An amateur’s response to various fallacious criticisms against Dr. Yan’s papers

By billwilliam

In her two groundbreaking reports, Dr. Li-meng Yan presented preponderant evidences that SARS-CoV-2 is an artificially manipulated virus and is likely a bioweapon developed by the Chinese military. However, several critics posted fallacious arguments against her. This article will recapitulate Dr. Yan’s evidences as a point-by-point response to their arguments.

1. Question:

Proponents of the nature origin theory often quote RaTG13, the pangolin coronaviruses, and RmYN02 bat coronavirus as potential ancestors of the current pandemic3-8. But Dr. Yan said these sequences are fraudulent. Her evidences will be presented below.

Answer:

Admittedly, Dr. Yan’s 2nd paper hadn’t been publicized by the time they raised this question. The major evidences in her 2nd paper will be summarized in an outline format to answer this question.

Why RaTG13 is a fabricated sequence:

  1. RaTG13 only exists as a sequence in the online database GenBank. No live virus has ever been isolated2.
  • In a fecal swab, bacterial genes typically account for 70-90% of the data in sample sequencing2. But in the case of RaTG13 sample, which is collected from bat feces, only 0.7% of the raw sequencing reads are from bacteria2. RaTG13 is a suspicious fecal sample containing extraordinarily little bacteria.
  • The receptor binding domain (RBD) of RaTG13 does not bind the ACE2 receptors of two kinds of horseshoe bats2. How can RaTG13 be a bat coronavirus if it cannot even infect bats?
  • In the case of RNA, a genome is a long sequence made of four building blocks Adenine (A), Guanine (G), Cytosine (C), and Uracil (U). The viral RNA genetic information is read in groups of three nucleotides called codons, which correspond to specific amino acids. For instance, the codons GAU, GAC, GAA, GAG all encode the amino acid alanine. As shown, the third nucleotide in the codon is usually interchangeable. Changes in the third nucleotide will still code for the same amino acid. Mutations in the first or second nucleotide will encode a different amino acid.

If a genetic code mutates but still encodes the same amino acid, this is called “synonymous mutation.” Conversely, mutation that produces a different amino acid is called “non-synonymous mutation.”

Unless in the case of massive recombination, viruses that arise through natural evolution should have standard ratios between synonymous and non-synonymous mutations.

When comparing SARS-CoV-2 with RaTG13, the ratio of syn/non-syn mutation is abnormally high, which means RaTG13 isn’t related to SARS-CoV-2 by natural evolution2. RaTG13 is likely a fabricated sequence.

  • The critical furin-cleavage site in SARS-CoV-2 is absent in RaTG132. The evolutionary origin of the furin-cleavage site is unknown.

Why the pangolin coronaviruses are fraudulent:

  1. In a 10-year study by Dr. Daszak, no coronavirus could be detected in Malayan pangolins9. How can pangolins be the intermediate host if they are not infected by coronaviruses?
  • If SARS-CoV-2 evolves from a coronavirus in pangolins, the Spike protein should have higher affinity to the pangolin ACE2 than to the human ACE2. However, two studies, one in silico and another in vitro, demonstrate that the Spike of SARS-CoV-2 binds tighter to human ACE2 than to pangolin ACE210,11. Pangolins couldn’t have been the immediate host.          
  • The pangolin coronaviruses also display abnormal ratios of syn/non-syn mutation when compared to SARS-CoV-22, so the pangolin coronaviruses are probably artificial.

Why RmYN02 is fraudulent:

  1. RmYN02 also fails the test of syn/non-syn mutation ratio2. It is not a natural sequence.
  • RmYN02 doesn’t contain a functional furin cleavage site. Its PAA site is neutrally charged and cannot be recognized by the furin protease, which requires polybasic sequences. The sequence of RmYN02 is also slightly out of alignment2. There is no way its PAA site could have evolved into the PRRA furin cleavage site in SARS-CoV-2.

2. Question:

In her first paper, Dr. Yan pointed out that the presence of two restriction enzyme sites surrounding the spike protein’s receptor binding motif (RBM) indicates artificial cloning. One reviewer questioned Dr. Yan’s analysis and asserted the restriction sites appear naturally by chance4.

Answer:

Restriction enzymes are enzymes that can recognize and cleave specific short sequences in a gene. Digestion by restriction enzyme is a classic technique used in gene cloning, in which a gene fragment can be cut out and then a new fragment can be inserted with ligation. Genes manipulated by such a technique contains two restriction sites flanking the sequence being cut out and swapped.

The reviewer’s claim is superficial at best, because he failed to address the abnormal locations of these restriction sites, which Dr. Yan presented in her 1st paper as the evidence of artificial manipulation. The restriction sites in SARS-CoV-2 Spike gene directly flank the RBM sequence that is responsible for binding with ACE2. Restriction enzyme digestion followed by ligation can be used to replace the original sequence with other RBM sequences (maybe artificially designed) so that the virus can be engineered to bind with and infect human cells. In fact, this is exactly what a group of virologists in China have been doing. Shi Zhengli actively pursued gain-of-function experiments in which her group swapped SARS-1 RBM into the Spike of a bat coronavirus to make it capable of infecting humans13. Li Fang, another scientist, swapped the RBM of SARS-CoV-2 into a SARS-1 Spike, and the resultant chimeric virus can bind with human ACE212. Stunningly, protein sequence alignment shows that the RBMs manipulated by Li and Shi are similar to the SARS-CoV-2 RBM flanked by restriction enzymes, as shown below in Figure 5C of Dr. Yan’s 1st paper1. Natural origin shouldn’t cause the location of these restriction sites to match almost perfectly with the locations used by Li and Shi for gain-of-function experiments. The only explanation is artificial manipulation.     

Figure 5C of Dr. Yan’s 1st paper1. The top SARS-CoV-2 RBM (orange) is defined by EcoRI and BstEII. The middle sequence (magenta) is the RBM swapped by Li Fang lab into a SARS-1 Spike protein. The bottom sequence (blue) is the RBM swapped by Shi Zhengli lab into the Spike protein of a different bat coronavirus.

3. Question:

A few reviewers and scholars claim the Spike protein of SARS-CoV-2 doesn’t resemble SARS-1 Spike5-7.

Answer.

At a cursory glance, they may appear dissimilar at the sequence level. But structural level analysis reveals that the receptor binding motif (RBM) amino acid residues critical for human ACE2 binding are all “kept.” For example, the essential residues responsible for disulfide bond formation (C467, C474) and electrostatic interactions (R444, E452, R453, D454) are preserved to maintain structural integrity of the RBM1. The essential residues for hydrophobic interaction are substituted by other hydrophobic residues (I428→L, L443→F, F460→Y, L472→F, Y484→Q)1. Therefore, the SARS-CoV-2 Spike binds with human ACE2 by the same interaction that SARS-1 binds with human ACE2. The Chinese military labs probably kept the essential residues for binding human ACE2 but mutated other non-essential proteins to cover up the traces of manipulation. The availability of high-resolution structures elucidates all essential residues for virus Spike binding with human ACE2 and thus facilitates intelligent design of the RBM. 

4. Question:

According to Dr. Yan’s 1st report, a furin-cleavage site was artificially inserted into the SARS-CoV-2 Spike protein to enhance its infectivity. She said this is the first instance a furin-cleavage site is found in this class of SARS-like coronavirus. Several critics attacked Dr. Yan’s analysis3-6, 8. Their reasoning is that furin-cleavage sites naturally occur in several viruses3-8. The critics cited MERS5,6 and sequence RmYN023 as examples containing furin-cleavage sites.

Answer.

First of all, Dr. Yan clearly stated on pg12 of her 1st report that “within the lineage B of β coronaviruses and with the exception of SARS-CoV-2, no viruses contain a furin-cleavage site at the S1/S2 junction1.” The furin-cleavage site is a unique feature to SARS-CoV-2 and is not present in other lineage B β coronaviruses, so SARS-CoV-2 couldn’t have evolved the furin-cleavage site through homologous recombination (a process by which organisms or viruses with very close genetic similarities can swap their genes), because other members of lineage B don’t contain the furin-cleavage site. MERS is a SARS-like virus but isn’t similar enough—it doesn’t belong to lineage B.

Second, RmYN02 is already debunked by Dr. Yan’s 2nd report as being fabricated2, and thus it couldn’t have been the ancestor of SARS-CoV-2. Despite sequence similarities, the Spike protein of RmYN02 contains a proline-alanine-alanine (PAA) sequence, whereas SARS-CoV-2 contains a proline-arginine-arginine-alanine (PRRA) sequence2. Furin protease only recognizes and cleaves specific polybasic protein sequences. Alanine is an uncharged amino acid, so PAA of RmYN02 sequence doesn’t have furin cleavage function. On the other hand, arginine is positively charged, and the PRRA sequence of SARS-CoV-2 is a legitimate furin cleavage site. In addition, according to Dr. Yan, “-PAA- in RmYN02 only partially resembles the -PRRA- insertion in SARS-CoV-2 and does not appear to be an actual insertion if properly aligned2.” So, the sequence of RmYN02 is out of alignment and isn’t a correct ancestor.

Third, even though some coronaviruses of other lineages do contain furin-cleavage sites, they don’t contain the same polybasic amino acid sequences as the one in SARS-CoV-2 (PRRAR/SVA)1. None of them can be the ancestor of SARS-CoV-2.   

Fourth, gene sequence alignment reveals that SARS-CoV-2 shares no more than 40% sequence identity at the Spike with other coronaviruses with furin-cleavage sites1. The low level of sequence identity precludes the possibility of homologous recombination. SARS-CoV-2 couldn’t have evolved the furin cleavage site by homologous recombination with other coronaviruses containing such sites.

Fifth, the two arginine residues (R) in the furin-cleavage site (PRRA) of SARS-CoV-2 are both encoded by the rare codon CGG1, which is the least used codon for arginine. Although different codons may encode the same amino acid (especially if the codons are only different in the third nucleoside), these synonymous codons don’t appear by the same frequency. The probability of having two rare codons in tandem is exceptionally low. This furin-cleavage site is abnormal because it is the only instance in the SARS-CoV-2 genome where the rare codons appear in tandem1. Dr. Yan suggested the choice of rare codons at the furin-cleavage site could have been the result of genetic manipulation to create a FauI restriction site1. Restriction fragment length polymorphism after FauI digestion is a convenient technique to check whether the weaponized virus preserves the furin-cleavage site in vitro1.   

5. Question:

Several critics claimed that restriction enzyme digestion and ligation is an outdated technique and that more modern techniques such as whole genome synthesis4-6 should have been used if this is a bioweapon.

Answer.

Restriction enzyme digestion is an old but classic technique that is widely used in genetic engineering. Such a technique is still effective1.

In bioweapon development, usually a set of candidates are created for selection and optimization. For example, in the case of SARS-CoV-2, several artificial RBM sequences could be created, and the one with the highest binding affinity to human ACE2 is selected to create the bioweapon. The presence of two restriction sites flanking the RBM sequence enables a convenient way to swap in different artificial sequences for trial1.

Creating a set of bioweapon virus candidates with the genome synthesis approach will require synthesizing the whole genome of every single candidate, which is tedious.   

6. Question:

Almost all critics argued that ZC45 shouldn’t be the template for SARS-CoV-2 because they are different by 3000 nucleotides or 10% of the genome3-6, 8.

Answer.

This is the most frequent argument by the critics, but this argument only focuses superficially on overall sequence identity while ignoring the stunning similarity in certain critical proteins. As described by Dr. Yan, SARS-CoV-2 and Zhoushan bat virus ZC45 are exceptionally similar in the amino acid sequence of these critical proteins: 94% identical in the nucleocapsid protein, 98.6% identical in the membrane protein, 95% identical in the S2 (2nd half) of the Spike protein, 94.2% identical in the Orf8 protein, and 100% identical in the E protein1.

The 94.2% identity in Orf8 and 100% identity in the E protein are very unusual. Orf8 in SARS-CoV-2 is a protein that helps the virus evade host immunity1. According to Dr. Yan, Orf8 is poorly conserved among coronaviruses. Strangely, the Orf8 of ZC45 shares a 94.2% identity with the Orf8 of SARS-CoV-2, whereas other coronaviruses don’t share more than 58% identity on that protein with SARS-CoV-21. In addition, the Envelope E protein, although conserved, is tolerant of mutations1. In fact, SARS-CoV-2 strains collected in April 2020 already displayed a few point mutations on the E protein1. If SARS-CoV-2 were of natural origin, it should have undergone significant mutations on the Orf8 and E proteins when it crossed the species barrier multiple times (purportedly from bat to bat, bat to human, bat to pangolin, or pangolin to human), but this is not what we observe. The natural origin theory cannot explain the abnormally high identity in Orf8 and E protein. The only explanation is that ZC45/ZXC21 is used as a template to create SARS-CoV-2.

The E protein of SARS-CoV-2 retained 100% identity to the E protein of ZC45 probably because the Chinese military labs chose it for its virulence. In addition to structural functions, the E protein of SARS coronavirus functions as an ion channel that enhances virulence14.

Another tell-tale phenomenon is the difference in sequence conservation between the S1 and S2 proteins. The S2 protein of SARS-CoV-2 shares 95% identity with that of ZC451. In comparison, the S1 protein of SARS-CoV-2 is much less conserved, retaining only 69% identity to that of ZC451. This contrast can be reconciled by the following reasoning: The S1 protein containing the RBM is solely responsible for binding with host ACE21, and an artificial RBM is swapped in to enable the virus to bind with human ACE2. The S2 protein, however, is responsible for maintaining trimeric form of the Spike and facilitating membrane fusion after binding1. Because S2 doesn’t participate in host recognition, the original sequence (in ZC45) is kept intact.  

Of course, differences in the sequences are probably introduced intentionally as a coverup. Between SARS-CoV-2 and ZC45, the ORF1b gene is less conserved1. The ORF1b gene encodes several proteins responsible for viral replication1. The ORF1b of any β coronavirus can be swapped in without hindering viral replication1. The Chinese military labs probably swapped in the ORF1b of another β coronavirus1 to make SARS-CoV-2 look less similar to the template ZC45.

In contrast, the E protein is kept intact (100% amino acid sequence identity) probably because it is a major virulence factor. The Orf8 protein (94.2% sequence identity), which allows the virus to shut down host immunity, is probably retained for this reason as well.     

References

1. Yan, L. et al. Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route.

2. Yan, L. et al. SARS-CoV-2 Is an Unrestricted Bioweapon: A Truth Revealed through Uncovering a Large-Scale, Organized Scientific Fraud.

3. Koyama, T. Review 1: “Unusual Features of the SARS-CoV2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route.” The MIT Press Journal. September 25, 2020.

https://rapidreviewscovid19.mitpress.mit.edu/pub/iqty3wru/release/1

4. Lauring, A. Review 2: “Unusual Features of the SARS-CoV2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route.” The MIT Press Journal. September 25, 2020.

https://rapidreviewscovid19.mitpress.mit.edu/pub/cly5o342/release/1

5. Gallo, R. Review 3: “Unusual Features of the SARS-CoV2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route.” The MIT Press Journal. September 30, 2020.

https://rapidreviewscovid19.mitpress.mit.edu/pub/eymbro7v/release/1

6. Reitz, M. Review 4: “Unusual Features of the SARS-CoV2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route.” The MIT Press Journal. October 4, 2020.

https://rapidreviewscovid19.mitpress.mit.edu/pub/loicw441/release/1

7. Andersen, K. The Proximal Origin of SARS-CoV-2. Nat Med 26, 450-455 (2020).

8. Brouillette, M. and Renner, R. Why misinformation about COVID-19’s origins keeps going viral. National Geographic website. September 18, 2020.

https://www.nationalgeographic.com/science/2020/09/coronavirus-origins-misinformation-yan-report-fact-check-cvd/?cmpid=org=ngp::mc=social::src=twitter::cmp=editorial::add=tw20200919science-coronavirusmisinformation::rid=&sf237963153=1#close

9. Lee, J. et al. No evidence of coronaviruses or other potentially zoonotic viruses in Sunda pangolins (Manis javanica) entering the wildlife trade via Malaysia. bioRxiv, https://doi.org/10.1101/2020.06.19.158717 (2020).

10. Piplani, S., Singh, P.K., Winkler, D.A. & Petrovsky, N. In silico comparison of spike protein-ACE2 binding affinities across species; significance for the possible origin of the SARS-CoV-2 virus. arXiv, arXiv:2005.06199 (2020).

11. Mou, H. et al. Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARSCoV-2. bioRxiv, https://doi.org/10.1101/2020.06.29.178459 (2020).

12. Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature (2020).

13. Ren, W. et al. Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. J Virol 82, 1899-907 (2008).

14. Nieto-Torres, J.L. et al. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog 10, e1004077 (2014).

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