturally occurring viruses throughout the world, mix and match them in the lab not necessarily with seamless ligation, put them throughout cell and animal passage Until they generated the perfect disease X to be unleashed into humans. All in the name of “saving wildlife”.
Hence the choice to release it at the Huanan Seafood Market. It was the scapegoat to usher in their radical environmentslist agenda.

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pangolin
5/30/2020 10:44:17 am

I perfectly agree with disease X possibly being SARS-CoV-2 but the market has nothing to do with the initial spread. The real boosters were the military games in Wuhan in October 2019. There are posts here on it.

Alexanderian TD Rocks
5/30/2020 09:13:24 am

From an early source. Explain the EcoHealth agenda very clearly.
The Coronavirus IS a bioweapon, albeit an unfinished one. Zhengli Shi have conducted gain-of-function researches on coronaviruses in three different papers--2013, 2015 and 2018. All three papers involve splicing a human ACE2-targeting domain into the spike protein of otherwise harmless coronaviruses and "proving" that the resulting chimeric strain could infect humans, to pandemic potential. An early stage of Bioweapons development. The bioweapon is not yet mature, and that's whi it does not have the common, military-targeting aspect of hypothetical bioweapons. In fact, no actual strain of known developing, or deployed bioweapons as already known genuinely had this trait engineered into the agent. Engineering the alleged age-group specifity and reduced infectivity without affecting leathality is just impossible with current technology, so all known historical or current bioweapons are really just strains that are known to be highly leathal, highly infectious, or have been engineered to be so. Strains that have been labeled as "Select agents" by the CDC.
https://www.selectagents.gov

Bioweapons are WMDs. Weapons that are intended to wipe out an entire population. An eitire country or ethnicity perhaps. specifity and fine-tuned control is of little concern in their development and will not really be something that hold back a user, especially that of a terrorist organization, from finding potential in, developing or using agents such like Ebola, SARS, Anthrax, HIV/AIDS or chimeric SL-Coronaviruses as their mean to achieve their goals. The official cover-up on this subject only strengthened this possibility.

Real World biological agents are deployed as soon as a vaccine is available to at least the military of the country thadt developed it-- and even if the strain is yet to be deployable they are almost always listed as Select Agents of great bioweapons potential/bioterrorism threat by the relevant authorities, like the CDC, primarily due to the potential catastrophic consequences such an agent could cause in case of a leak.

This is an act of ecoterrorism of a rogue actor within one of the many acedemic, social and environmental groups. groups which are known to ardently stick to the depopulation agenda and hold firm beliefs that humanity is to be somehow culled, if not eliminated. Especially that of the acedemic researcher Zhengli Shi, a known worshiper of nature who had fallaciously attributed an animus to nature, something that a well-disciplined acedemic would never do, just a month ago at 1st feburary in China. Who have been studying SARS and SL-CoVs since 2003 and have published multiple papers on engineering more infectivity and leathality into them, even to the point of openly proving the effectiveness of the agent on her 2015 Nature article https://www.nature.com/articles/nm.3985

through animal tests on genetically engineered mice bearing Human ACE2 receptors and ferrets, which had ACE2 receptors almost identical to that of humans.

The virus fits nearly perfect to the environmentalist narrative of "eating wild animals are bad, cause sickness, et cetera" when there are absolutely NO evidence of either a confirmed case of Covid-19 contracting it from an animal (After it have been confirmed that the Wuhan Seafood market was NOT the source of the outbreak

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30183-5/fulltext

), nor there have been any known epidemics caused by eating wild animals even thouth tropical people have been hunting and eating wild animals for more than 50000 years, since the dawn of history. For example, the Indonesians have eaten fruit bats for more than 1500 years yet not even a single confirmed new human-transmissible virus in indonesians have been solidly confirmed to have came from a bat. If a specific group is to be blamed, it was the environmentalists.

Who had the perfect motive in an desperate attempt to end their percieved "uncivilized" habit of eating wild animals (all highly civilized western countries hunt all kinds of wild animals and eat their game, the only difference being a official excemption in the form of a liscence/permit which disprove this narrative).

The perfect technology for doing so, with the exact reverse genetic system of engineering deadly SARS-like coronaviruses from bat genetic materials being developed as early as back in 2012

journals.plos.org › plosone › article › journal.pone.0032857

by the same cluster of potential eco-terrorist acedemics that have produced such deadly viruses in at least three different occcasions.

They have a perfect delivery system, with virus samples developed in the U.S. and Cannada being stolen and sent to chinese labs of which bats were tested and experimented on all the time

https://nationalpost.com/news/canadian-lab-immersed-in-rcmp-probe-sent-ebola-and-another-deadly-virus-to-china-health-agency

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Babstar
5/30/2020 01:04:39 pm

Alina Chan just tweeted ?
2006 study inserts furin cleavage site into SARS-CoV spike at the exact residue where SARS-CoV-2's PRRA(R) site is - and demonstrates enhanced cell-to-cell fusion.
https://twitter.com/Ayjchan/status/1266805310313967617?s=20

Direct link to the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111780/

The noose tightens.

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Henri
6/1/2020 07:47:19 pm

Why does the noose tighten? No one denies such reearch has been performed.

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George Formby
5/30/2020 10:48:01 pm

This first appeared as a pre-print at the end of March, published on May 29th.

"Emergence of SARS-CoV-2 through recombination and strong purifying selection"

https://advances.sciencemag.org/content/early/2020/05/28/sciadv.abb9153

Comments?.

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Nerd has power
6/2/2020 06:15:44 am

Thank you for bringing up this paper. I think their evidence proves the opposite. They mainly argued that because all the suspicious elements of SARS-CoV-2 (RBM, furin-cleavage site) are hinted to exist in nature, then recombination must be responsible for the appearance of SARS-CoV-2. Of course, in my eyes, they used all the wrong sequences, RaTG13, pangolin coronaviruses, YN302, to do their analysis and thus support their "conclusion". But I think their data revealed the opposite. When they analyzed the syn/non-syn mutations, they showed extreme high purifying pressure for SARS-CoV-2, RaTG13, pangolin viruses in S gene and in other genes (figure 4 and 5). Unfortunately, they did not always include a similar analysis of another group. I think they probably did those analysis in figure 5 and I suspect that the results are too daunting and not aligned with their "conclusion" of natural recombination being the source. I will try to find the time to do that comparison for them.

The strong purifying pressure thing is self-contradicting. The virus is jumping in between species in group A of figure 4. If anything, they should see less purifying pressure than group B, which are viruses that have comfortably settled down in bats. Honestly, this claim itself should make people sick. I don't know how they managed to say it in the paper and how the reviewers let it pass. On the other hand, this saying is in line with their analysis. You can't argue against your own results, I guess. Also, if their conclusion is that the virus is not of natural origin. then their university (Jilin Univ) would not let the manuscript out because the Chinese government strictly screens manuscript looking into the origin of the virus.

Anyways, this paper has some interesting hints and needs to be figged further.

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John F. Signus.
6/8/2020 07:06:18 am

Also, Here is a complete, detailed analysis on why RmYN02 itself--Not only it have no furin site, but it was also Not viable!
https://drive.google.com/file/d/1CuWg0h2rJ1kTGJDP04u1LUNkGk7AGgA3/view?usp=sharing
Either a R!Polar residue TMPRSS2 site, a RxxR furin site, or a KxR tryptin site, is required for coronavirus S1-S2 cleavage and Spike function. However, In RmYN02 neither of these three sites were present at the S1/S2 junction--indicating that the Spike will not be claeved in a host, nomatter which stage of infection it was currently in. This kind of S1/S2 deactivation is only ever found in highly attenuated coronavirus varients only able to infect cell cultures--it is not viable in a real bat.

Javier
6/2/2020 12:59:54 pm

ZXC21 and ZC45 : strong purifying selection and same habitat (in caves collected by a military research lab of the CCP)
Wuhan-Hu-1 and RaTG13: strong purifying selection ¿and same habitat (WIV lab)?

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Henri
5/31/2020 06:09:21 pm

Open request to NerdHasPower:

For the third time, I ask NerdHasPower to check the technical portion of my Letter to Nature (reproduced below) and make any changes that may be needed. Otherwise I will assume I have presented you arguments correctly and submit the letter by mid-day TODAY.

TECHNICAL PORTION OF MY LETTER TO NATURE:

The author of the website wishes to remain anonymous. I am therefore acting as an intermediary.

Amongst others, the main 'anomalies' raised by the website's author are as follows:

A) SARS-COV2 has overall 95% sequence similarity to bat corona viruses ZC45 and ZXC21. However, this similarity is not evenly spread over the genome. It ranges from 94 -100% for the nucelocapsid, S2, membrane and E proteins, but is only 69% for the S1 portion of the spike protein. This pattern extremely rarely occurs naturally but is common in laboartory chimeric viruses.

B) The E protein has a structural role and can withstand repeated mutation without loss of function. It is highly unusual that the E protein found in the earlier identified SARS-COV2 has an identical amino acid sequence to a distantly related virus, RaTG13. Moreover, later isolates of SARS-COV2 have already shown mutation in this region in just a few months.

C) The synomonous (s) to non-synonomous (ns) mutation ratio remains at the typical 5:1 ratio when ZC45 is compared to ZXC21 I (Fig. 1a). In contrast, comparison of SARS-COV2 with RaTG13 shows an unusual pattern (Fig. 1b), including a flat region for the ns mutations leading to an unusually high s/ns ratio which is highly unlikely to occur in nature.

D) In an interview with the Insitute's head, which can be found online (7), it is admitted that the laboratory does not have an isolate of RaTG13 and is no longer in possession of the sample taken in 2013 from which the sequence was derived. Thus, significant conclusions about the origins of SARS-COV2 is relying upon a computer-generated sequence and not on the characteristics of an established virus.

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yano
5/31/2020 09:16:52 pm

E) At the beginning of the 2019 pandemic SARS-CoV-2 was better adapted to infect humans than any other animal. No selective mutations or adaption to humans was needed (or observed). This suggests the virus was already well adapted to human cells and did not come from animals

As a comparison, 2003 SARS-CoV-1 jumped from animals to humans, then mutated to better infect humans as the pandemic progressed in time. See Ref. above

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Henri
6/1/2020 03:41:43 am

Letter submitted. Let's see what happens.

Nerd has power
6/2/2020 05:08:10 am

Sorry for not responding earlier. Did not find the time to pay a visit here for two days. You were saying that you needed 1 or 2 weeks to draft the letter. So I didn't think you have it ready already. Anyways, the letter looks very good to me. Point C did not explicitly state that comparison was done for the Spike, but that does not hurt the argument or make anything incorrect. Thanks again for doing it. Please keep us posted with the results.

John F. Signus
6/8/2020 06:59:41 am

Also YNLF31C/SARS.

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MagmaHombre999
6/2/2020 12:40:07 am

Have you considered the possibility that rather than the changes in the ratios of synchronous to non-synchronous mutations were in the published "RaTG13" genome were an intentional signal from Dr. Shi as she was probably ordered to conduct the cover-up??? Could there be a coded message in the changes???

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Nerd has power
6/2/2020 06:59:36 am

I don't know, but I do hope that's the case. If one day she would be able to come out and tell the truth to the public, it would be good for the world and for herself. Maybe she desires for such an opportunity too.

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Henri
6/2/2020 07:20:09 pm

The Letter to Nature was rejected outright. I appealed. The appeal failed. I now intend to take the matter up with the Editor-in-Chief. If that fails to get the letter published, we must discuss our next move.

Henri
6/4/2020 03:32:54 am

Radio silence form the Editor in chief and Nature. So, NerdHasPower, where we do we go from here? I have a couple of ideas. Your thoughts please.

Lilian
6/2/2020 10:41:35 am

The Case Is Building That COVID-19 Had a Lab Origin

https://www.independentsciencenews.org/health/the-case-is-building-that-covid-19-had-a-lab-origin/

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Javier
6/2/2020 12:42:37 pm

Three well done summaries, reported by Claire Robinson

"Evidence that the SARS-CoV-2 virus is genetically engineered" (May 12)
https://www.gmwatch.org/en/news/latest-news/19396
"Chinese and US scientists genetically engineered bat coronaviruses in dangerous gain-of-function research stretching back years" (May 26)
https://www.gmwatch.org/en/news/latest-news/19410-chinese-and-us-scientists-genetically-engineered-bat-coronaviruses-in-dangerous-gain-of-function-research-stretching-back-years
"Lab escape theory of SARS-CoV-2 origin gaining scientific support" (May 28)
https://www.gmwatch.org/en/news/latest-news/19412-lab-escape-theory-of-sars-cov-2-origin-gaining-scientific-support

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Lila Rajiva link
6/3/2020 08:33:59 am

Hello Nerd,

Wonderful, lucid analysis. This pulled it together for me.

As a political observer, I concluded a long time ago that this was either a lab accident being covered up or an engineered virus intended as the pretext for an economic shut-down that provides cover for the long-heralded global collapse.

One question. How does your argument bolster or weaken the other arguments made in favor of a man-made origin?

Specifically, I am thinking of the preprint paper published and then withdrawn under international pressure by a group of Indian scientists that noted the similarity of SARS-CoV-2 to the AIDS virus and speculated that it had been created in the quest for an AIDS vaccine.

This was shouted down by all the usual suspects in February. But then in April, the French doctor who won the Nobel Prize for discovering the AIDS virus, came out in support of the Indian paper, drawing on similar conclusions by a French statistical researcher. And another French scientist also concluded the virus was created in imitation of the AIDS virus.

Wasn't it yet another French scientist who created a furor a decade ago with his gain-of-function research involving the flu virus and ferrets? And didn't the French have a large input into the Wuhan Institute?

Regarding your question of how various experts covering up the lab origin could all be coordinating their responses, perhaps you should research a bit more into the ties of the Wuhan Institute of Virology with WHO, CDC, NIH, and the role of the foundations (especially the Gates Foundation) in funding and supporting these bodies. The CDC for instance receives money through its private foundation, the CDC Foundation, whose private partners include major corporations and foundations like the Gates Foundation. Gates funds a frightening number of media outlets, universities, research labs, and public health bodies all over the world. If you trace the connections, the experts covering up this bio-weapon all go back to this network.

This stuff goes right to the top - to participants in the World Economic Forum, to Davos, to the biggest players.

It would be nothing for a transnational intelligence agency to coordinate a global media cover-up. They do it all the time!

Thanks for your work.

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Henri
6/3/2020 07:20:51 pm

You seem to have misunderstood my position. I am on the side of 'it was NOT made in a lab'. NerdHasPower has put forward SOME arguments that RaTG13 is fake. As a scientist, I believe his arguments summarised in my letter require addressing by the Wuhan group. I believe there are satisfactory answers in scientific terms.

Since NetdHasPower wishes to remain anonymous, I have offered to act as an intermediary and submit a letter to Nature on his / her behalf. Whilst I am on the side side of 'not lab made' I am also on the side of 'Nature magazine has a responsibility to further investigate possible scientific fraud'. Consequently, I am pissed off that they have rejected my letter outright and have refused to publish it.
As for the Indian group and HIV, again it is an incorrect analysis. The HIV sequences are so short that just by chance they occur in many species, even fish. I did my own BLAST search and many, many organisms came up with the same short sequences. Why Montagnier jumped onto this bandwagon is beyond me. To add to his embarrasment, many
sequences he published are not even in-frame. That is, they would not code for HIV amino acid sequences.

I hope this clarifies my position. I still believe that my letter should be published by Nature and I trust you will support my efforts to get it done so in the near future. I can already see that Nature arr going ti be stubborn about this and this might lead to allegations of a cover-up. I hope it does not get to that stage but, if it does, they only have themselves to blame.


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Robert Cartwright
6/4/2020 02:17:44 am

Here the new paper of Perez and Montagnier

https://osf.io/tgw2d/

Lila Rajiva link
6/4/2020 02:42:14 am

Sorry, I meant to address the blog owner and the author of the original post.

Also, it is not just Montaigner. There are two other French scientists.

Lila

Robert Cartwright
6/4/2020 02:42:39 am

I think Henri's comment about Montagnier and Perez is exactly the same as that of the mainstream scientific community: many, many organisms came up with the same short HIV sequences. This, in my opinion, completely misses the point. The problem is not that the sequences are short and common but where they are located, and what mutations it induces subsequently (i.e. the S1 protein). This, I seem to understand, has been deduced by artificial intelligence simulations and, contrary to what Henri thinks, is the most sensible research direction to explore. This hypothesis, in the absence of information from Wuhan) is a hypothesis that can be verified ex-post only by analyzing whether the evolution of the virus deletes the exogenous RNA Sequences. The new Perez and Montagnier show that this is happening

Nerd has power
6/4/2020 09:52:09 pm

Thank you, Lila, for the comment and thoughts. I have answered the question of whether or not HIV sequences have being inserted into the SARS-CoV-2 genome. But I understand that it is very hard to screen all the comments now. My short answer is that I'm not convinced that such insertions are true. Or I should say that my own analysis failed to convince me in believing so. My conclusion that the virus is man-made is apparently based on other evidence and reasoning.

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Greg Felton
8/21/2020 02:02:45 pm

Hi Nerd:
I have seen arguments pro and con about HIV being the source of the insert, and have to agree with you that evidence is lacking. I have also seen claims for MERS and Ebola. Is there any evidence for any one of them?

Lilian
6/3/2020 03:12:53 pm

Another article worth to read

https://ccnationalsecurity.org/a-new-scientific-study-provides-evidence-of-genetic-manipulation-in-covid-19/

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Wilson Volleyball
6/4/2020 01:06:12 am

Exclusive: Coronavirus began 'as an accident' in Chinese lab, says former MI6 boss

https://www.telegraph.co.uk/news/2020/06/03/exclusive-coronavirus-began-accident-disease-escaped-chinese/

In an interview with The Telegraph, Sir Richard Dearlove, former director of British intelligence said he had seen an "important" new scientific report suggesting the virus did not emerge naturally but was man-made by Chinese scientists.

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Andrew M
6/4/2020 03:41:02 am

Some of the commenters don't leave facility for a direct reply - I don't know why that is. So, a general post about HIV, Montagnier and Perez. And Dearlove.

This HIV material really just detracts from Nerd's thesis and he has stated above he does not endorse it. Montagnier is well into his dotage and Perez is a quack. Their paper is cod science, written at too sophisticated a level for the majority of readers to appreciate that. Perez believes in numerological analysis of DNA - a golden ratio hidden in the code - that kind of thing (here's his blog: http://golden-ratio-in-dna.blogspot.com/). He is a numerologist (which is to a mathematician what an astrologer is to an astronomer). This is why the mainstream scientific community does not even bother to respond - their claims are not worthy of a scientific response.

On Richard Dearlove: have we got to the point now of taking as evidence a newspaper article referring to a scientific paper which someone says they have seen? Let's see the scientific paper itself and then decide for ourselves if there's anything in it worth considering. Not much that appears in The Daily Telegraph can be relied upon. NB of the 2 authors of the scientific paper, the British one has been a candidate for a right-wing political party but never a virologist. The Norwegian one has been an HIV researcher - so maybe a little bit more credibility there.

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Henri
6/4/2020 03:56:25 am

I agree. That is why in my letter to Nature I have mentioned only what I feel are the most powerful points made by NerdHasPower. I still have faith that there is a satisfactory explanation. But, I also feel Nature has a public duty to investigate the claims. They have remained stubborn about not publishing the letter. No real reason given. So, what next? Any suggestions?

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pangolin
6/4/2020 03:59:35 am

I would try to find another journal not controlled by EcoHealth Alliance. But it might be hard.

Andrew M
6/4/2020 06:17:01 am

Write it up formally as a paper and find a journal willing to publish it as a pre-print, inviting peer review?

Find an academic (a genuine, practising virologist) willing to address the points?