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More than eight months after SARS-CoV-2 emerged as a global threat, there is still no clarity about its origins. Those who suspect that the virus was developed in a laboratory are frequently dismissed as conspiracy theorists, but there is growing evidence to support the suggestion that gain-of-function research has made SARS-CoV-2 particularly virulent.
While some scientists still argue that SARS-CoV-2 is a product of natural evolution, others consider an accidental or deliberate leak from a laboratory to be a valid hypothesis that merits further investigation.
For decades, gain-of-function research, which alters viruses to increase their transmissibility, pathogenicity, virulence or lethality, has been carried out by American and Chinese scientists working in collaboration. There have been numerous ‘leaks’ of viruses from laboratories, including during the severe acute respiratory syndrome (SARS) outbreak that occurred in 2003–2004.
Those who suggest that SARS-CoV-2 may well have originated in a laboratory include the Norwegian virologist Birger Sørensen, the French scientist and Nobel prize winner Luc Montagnier, and the exiled Chinese scientist Li-Meng Yan, who says that SARS-CoV-2 is an “unrestricted bioweapon” and there has been “large-scale, organised scientific fraud” in covering up the truth.
Yan and others say there is evidence within the spike protein of the SARS-CoV-2 genome that suggests it is a product of genetic manipulation.
Proponents of the natural spillover theory allege that the virus jumped species, possibly via an intermediary host, to cross over to humans via the wildlife trade or by other means.
The main research that is cited in defence of the natural-origin hypothesis is that conducted by Kristian G. Andersen et al. who say it is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related coronavirus. Andersen et al’s findings have been challenged, however.
Working behind the scenes, there is a team of scientists, journalists, and other independent researchers who refer to themselves collectively as DRASTIC (Decentralised Radical Autonomous Search Team Investigating Covid-19). They investigate anomalies in the narratives about SARS-CoV-2, collect and present evidence, and put forward questions and hypotheses.
One of the issues raised by the DRASTIC team is the fact that a database containing unpublished information about the sequencing of samples collected by the Wuhan Institute of Virology (WIV) on trips to an abandoned copper mine in Yunnan has been taken offline.
Six of the men who were working in the mine, removing bat faeces from a cave, suffered a severe pneumonia-like illness in 2012. Three of them died.
The miners had a high fever, dry cough, sore limbs and, in some cases, headaches – all symptoms that are now associated with Covid-19.
“The WIV’s wild animal virus database and its password-protected section containing unpublished virus sequences are no longer available publicly, and even the pages describing it have now been taken offline,” the DRASTIC team said.
There has been much discussion – and speculation – about a bat coronavirus that was reportedly discovered in a faecal sample collected in the abandoned mine in Yunnan in 2013.
The virus was initially referred to as RaBtCoV/4991, but later, when its whole genome was sequenced, it was renamed RaTG13. The genomic sequence of RaTG13 is reported to be 96% identical to that of SARS-CoV-2.
The director of the Centre for Infectious Diseases at the Wuhan Institute of Virology (WIV), Shi Zhengli, says that RaTG13 is the same sample as the one referred to as RaBtCoV/4991 in a scientific paper published by researchers from the WIV in 2016.
Zhengli says RaTG13 was never cultured in her laboratory and insists that SARS-CoV-2 did not originate there.
There is, meanwhile, a hypothesis put forward by two American researchers – Jonathan Latham and Allison Wilson – that a bat virus evolved into SARS-CoV-2 inside the bodies of the miners who became ill in Yunnan and that researchers at the WIV worked on samples of that virus, which then leaked from a WIV lab.
A group of Chinese scientists that includes Li-Meng Yan say there is no evidence for Latham and Wilson’s hypothesis and it makes no sense.
Scientists point to ‘suspicious’ SARS-CoV-2 properties
In an interview published in Minerva in Norway on July 2, Birger Sørensen said it was more than 90% certain that SARS-CoV-2 originated in a laboratory.
Sørensen and his fellow scientists Angus Dalgleish and Andres Susrud have done their research with a view to producing a vaccine.
Sørensen’s work came to international attention in 2008 when he launched a new immunotherapy for HIV. Dalgleish is the professor at St George’s medical school at the University of London who became world famous in 1984 after he discovered a novel receptor that the HIV virus uses to enter human cells.
Sørensen (pictured left) told Minerva that he and his colleagues discovered that SARS-CoV-2 was exceptionally well adjusted to infect humans, to the degree that it was suspicious.
He said he and his colleagues had discovered properties in SARS-CoV-2 that enable it to use an additional receptor and create a binding to human cells in the upper respiratory tract and the intestines that is strong enough to produce an infection.
Sørensen says the SARS-CoV-2 spike protein is very stable and this points to it being “a fully developed, almost perfected virus for infecting humans”. This, he says, indicates that the structure of the virus cannot have evolved naturally.
He also points to the inserts in SARS-CoV-2, several of which do not, he says, exist in other coronaviruses. Sørensen told Minerva that it’s possible for a virus to attain these properties in nature, but it’s not likely.
“If the mutations had happened in nature, we would have most likely seen that the virus had attracted other properties through mutations, not just properties that help the virus to attach itself to human cells,” he said.
“What we see is that an area that you could observe in the first SARS coronavirus has been moved, so that the parts of the virus that are particularly well suited to attach to humans have become part of the spike protein that the virus uses to penetrate human cells.”
Sørensen told Minerva that the properties seen in SARS-CoV-2 had yet to be discovered anywhere in nature. If the virus came from nature, he says, there should also be many animals infected with it.
“The only place we are aware of where an equivalent virus to that which causes Covid-19 exists is in a laboratory,” Sørensen told Minerva. “So the simplest and most logical explanation is that it comes from a laboratory. Those who claim otherwise, have the burden of proof.”
In an article published on July 13, Sørensen, Dalgleish, and Susrud present detailed reasoning for their argument that the origin of SARS-CoV-2 is not natural.
“SARS-CoV-2 is possessed of dual action capability. In this paper we argue that the likelihood of this being the result of natural processes is very small,” the authors state.
“The spike has six inserts which are unique fingerprints with five salient features indicative of purposive manipulation.”
Sørensen, Dalgleish, and Susrud analyse four research projects which, they suggest, show by deduction “how, where, when, and by whom the SARS-CoV-2 spike acquired its special characteristics”.
They state: “This reconstructed historical aetiology meets the criteria of means, timing, agent and place to produce sufficient confidence to reverse the burden of proof.
“Henceforth, those who would maintain that the Covid-19 pandemic arose from zoonotic transfer need to explain precisely why this more parsimonious account is wrong before asserting that their evidence is persuasive, most especially when, as we also show, there are puzzling errors in their use of evidence.”
Sørensen, Dalgleish, and Susrud challenge the arguments put forward by Andersen et al. in their controversial article entitled ‘The proximal origin of SARS-CoV-2’, published on March 17 in Nature Medicine.
The three scientists say the contention of Andersen et al. that it is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus because the ACE2 binding is not ideal “is weakened because Andersen et al. cite two authorities which actually say the reverse of what they say that they say”.
ACE2 (the Angiotensin-converting enzyme 2) is a protein found on the surface of human cells, as well as in soluble form in the blood, that has been identified as the receptor for the SARS-CoV-2 viral entry.
Sørensen, Dalgleish, and Susrud have identified five features of SARS-CoV-2 that individually, they say, seem unlikely to be the result of natural evolution and which, taken together, make natural evolution a less likely explanation than purposive manipulation, “specifically for gain of function”.
Sørensen and his colleagues say that a major part of the SARS-CoV-2 spike protein has human-like domains with matured transmission adaption.
“Blasting the spike protein with a rolling window of six amino acids showed that 78.4% of six amino acid windows are human like,” they write.
Nearly 80% of the spike protein has a built-in stealth property by having high human similarity, they add.
“Therefore, it is remarkably well-adapted virus for human co-existence,” the scientists write. “Such high human similarity also implies a high risk for the development of severe adverse events/toxicity and even Antibody Dependent Enhancement (ADE) unless specific precautions are taken when using the spike protein in any vaccine candidate.”
Sørensen and his co-authors say their discovery that the SARS-CoV-2 spike displays new amino acid inserts “with condensed cumulative charge, all of which are surface exposed” is particularly significant.
“Being physically located on the surface of the spike protein greatly increases the infectivity and pathogenicity of the virus, enabling these inserts to participate in binding to co-receptors/negatively charged attachment receptors or even, as we have discovered, to the negatively charged phospholipid heads on the cell membrane,” they write.
“Such a result is typically the objective of gain of function experiments to create chimeric viruses of high potency. Therefore this is a strong indicator of manipulation.”
Sørensen, Dalgleish, and Susrud point to articles by Chinese and American researchers in which, they say, “those researchers demonstrate and discuss how they have manipulated new chimeraviruses into existence, with SARS-coronavirus as a starting point”.
A comprehensive review of the relevant literature shows that a substantial amount of directly relevant gain-of function research has been undertaken, the three scientists say.
“Four studies are especially noteworthy. They are linked in two ways: scientifically, in that the third and fourth build upon the results of the first and second, and in continuity of the institution and personnel across all four.”
Sørensen and his colleagues note that the WIV was a key collaborator in all the projects.
The first project, in 2008, successfully demonstrated technical capabilities to interchange receptor-binding domains (RBDs) between bat SARS-like and human SARS viruses, Sørensen and his co-authors say.
Sørensen and his colleagues also say that, in 2010 scientists from the ‘Special Viruses’ section of the WIV were engaged in gain-of-function experiments, jointly with international collaborators, to increase SARS-CoV infectiousness for humans.
“They used an HIV pseudovirus to express seven bat ACE2 receptors and compared their binding properties to human ACE2 receptors in order to pick the best for further optimising a SARS-like coronavirus’s ability to bind to human cells”, Sørensen and his co-authors write.
“They also found that some bat ACE2 receptors are very close to human ACE2 receptors. This study provided a model system for testing the most infectious of SARS-CoV-like viruses which already had been selected in a vast survey of Chinese bat populations between 2005–2013.”
Sørensen and his co-authors also highlight the gain-of-function research carried out in 2015.
“In 2015 scientists from the ‘Special Viruses’ section of the Wuhan Institute of Virology were engaged in ‘gain-of-function’ experiments jointly with a majority team from the University of North Carolina Chapel Hill,” they write.
“Together, they manipulated bat viruses to create a mouse adapted chimeric virus SHC014-MA15 which binds to and can proliferate on human upper airway cells.”
Sørensen and his colleagues detail the way the SHC014 spike, in a wild-type backbone, can “efficiently utilise multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV” and say that what are described are “precisely SARS-CoV-2 properties”.
The three scientists say that those who reported on the research carried out in 2015 “were well aware that the chimeric virus which they had created was very dangerous because they discussed this fact”.
Sørensen and his colleagues added: “It is certainly the case that this experiment created a chimeric virus with very high infectivity potential targeted to the human upper respiratory tract.”
Sørensen, Dalgleish, and Susrud say that when the in vivo experiments were carried out at Chapel Hill, and the chimeric virus replicated in mouse lung showed significant pathogenesis, this was the opposite of the result the researchers had expected.
“The creation of chimeric viruses like SHC014-MA15 was not expected to increase ‘pathogenicity’,” Sørensen and his colleagues write.
In summary, the scientists say the work done in 2010 built upon the research carried out in 2008. “The 2010 work (Hou et al., 2010) perfected the ability to express receptors on human cells.
“On these foundations, the central gain of function work that underpins the functionalities of SARS-CoV-2 took place, carrying the WIV spike and plasmid materials to bond successfully to a UNC Chapel Hill human epithelial cell-line.”
‘The idea of an animal intermediate is speculation’
In an article published in Independent Science News on June 2, Jonathan Latham and Allison Wilson, who are co-founders of the non-profit Bioscience Resource Project, which is based in Ithaca, New York, write: “Unfortunately, in the US at least, the question of the pandemic’s origin has become a political football; either an opportunity for Sinophobia or a partisan ‘blame game’.
“But the potential of a catastrophic lab release is not a game and systemic problems of competence and opacity are certainly not limited to China (Lipsitch, 2018). The US Department of Homeland Security (DHS) is currently constructing a new and expanded national Bio and Agro-defence facility in Manhattan, Kansas.”
The DHS has put the 50-year risk of a release from its laboratory at 70%, Latham and Wilson say.
Citing the comment of the Australian virologist Nikolai Petrovsky, from Flinders University, that no natural virus matching SARS-CoV-2 has been found in nature despite an intensive search to find its origins, Latham and Wilson said: “The idea of an animal intermediate is speculation. Indeed, no credible viral or animal host intermediaries, either in the form of a confirmed animal host or a plausible virus intermediate, has to date emerged to explain the natural zoonotic transfer of Sars-CoV-2 to humans (e.g. Zhan et al., 2020).”
Petrovsky (pictured left) was the lead author of a preprint paper published on the arXiv server on May 13.
The Flinders University scientists found that SARS-CoV-2 targeted humans more potently than any of the tested animal species.
They said: “The binding energy between SARS-CoV-2 spike protein and ACE2 was highest for humans out of all species tested, suggesting that SARS-CoV-2 spike protein is uniquely evolved to bind and infect cells expressing human ACE2.
“This finding is particularly surprising as, typically, a virus would be expected to have highest affinity for the receptor in its original host species, e.g. bat, with a lower initial binding affinity for the receptor of any new host, e.g. humans.
“However, in this case, the affinity of SARS-CoV-2 is higher for humans than for the putative original host species, bats, or for any potential intermediary host species.”
Latham and Wilson note that, in 2014, just before a ban on gain-of-function research went into effect in the US, Zhengli did work with researchers from Ralph Baric’s laboratory in North Carolina, where gain-of-function research on bat coronaviruses was carried out, and published a paper.
The researchers combined the spike of the bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone into a single engineered live virus, Latham and Wilson write.
“The spike was supplied by the Shi lab. They put this bat/human/mouse virus into cultured human airway cells and also into live mice. The researchers observed ‘notable pathogenesis’ in the infected mice.”
Researchers in Shi Zhengli’s laboratory produced recombinant bat coronaviruses and placed these in human cells and monkey cells, Latham and Wilson note. “All these experiments were conducted in cells containing human or monkey ACE2 receptors,” they said.
Reports by exiled Chinese scientist
Exiled scientist Li-Meng Yan (pictured left) and three of her colleagues at the Rule of Law Society and Rule of Law Foundation in New York in the US have published two reports about the possible origins of SARS-CoV-2.
In the first report, published on September 14, Yan, Shu Kang, Jie Guan, and Shanchang Hu lay out arguments that suggest that SARS-CoV-2 originated in a laboratory in China.
Yan, who specialised in virology and immunology at the Hong Kong School of Public Health, but fled to the US in April, says that the Chinese government and the WHO knew about person-to-person transmission of Covid-19 much earlier than was reported.
She says that her supervisors ignored research that she conducted at the beginning of the pandemic that she believes could have saved lives.
In their first paper, which was published on the preprint website Zenodo, Yan, Kang, Guan, and Hu say genetic evidence within the spike gene of the SARS-CoV-2 genome exists that suggests that the genome is a product of genetic manipulation.
“Furthermore, the proven concepts, well-established techniques, and knowledge and expertise are all in place for the convenient creation of this novel coronavirus in a short period of time,” Yan et al. state.
The scientists say that the genome sequence of SARS-CoV-2 has likely undergone genetic engineering, through which the virus has gained the ability to target humans with enhanced virulence and infectivity.
“The characteristics and pathogenic effects of SARS-CoV-2 are unprecedented. The virus is highly transmissible, onset-hidden, multi-organ targeting, sequelae-unclear, lethal, and associated with various symptoms and complications,” they state.
The four scientists say that the theory that SARS-Cov-2 has a natural origin, although widely accepted, lacks substantial support.
“The alternative theory that the virus may have come from a research laboratory is, however, strictly censored on peer-reviewed scientific journals. Nonetheless, SARS-CoV-2 shows biological characteristics that are inconsistent with a naturally occurring, zoonotic virus.
“In this report, we describe the genomic, structural, medical, and literature evidence, which, when considered together, strongly contradicts the natural origin theory.”
Yan et al. say there is evidence indicating that SARS-CoV- 2 is a laboratory product created by using bat coronaviruses ZC45 and/or ZXC21 as a template and/or backbone.
“Evidently, the possibility that SARS-CoV-2 could have been created through gain-of-function manipulations at the WIV is significant and should be investigated thoroughly and independently,” they write.
The four researchers present three main arguments to support their contention that SARS-CoV-2 was manipulated in a laboratory.
They say that the genomic sequence of SARS-CoV-2 is “suspiciously similar” to that of a bat coronavirus discovered in military laboratories in the Third Military Medical University in Chongqing, China, and the Research Institute for Medicine of Nanjing Command.
ZC45 and ZXC21 were discovered (between July 2015 and February 2017), isolated, and characterised by researchers at the military laboratories in the Third Military Medical University and the Research Institute for Medicine of Nanjing Command, Yan et al. say. The data and associated work were published in 2018.
The researchers also say that the receptor-binding motif (RBM) within the spike protein of SARS-CoV-2 cannot be born from nature. They say that the RBM of the spike resembles that of SARS-CoV from the 2003 epidemic “in a suspicious manner”. Genomic evidence suggests that the RBM has been genetically manipulated, they say.
“The way that SARS-CoV-2 RBM resembles SARS-CoV RBM and the overall sequence conservation pattern between SARS-CoV-2 and ZC45/ZXC21 are highly unusual,” Yan et al. write. “Collectively, this suggests that portions of the SARS-CoV-2 genome have not been derived from natural quasi-species viral particle evolution.”
The scientists further state that SARS-CoV-2 contains a unique furin cleavage site in its spike protein, “which is known to greatly enhance viral infectivity and cell tropism”.
This cleavage site is completely absent in this particular class of coronaviruses found in nature, the scientists say.
“Within the lineage B of β coronaviruses and with the exception of SARSCoV-2, no viruses contain a furin cleavage site at the S1/S2 junction [of the spike protein],” they write.
“In addition, rare codons associated with this additional sequence suggest the strong possibility that this furin cleavage site is not the product of natural evolution and could have been inserted into the SARS-CoV-2 genome artificially by techniques other than simple serial passage or multi-strain recombination events inside co-infected tissue cultures or animals.”
A codon is a trinucleotide sequence of DNA or RNA that corresponds to a specific amino acid.
Yan et al. say that, in a discovery that is consistent with the RBM engineering theory, they have identified two unique restriction sites, EcoRI and BstEII, at either end of the RBM of the SARS-CoV-2 genome.
“These two sites, which are popular choices of everyday molecular cloning, do not exist in the rest of this spike gene,” the scientist say.
“Such EcoRI and BstEII sites do not exist in the spike genes of other β coronaviruses, which strongly indicates that they were unnatural and were specifically introduced into this spike gene of SARS-CoV-2 for the convenience of manipulating the critical RBM.”
A restriction site is a sequence of approximately six–eight base pairs of DNA that binds to a given restriction enzyme. A restriction enzyme is a protein that recognises a specific, short nucleotide sequence and cuts the DNA only at a specific restriction site. The natural function of restriction enzymes is to inactivate invading viruses by cleaving the viral DNA.
Yan et al. say that once restriction sites are successfully introduced, the RBM segment can be swapped conveniently using routine restriction enzyme digestion and ligation.
The feasibility of this RBM-swap strategy has been proven, the researchers add. In 2008, they say, Shi Zhengli’s group swapped a SARS RBM into the spike proteins of several SARS-like bat coronaviruses after introducing a restriction site into a codon-optimised spike gene. They then validated the binding of the resultant chimeric spike proteins with the human ACE2 receptor (hACE2).
Yan et al. refer in their report to the work of Zhengli’s collaborator, Fang Li. “Dr Li was the first person in the world to have structurally elucidated the binding between SARS-CoV RBD and hACE238 and has been the leading expert in the structural understanding of spike-ACE2 interactions,” the researchers said.
“The striking finding of EcoRI and BstEII restriction sites at either end of the SARS-CoV-2 RBM, respectively, and the fact that the same RBM region has been swapped both by Dr Shi and by her long-term collaborator, respectively, using restriction enzyme digestion methods are unlikely a coincidence.
“Rather, it is the smoking gun proving that the RBM/Spike of SARS-CoV-2 is a product of genetic manipulation.”
Although it may be convenient to copy the exact sequence of SARS RBM, it would be too clear a sign of artificial design and manipulation, Yan et al. say.
“The more deceiving approach would be to change a few non- essential residues, while preserving the ones critical for binding … Importantly, changes might have been made intentionally at non-essential sites, making it less like a ‘copy and paste’ of the SARS RBM.”
Yan et al. say that SARS-CoV-2 could have been created in a laboratory in a period of about six months.
The four scientists question the existence in nature of the RaTG13 virus.
“While suggesting a natural origin of SARS-CoV-2, the RaTG13 virus also diverted the attention of both the scientific field and the general public away from ZC45/ZXC21,” Yan et al. say.
They say that researchers from a Chinese BSL-3 lab (the Shanghai Public Health Clinical Centre), published an article in Nature in which they reported a conflicting close phylogenetic relationship between SARSCoV-2 and ZC45/ZXC21 rather than with RaTG13, but the article “was quickly shut down for ‘rectification’”.
They add: “It is believed that the researchers of that laboratory were being punished for having disclosed the SARS-CoV-2–ZC45/ZXC21 connection.
“On the other hand, substantial evidence has accumulated, pointing to severe problems associated with the reported sequence of RaTG13 as well as questioning the actual existence of this bat virus in nature.”
Yan et al. say that a very recent publication indicates that the RBD of the RaTG13’s spike protein could not bind to the ACE2 of two different types of horseshoe bats.
They say that their finding further substantiates the suspicion that the reported sequence of RaTG13 could have been fabricated “as the spike protein encoded by this sequence does not seem to carry the claimed function”.
They add: “The fact that a virus has been fabricated to shift the attention away from ZC45/ZXC21 speaks for an actual role of ZC45/ZXC21 in the creation of SARS-CoV-2.”
Yan et al. say genomic sequence analysis reveals that bat coronavirus ZC45 is the closest match to SARS-CoV-2.
“When SARS-CoV-2 and ZC45/ZXC21 are compared on the amino acid level, a high sequence identity is observed for most of the proteins,” they say.
“The nucleocapsid protein is 94% identical. The membrane protein is 98.6% identical. The S2 portion (2nd half) of the spike protein is 95% identical. Importantly, the Orf8 protein is 94.2% identical and the E protein is 100% identical.”
Yan et al. say sequence blast analysis indicates that, with the exception of SARS-CoV-2, no known coronaviruses share 100% amino acid sequence identity on the E protein with ZC45/ZXC21.
“Although 100% identity on the E protein has been observed between SARS-CoV and certain SARS-related bat coronaviruses, none of those pairs simultaneously share over 83% identity on the Orf8 protein.”
Yan et al. say that the 94.2% identity on the Orf8 protein, 100% identity on the E protein, and the overall genomic/amino acid-level resemblance between SARS-CoV-2 and ZC45/ZXC21 are therefore highly unusual.
“Such evidence, when considered together, is consistent with a hypothesis that the SARS-CoV-2 genome has an origin based on the use of ZC45/ZXC21 as a backbone and/or template for genetic gain-of-function modifications,” they write.
They add that the high sequence identity between SARS-CoV-2 and ZC45/ZXC21 on various proteins (94-100% identity) do not support the scenario of an ancient recombination event followed by convergent evolution and clearly indicates that SARS-CoV-2 carrying such an RBM cannot come from a ZC45/ZXC21-like bat coronavirus through this convergent evolutionary route.
Yan et al. say that, if a natural recombination event is responsible for the appearance of SARSCoV-2, then the ZC45/ZXC21-like virus and a coronavirus containing a SARS-like RBM would have to recombine in the same cell by swapping the S1/RBM, which is a rare form of recombination.
“Furthermore, since SARS has occurred only once in human history, it would be at least equally rare for nature to produce a virus that resembles SARS in such an intelligent manner – having an RBM that differs from the SARS RBM only at a few non-essential sites.”
The possibility that this unique SARS-like coronavirus would reside in the same cell with the ZC45/ZXC21-like ancestor virus and the two viruses would recombine in the “RBM-swapping” fashion is extremely low, the researchers say. Also, such a recombination event would have to happen to produce a spike as seen in SARS-CoV-2.
Yan et al. say that, judging from the evidence that they and others have gathered, there should be an independent audit of the WIV P4 laboratories and the laboratories of close collaborators of the WIV researchers.
“Such an investigation should have taken place long ago and should not be delayed any further,” Yan et al. say in their report.
“We also note that in the publication of the chimeric virus SHC015-MA15 in 2015, the attribution of funding of Zhengli Shi by the NIAID was initially left out. It was reinstated in the publication in 2016 in a corrigendum, perhaps after the meeting in January 2016 to reinstate NIH funding for gain-of-function research on viruses.”
This, the four scientists say, is unusual scientific behaviour, which needs explaining.
The researchers also say a critical look should be taken into certain recently published data, “which, albeit problematic, was used to support and claim a natural origin of SARS-CoV-2”.
Yan, Kang, Guan, and Hu published a second report on October 8 in which they say SARS-CoV-2 is an “unrestricted bioweapon” and there has been “large-scale, organised scientific fraud”.
They allege that records indicate that “the unleashing of this weaponised pathogen should have been intentional rather than accidental”.
The current pandemic, they say, is “a result of unrestricted biowarfare”.
In the new paper, published on the preprint website Zenodo, Yan, Kang, Guan, and Hu write: “The fact that data fabrications were used to cover up the true origin of SARS-CoV-2 further implicates that the laboratory modification here is beyond simple gain-of-function research.
“The scale and the coordinated nature of this scientific fraud signifies the degree of corruption in the fields of academic research and public health.
“As a result of such corruption, damages have been made both to the reputation of the scientific community and to the well-being of the global community.”
Yan et al. say that, while SARS-CoV-2 meets the criteria of a bioweapon specified by the People’s Liberation Army (PLA) in China, “its impact is well beyond what is conceived for a typical bioweapon”.
In their new paper, Yan et al. write about the novel animal coronaviruses they say were reported by researchers in laboratories in China after the start of the outbreak of SARS-CoV-2.
“While no coronaviruses reported prior to 2020 share more than 90% sequence identity with SARS-CoV-2, these recently published, novel animal coronaviruses (the RaTG13 bat coronavirus, a series of pangolin coronaviruses, and the RmYN02 bat coronavirus) all share over 90% sequence identities with SARS-CoV-2,” they write.
“As a result, these SARS-CoV-2-like viruses have filled an evolutionary gap and served as the founding evidence for the theory that SARS-CoV-2 has a natural origin.
“In this report, we provide genetic and other analyses, which, when combined with recent findings, prove that these novel animal coronaviruses do not exist in nature and their genomic sequences are results of fabrication.”
Yan et al. say the RaTG13 virus has served as the founding evidence for the theory that SARS-CoV-2 must have a natural origin, but no live virus or intact genome of RaTG13 has ever been isolated or recovered.
“Therefore, the only proof for the ‘existence’ of RaTG13 in nature is its genomic sequence published on GenBank,” they write.
Yan et al. refer to an article by Yong-Zhen Zhang et al., published in Nature magazine on February 3, in which they make no mention of RaTG13 and show that, evolutionarily, SARS-CoV-2 is closest to two bat coronaviruses, ZC45 and ZXC21.
Both of these viruses “were discovered and characterised by military research laboratories under the control of the CCP government,” Yan et al. say.
“Immediately after the publication of this article, Dr Zhang’s laboratory was shut down by the CCP government with no explanations offered,” they write.
In order to have the sequence of a viral genome successfully uploaded onto GenBank, submitters have to provide both the assembled genomic sequence (text only) and raw sequencing reads.
“However, due to the huge amount of work involved in assembling raw reads into complete genomes, no sufficient curation is in place to ensure the correctness or truthfulness of the uploaded viral genomes,” Yan et al. write.
Therefore, Yan et al. say, an entry on GenBank is not definitive proof that the viral genome is correct or real.
“Clearly, a viral genomic sequence and its GenBank entry can be fabricated if well-planned.”
The RaTG13 virus and its published sequence are suspicious and show signs of fabrication, Yan et al. say.
“The evidence presented both here and from recent literature collectively prove that RaTG13 does not exist in nature and its sequence has been fabricated,” they add.
“If the RaBtCov/4991 virus is equivalent to RaTG13, then RaBtCoV/4991 must be fraudulent as well.”
Yan et al. suggest that the fabrication of RaTG13 was planned and executed in coordination with the laboratory creation of SARS-CoV-2.
The four researchers say in their new report that the complete genomic sequence of RaTG13 was first submitted to GenBank on January 27, 2020, and the raw sequencing reads were made available on February 13.
“However, the sequencing data for gap filling, which is indispensable in assembling a complete genome, was only made available on May 19th, 2020 … The timing and the reversed order of events here are strange and suspicious.”
According to Yan et al., the raw sequencing reads of RaTG13 have multiple abnormal features.
“No independent verification of the RaTG13 sequence seems possible because, according to Dr Zhengli Shi, the raw sample has been exhausted and no live virus was ever isolated or recovered,” they add.
“However, judging from Shi’s published protocol, exhaustion of the faecal swab sample is highly
Yan et al. write: “Intriguingly, despite the pivotal role of RaTG13 in revealing the origin of SARS-CoV-2, the information provided for its discovery was surprisingly scarce with key points missing (location and date of sample collection, previous knowledge and publication of this virus, etc).”
They added: “Only in the source section of the NCBI entry for RaTG13 (GenBank accession code: MN996532.1), one could find that the original sample was a ‘faecal swab’ collected on ‘July 24th, 2013’.”
According to Yan et al., there are discrepancies between what Shi Zhengli suggested in the article published in Nature in January 2020 (that the sequencing of the full genome of RaBtCoV/4991 was not done until 2020) and what Zhengli said later in emailed replies to Science magazine.
Yan et al. say that, in June 2020, file names of the raw sequencing reads for RaTG13 uploaded were found, which indicated that the sequencing experiments were done in 2017 and 2018.
“Likely responding to this revelation, in her email interview with Science, Dr Shi contradicted her own description in the Nature publication and admitted that the sequencing of the full genome of RaTG13 was done in 2018.”
The researchers say no follow-up work on RaTG13 has been reported by Zhengli and her colleagues.
“Upon obtaining the genomic sequence of a SARS-like bat coronavirus, the Shi group routinely investigate whether or not the virus is capable of infecting human cells. This pattern of research activities has been shown repeatedly.
“However, such a pattern is not seen here despite that RaTG13 has an interesting RBM and is allegedly the closest match evolutionarily to SARS-CoV-2.”
There were, Yan et al. say, deviations from normal research activities and logical thinking that are difficult to reconcile or explain.
Yan et al. say investigations should be carried out “on the suspected government and individuals and the responsible ones be held accountable for this brutal attack on the global community”.
Yan has told television host in the US Tucker Carlson that her 63-year-old mother has been detained in China.
Alina Chan, who is a postdoctoral fellow at the Broad Institute of MIT and Harvard in the US, has provided a lengthy critique on Twitter of Yan et al.’s first report. Chan is herself the focus of a major article published earlier this month in Boston magazine entitled ‘Could COVID-19 Have Escaped from a Lab?’
Chan, (pictured left) who describes herself as a “human frontier science programme fellow with 12+ years of research training in medical genetics, biochemistry, synthetic biology, and vector engineering” describes the claims that RaTG13 is fabricated and that SARS-CoV-2 is derived from the Zhoushan viruses as “gut speculations” by Yan.
My position on this: I don’t know. Someone should be looking into the origins of RaTG13 and GD pangolin CoV. What was the sampling+sequencing process? Are there still ways to verify these samples (the former has disintegrated)? Were similar viruses discovered but not reported?
— Alina Chan (@Ayjchan) September 17, 2020
Chan added: “What I do know: claiming SARS2 was derived from the Zhoushan viruses that are >3000 mutations different – this has destroyed the report’s credibility, and, more importantly, diverted attention away from RaTG13, miners, and the missing WIV virus database …”
Referring to Claim 2 made by Yan et al. that the receptor-binding motif of the SARS-CoV-2 spike was genetically manipulated, Chan says that this claim relies on Claim 1 (that SARS-VoV-2 is similar to the Zhoushan viruses isolated and studied in Chinese military labs) being true. This, Chan says, is a serious weakness.
“Instead of just going with ‘RBD was copied from another virus’, Yan et al. perform enzymatic gymnastics to figure out how it could have gotten into the Zhoushan virus,” Chan tweeted.
Chan (pictured left) says that what Claim 2 “kind of gets correct” is that laboratories, including the WIV, have been codon optimising spikes and swapping in RBMs to study receptor binding for more than a decade.
She says that “this fixation on cloning sites is irrelevant to determining whether SARS2 was ever manipulated in a lab”.
Chan tweeted: “Scientists have been able to clone coronavirus genomes seamlessly for years. They introduce cloning sites to show you that a genome has been manipulated. Another reason to retain a cloning site could be to monitor a feature, e.g. FCS [furin cleavage site], that tends to be lost during cell passage.”
Referring to the claim by Yan et al. about the S1/S2 FCS being artificially inserted, Chan tweeted that “the fixation on whether there is a cloning site surrounding the FCS is unhelpful”.
Chan says that Yan et al. refused to incorporate any of the newly published SARS2-like viruses in their analysis.
“This is a major weakness of the report that has been pointed out by numerous experts. I would argue that at least the 4991 RdRp [RNA-dependent RNA polymerase] fragment should be considered,” Chan tweeted.
Chan says the ripple effect of Yan et al.’s report “has done more to discredit the lab origins hypothesis than all of the peer-reviewed natural origins papers combined”.
[UPDATE] In an extremely detailed preprint published in September, four Indian molecular biologists say that there are inadequate grounds to consider that RaTG13 is the “ancestral pool of SARS-CoV-2”.
Mohit Singla from the All India Institute of Medical Sciences, New Delhi, and Saad Ahmad, Chandan Gupta, and Tavpritesh Sethi from the Indraprastha Institute of Information Technology Delhi say that “the currently specified level of details are grossly insufficient to draw inferences about the origin of SARS-CoV-2”.
They write: “We analysed the sequence and found that the data quality issues along with the lack of sufficient experimental details preclude reliable inference of the origins of SARS-CoV-2.”
Singla et al. say there is strong evidence pointing to DNA contamination in the sample “that may preclude inferences”.
They write: “We conjecture that the sample has gross contamination issues while handling, with RNA and DNA mixed in the same sample. However, in the absence of details about the experiments we do not rule out any steps that we may have missed, in which case the original studies should make these known.”
The four scientists say there is a need for a dispassionate review of the data relating to the RaTG13 genome.
They say that the authors reporting the existence of RaTG13 need to expeditiously detail the experimental procedure adopted.
They also call upon the scientific community to refrain from citing the genome of RaTG13 until the entirety of the genome is established as being fully supported by the data by independent reviews.
“This work is a call to action for the scientific community to better collate scientific evidence about the origins of SARS-CoV-2 so that future incidence of such pandemics may be effectively mitigated,” Singla et al. add.
The editors of the journal Rapid Reviews: COVID-19, which is produced at the University of California in Berkeley in the US, published four reviews of Yan et al.’s first paper.
Robert Gallo, who co-founded and directs the Institute of Human Virology at the University of Maryland School of Medicine and is best known for co-discovering the human immunodeficiency virus (HIV) as the infectious agent responsible for acquired immune deficiency syndrome (AIDS), notes that the “military virus” Yan et al. write about as the predecessor of SARS-CoV-2 “is over 3,000 nucleotides different from SARS-2”. This, Gallo says, “is a long way off”.
Gallo says that Yan et al.’s assertion that the RBD of ZC45 is “suspiciously close to SARS-1” is untrue. “The SARS-CoV-2 RBD sequence is nearly 100% homologous with that of the pangolin sequence,” Gallo says.
He also challenges Yan et al.’s assertion that the furin cleavage sites do not occur in “other viruses of this class”.
Gallo writes: “By class, she means SARS-like. This is not true, as such sites are present in some coronaviruses and are subject to the whims of Mother Nature’s evolutionary bent. In fact, MERS has two such sites and a chicken coronavirus also has two.”
Adam Lauring, who is an assistant professor in the departments of internal medicine, microbiology and immunology, and ecology and evolutionary biology at the University of Michigan, says Yan et al.’s paper is “not scientific” and reads like an opinion piece.
“The authors tie various threads together into a story,” Lauring writes. “However, they are only able to do this by focusing on the most sinister interpretation of just a subset of evidence.
“Much data is ignored or discounted. Perhaps most important, the argument or hypothesis made by the authors is neither provable nor falsifiable.”
Lauring writes: “The authors speculate – without evidence – that SARS-CoV-2 was engineered from the backbone of ZC45 or ZXC21 bat coronaviruses. This appears to be convenient for their argument, as these viruses have some linkage, per the authors, to the Chinese government or military.”
Using ZC45 or ZXC21 as starting points for genetic engineering makes little sense, Lauring says. “These viruses differ from SARS-CoV-2 at approximately 10% of the positions in the genome. If someone were to engineer a virus like SARS-CoV-2, they would start with a more closely related virus. It is much simpler.”
Lauring also says that attaching significance to a restriction enzyme site near the receptor binding domain “does not make a lot of scientific sense”.
“This site is a 6 nucleotide recognition sequence and would occur by chance once every 4096 bases in a genome sequence. In SARS-CoV-2, which is approximately 30,000 bases, one would expect to find this particular sequence 7-8 times by chance.”
The existence of a furin-like cleavage site is, in and of itself, not evidence for an engineered origin for SARS-CoV-2, Lauring says. “It should be noted that many viruses have these cleavage sites.”
Marvin Reitz, who is an adjunct professor at the University of Maryland’s Institute of Human Virology, says Yan et al.’s report is “composed entirely of opinion and innuendo”. He says Yan et al. are incorrect in stating that the natural origin theory lacks substantial support.
Reitz says that Yan et al.’s claim that SARS-CoV-2 shows biological characteristics that are inconsistent with a naturally occurring, zoonotic virus is dubious.
Yan postulates a synthetic route that is “partly doable but tedious, and is otherwise logically impossible”, Reitz says.
Takahiko Koyama, who is a lead scientist at the Thomas J. Watson Research Centre in Yorktown Heights, New York, says there is no concrete evidence to support Yan et al.’s claim that the bat coronavirus RmYN02 is likely to be fraudulent.
Koyama says Yan et al.’s manuscript “does not demonstrate sufficient scientific evidences to support genetic manipulation origin of SARS-CoV-2”. [ENDS UPDATE]
The second report by Yan et al. is “littered with errors”, Alina Chan says. She tweeted: “… I do wonder why there hasn’t been international impetus to investigate the source of these SARS2-like viruses. Why not go to the Yunnan mine to look for more RaTG13s? Why not investigate the miners – what actually happened in 2012?”
She added: “I’m glad Yan is shining the spotlight on these research integrity issues. But I worry that framing the verifiable misdirection surrounding RaTG13/pangolins within a specious article may actually hurt the legitimacy of research integrity inquiries regarding SARS2-like viruses.”
Chan tweeted: “Yan et al. claim because a cover-up was ‘orchestrated by the CCP’ … ‘the unleashing of the virus must be a planned execution rather than an accident’. I disagree. Just because something is accidental, doesn’t mean it has no consequences & will be admitted openly.”
Yan, Chan says, “goes too far by asserting that SARS-CoV-2 is an ‘unrestricted bioweapon'”.
I urge a shift away from bioweapons speculation back onto solid ground: the research integrity concerns surrounding some of the closest viruses to SARS2.
These are discussed in Yan’s report, but completely overshadowed by claims about SARS2 being an “unrestricted bioweapon”.
— Alina Chan (@Ayjchan) October 11, 2020
Chan recently tweeted about the Joint Hearing: “Strengthening Biological Security: Traditional Threats and Emerging Challenges”, held on October 2 and organised by the House Committee on Armed Services in the US.
The hearing involved the Subcommittee on Intelligence and Emerging Threats and Capabilities and the House Foreign Affairs Committee’s Subcommittee on Asia, the Pacific, and Nonproliferation.
Congressman Brad Sherman asked about the US administration’s “operating assumptions”, the “percentage likelihood” it would accord to the four possible origins of the Covid-19 pandemic.
Sherman cited the Huanan wet market and three possibilities involving the Wuhan lab. “It may have come from the Wuhan lab, which might have been engaged in entirely peaceful activities and had a tragic release; it could have come from a Wuhan lab that was engaged in military activities, but had an unintentional release; and I think least likely … it could have come deliberately from a Wuhan lab,” Sherman said.
The response to Sherman was that SARS-CoV-2 is a “virus of zoonotic origin” and had been “identified as existing in animals”.
Chan tweeted: “The answer was noncommittal but it looks like leaders are still asking: where did SARS-CoV-2 come from? Nature, accident, or deliberate?”
The Mojiang mine
In a preliminary abstract published on September 17 in Frontiers in Public Health, Indian researchers Monali C. Rahalkar (pictured left) and Rahul A. Bahulikar say that RaTG13 (RaBtCoV/4991) was collected from the Tongguan mineshaft in Mojiang, Yunnan, in 2013.
“Surprisingly, the same mineshaft was also associated with a severe pneumonia-like illness in miners in 2012 killing three of the six miners,” Rahalkar and Bahulikar write.
“A Master’s thesis (in the Chinese language) was found on the cnki.net website which described in detail the severe illness in miners. The thesis concluded that a SARS-like CoV originating from Chinese horseshoe bats (Rhinolophus) was the predicted causative agent.”
The thesis was written by the Chinese doctor, Li Xu, who treated the miners and sent their tissue samples to the WIV for testing, and was published in May 2013.
Rahalkar and Bahulikar explain that the cases were remotely monitored by a prominent pulmonologist in China.
“The retrospective analysis of the pneumonia cases shows striking similarities with Covid-19. Bilateral pneumonia, vascular complications like pulmonary thromboembolism, and secondary infections are the main similarities,” Rahalkar and Bahulikar write.
‘The treatment regimes were similar to the currently given treatment for Covid-19. We propose that the Mojiang mineshaft and the miners’ illness cases could provide important clues to the investigation of the origin of SARS-CoV-2.”
[UPDATE] In their full, peer-reviewed article, published in Frontiers in Public Health on October 20, Rahalkar and Bahulikar write that the striking similarities between the Mojiang pneumonia cases and Covid-19 are noteworthy, “as is the fact that RaTG13/CoV4991, the next genomic relative of SARS-CoV-2 was found in the same mineshaft”.
The two researchers write: “Although we cannot say that RaTG13 or SARS-CoV-2 infected the miners, there is a high chance that it could be a virus quite similar in genetic composition to these two.
“The coincidence between the 2012 illness in Mojiang miners, the subsequent samplings, and finding the nearest SARS-CoV-2 relative from this single mine warrants further inquiry, and the data along with the full history of this incident would be invaluable in the context of the current pandemic.”
Rahalkar and Bahulikar go into detail about Li Xu’s Master’s thesis, in which he concluded that “the pneumonia cases were due to viral pneumonia, primarily from SARS-like coronaviruses originating from horseshoe bats”.
They write: “According to the Master’s thesis, in April 2012, six miners were given a job of clearing bat waste and bat faeces from a copper mineshaft in Tongguan, Mojiang, Yunnan. After working for ~14 days in the case of four miners, and 4–5 days in the case of the last two miners, they started facing breathing problems, cough, and fever which required immediate admission to the Kunming hospital in late April and early May.
“Three of the miners died in the course of ~100 days and three survived. The thesis featured medical reports, radiological images such as CT scans, and detailed information regarding the diagnosis and treatment of the miners.”
Radiography showed interstitial pneumonia, ground-glass opacities, and severe acute respiratory distress syndrome (ARDS) in some of the patients and some showed clotting complications such as pulmonary thromboembolism or thrombosis and elevated D-dimer values.
Dr Zhong Nanshan, an expert in respiratory diseases and a national advisor about the SARS and Covid-19 epidemics, provided remote consultation for the two patients who had the most serious illness.
Nanshan’s conclusion that the Mojiang miners’ pneumonia appeared to be primarily viral and that it was most probably due to bat-related coronaviruses, is noteworthy, Rahalkar and Bahulikar say.
“Based on the detailed evidence presented in the Master’s thesis and the PhD thesis and the discussion presented here, we do not think that fungus was the primary reason for the illness,” they add.
“We think that if it was a fungal disease, only antifungals could have cured the illness. Vascular complications such as elevated D-dimer and thromboembolism are not common in fungal disease and have been observed in the miners’ illness and COVID-19. Elevated SAA (serum amyloid A) and declined lymphocytes are indicative of the fact that it was primary viral pneumonia.”
The two researchers add that, according to a translation of a PhD thesis by Canping Huang, supervised by George Gao, the “blood test results of four cases showed that: four people carried SARS virus IgG antibodies, of which two were discharged with higher antibody levels … and two which were hospitalised had lower antibody levels …”.
Rahalkar and Bahulikar pose numerous questions: “We are curious to know what kind of samples the WIV received from the Mojiang miners, along with other questions, such as whether the samples are still stored in WIV, and whether they are available for study by other researchers.
“It would also be of particular value to know whether any viruses were isolated and if there is any DNA/RNA available from these samples. It would also be useful to know if PCR was performed on the miners’ samples and available sequences.”
Other questions include ‘Why were the severe pneumonia cases in 2012 not mentioned in any of the WIV publications before 2020?’ and ‘Were any SARS-like CoV isolated from the bat faecal samples collected in 2012–13?’.
Rahalkar and Bahulikar also ask why the Mojiang miners pneumonia cases in 2012 were not reported to any public health agency such as the WHO and why programmes like PREDICT didn’t mention the lethal pneumonia cases as a mini-outbreak.
“Why was the Mojiang mine being visited by researchers until October 2014?,” the researchers ask. “Questions also remain as to why Dr Shi attributed the outbreak in Mojiang to a fungus in the interview with Scientific American. Was the mine open for researchers and were any samples brought after 2014?
“Did any of the researchers who visited the Mojiang mineshaft get infected by any coronavirus between 2012 and 2019? Are there any whole genome sequences available for SARS-like CoV originating from this mine?” [ENDS UPDATE]
A member of the DRASTIC team, who tweets under the handle @TheSeeker268, says that, in July 2012, a few months after the pneumonia outbreak among the miners in Mojiang, there was a disease-control operation in the area that lasted for six months.
“Oddly enough, the atypical pneumonia cases among the miners didn’t make it to the official CDC statistics for 2012, which definitely suggests a cover-up to me,” @TheSeeker268 tweeted.
@TheSeeker268 also tweeted about the case of a Thai tourist who was visiting Yunnan in 2013 and died of multiple organ failure caused by “unexplained pneumonia”.
Around the same time, China’s Ministry of Science & Technology initiated a project (2013FY113500) to identify and investigate viral pathogens and their relation with major infectious diseases, @TheSeeker268 also tweeted.
The project was initiated in May 2013, just two months before Shi Zhengli sampled RaBtCoV/4991, @TheSeeker268 noted. The first project meeting took place on May 31 in Wuhan.
“To add to this mystery, the viral database for Project 2013FY113500 has been taken down,” @TheSeeker268 also notes.
In a paper entitled ‘Proposed SARS-CoV-2 Spillover During 2019 Review of Samples from a Mineshaft in Mojiang, Yunnan Province, China’, which was published on Zenodo on September 14, a researcher who prefers to remain anonymous, but who credits the members of DRASTIC, reports on activity at the WIV in late 2019, when samples from the Mojiang mineshaft were being reviewed.
‘Anon’ poses the question ‘Did a Review of Samples Collected from a Mineshaft Cause the Covid-19 Pandemic?’ and proposes that spillover occurred during the review of samples of the RaBtCoV/4991 virus, which, ‘Anon” notes, is only 1% different to SARS-CoV-2 in its RdRp.
There is evidence in documents from China’s Ministry of Science and Technology that WIV staff were handling samples and specimens containing BtCoV/4991 (RaTG13) and related viruses around the time of the SARS-CoV-2 outbreak, ‘Anon’ says.
“Several Wuhan laboratories conducted research into SARS or SARS-related coronaviruses in the years prior to the pandemic,” ‘Anon’ notes. “These include facilities at Huazhong University, the Wuhan Centre for Disease Control, the Wuhan Institute of Virology, and Wuhan University (WU).
“This research focuses on a programme connecting these institutions and presents information supporting a potential spillover event due to mishandling of a sample or specimen stored at the WIV in late 2019.
“This is proposed to have taken place during a well-documented review of samples and specimens collected under the multiyear programme that identified the closest known virus to SARS-CoV-2.”
‘Anon’ says that the WIV filed its only patent for a device to protect against accidental virus transmission in a biosafety laboratory on November 15, 2019. This, ‘Anon’ says, shows that accidental transmission was a concern at the time of the SARS-CoV-2 outbreak.
The WIV is not the only research institute with safety issues, ‘Anon’ points out. Researchers at WU also worked on the programme that identified RaTG13 and the university operates its own Animal Biological Safety Level 3 (ABSL-3) facility.
WU facilities were being inspected in late 2019, ‘Anon’ says. This was to check that problems identified earlier had been rectified.
“These problems included: hazardous waste being exposed; no separation of the experiment area; students not wearing lab coats; no eyewash; a crowded experiment area clutter,” ‘Anon’ writes.
“The facts that WU staff worked on the same programme that identified RaTG13, and WU had such a poor track record of lab safety adds to the plausibility of a WIV staff member on the programme mishandling a sample or specimen.”
WIV and WU staff often work together, and move between the institutions.
‘Anon’ notes that much initial focus was on the wild animal trade at the Huanan seafood market in Wuhan, where the first reported cases of infection by SARS-CoV-2 were publicly confirmed.
“However, the earliest publicly reported case using unclassified data had no exposure to the Huanan seafood market and developed symptoms on 1 December 2019, nine days before the first patient connected to the market developed symptoms,” ‘Anon’ writes.
“The virus strains sampled in the market were shown to be already adapted to human transmission, indicating that the virus had jumped species earlier.”
‘Anon’ notes that the theory that the pangolin is the animal source of SARS-CoV-2 relies on data made public by “the State Key Laboratory of Pathogen and Biosecurity, under the Beijing Institute of Microbiology and Epidemiology, under the People’s Liberation Army’s Academy of Military Sciences”, on 22 January 2020.
“This lab was the single source of pangolin data used in multiple research papers, without this single source being disclosed,” ‘Anon’ writes.
“While the data from this lab was reportedly from smuggled pangolins, no evidence has been found of coronaviruses in Sunda pangolins entering the wildlife trade via Malaysia.
“Additionally, as the pangolin ACE2 receptor has a low binding affinity for the SARS-CoV-2 receptor- binding domain (RBD), it appears unlikely to be the intermediary host.”
According to ‘Anon’, there is evidence suggesting that SARS-CoV-2 was circulating in Wuhan months before the outbreak was reported publicly. This includes reports that athletes competing in the Wuhan Military Games in October 2019 fell ill after arriving in the city. Six of the athletes are reported to have later tested positive for SARS-CoV-2 antibodies.
‘Anon’ suggests that a change in guidelines about influenza in November 2019 may have increased the chance of misdiagnosis of Covid-19 cases as influenza and this could partly explain how SARS-CoV-2 may have spread undetected before December 2019. From November 2019, virus specimen isolation was no longer recommended in the case of patients with respiratory disease who tested negative for influenza.
“The Covid-19 outbreak was made public by the isolation of virus specimens from patients testing negative for influenza, against the recommendation of the 2019 influenza guidance. The results of this specimen isolation were shared with Dr Li Wenliang, who shared the results with others who shared them with the world.”
‘Anon’ notes that, under the 2013FY113500 programme (‘Investigation of viral pathogens of major natural hosts and vector insects in China’), researchers at the WIV investigated the main natural virus hosts and vectors in China, taking samples from bats, birds, mosquitoes, rodents, and ticks.
“The WIV had collected over 15,000 such samples from bats, over 1,400 live viruses and over 60,000 strains,” ‘Anon writes. “Data from over 20,000 samples and specimens collected on such trips were stored on an WIV database, and the samples themselves were stored at -80°C.
“Research into ACE2 receptors and spike proteins of SARS-related coronaviruses and vaccines was funded under the programme. SARS vaccine research had been carried out at Wuhan University and other institutions.”
‘Anon’ notes that the WIV collected samples containing RaBtCoV/4991 under the 2013FY113500 programme. “The partial BtCoV/4991 sequence published in 2016 is a 98.9% match to SARS-CoV-2. The complete RaTG13 genome published by the WIV after the Covid-19 outbreak is a 96.1% match to SARS-CoV-2.”
It has been reported in a comment in a PhD thesis that four of the six miners who were tested after becoming ill were shown to have SARS antibodies.
“These cases were not reported in China’s unknown pneumonia statistics, despite the PhD thesis being supervised by now head of China’s CDC George Gao, and samples being sent to SARS expert Zhong Nanshan’s laboratory,” ‘Anon’ writes.
“The 2005 international health regulations state that the WHO should be notified of cases matching the clinical definition of SARS.”
‘Anon’ highlights the inconsistencies in statements made about RaBtCoV/4991 (RaTG13).
“The sequence of events described by Shi Zhengli and colleagues in a 2020 paper implied that RaTG13 was sequenced after the WIV found that SARS-CoV-2 matched the short BtCoV/4991 RdRp.”
This, ‘Anon’ notes, was corroborated by the president of the US-based EcoHealth Alliance, Peter Daszak, who said that the Wuhan team had worked on the sample in 2013, but did no more on it until the Covid-19 outbreak because it had not been a close match to SARS. Daszak said the sample was just put in the freezer.
“This is contradicted by the 2017–18 dates present in the filenames of the RaTG13 amplicon and swab sequences,” ‘Anon’ writes. “Shi Zhengli later issued a statement saying that the WIV fully sequenced RaTG13 in 2018.
“RaTG13 was initially uploaded accompanied by a statement saying that it had been extracted from bronchoalveolar lavage fluid, which is inconsistent with it being a bat faecal swab sample.”
It is stated in Li Xu’s thesis that the Kunming Institute of Zoology confirmed that a Rhinolophus sinicus bat was the source of the mine workers’ virus, ‘Anon’ points out. “It was clear to the zoology institute that the virus came from a Rhinolophus bat, known as a reservoir of SARS-like viruses, even before the WIV sampled the only SARS-like virus they reported finding in the mineshaft.”
The hypothesis presented by Jonathan Latham and Allison Wilson that RaTG13 (RaBtCoV/4991) caused the death of the Yunnan miners has been disputed by a group of Chinese scientists.
In presenting their hypothesis, Latham and Wilson rely heavily on a translation of Li Xu’s thesis.
Latham and Wilson suggest that RaTG13, or a very similar virus, evolved into SARS-CoV-2 inside the bodies of the miners and that researchers at Zhengli’s lab used medical samples taken from the miners and sent to them from Kunming University Hospital.
“It was this human-adapted virus, now known as SARS-CoV-2, that escaped from the WIV in 2019,” Latham and Wilson assert. “We refer to this Covid-19 origin hypothesis as the Mojiang Miners Passage (MMP) hypothesis.”
Passaging is a standard virological technique for adapting viruses to new species, tissues, or cell types.
“It is normally done by deliberately infecting a new host species or a new host cell type with a high dose of virus,” Latham and Wilson explain. “This initial viral infection would ordinarily die out because the host’s immune system vanquishes the ill-adapted virus. But, in passaging, before it does die out, a sample is extracted and transferred to a new identical tissue, where viral infection restarts.”
Li-Meng Yan said during a video conversation in March with the founder of Lude Media, Wang DingGang, that there had been no successful biopsy or autopsy examinations in the case of the six miners, which, Yan says, means that the whole virus that infected them could not be isolated and sequencing could not be done to identify it.
Yan’s colleague Shu Kang says it’s not possible for the 4991 bat coronavirus to be SARS-CoV-2.
“If it was exactly the same virus it would have caused a pandemic, or even an epidemic. There’s no way they could limit the infection to very local small cluster,” Kang said during the video conversation.
“Even if they say that 4991 evolved into SARS-CoV-2 slowly in the human population it’s also not possible because you would have then detected such viruses in the population previously. However, we all know that, before late 2019, there were no cases.”
Jie Guan said in the same video conversation that there was insufficient evidence that four of the miners tested positive for SARS antibodies. He also said that, in the case of RaBtCoV/4991, there was only 440-bp sequencing, which, Guan says, is sequencing only the equivalent of 1.5% of the coronavirus genome. (Bp is a reference to the number of base pairs sequenced from a DNA fragment.)
Guan says that, in Zhengli’s original paper about RaTG13, she doesn’t mention the source of RaBtCoV/4991 and doesn’t even cite her previous paper about RaBtCoV/4991.
“Suddenly, after all the questioning, she claimed that RaTG13 is basically the same sample as CoV/4991 … This doesn’t make any sense.”
Zhengli is trying to link RaTG13 with the case of the six miners in 2012, Guan says, but there is a lack of evidence, and there are flaws in her logic.
Retired US Army Reserve colonel Lawrence Sellin, who worked at the US Army Medical Research Institute of Infectious Diseases and conducted research in the pharmaceutical industry, writes, in an opinion piece for the WION (World is One News) television channel in India, that there are several weak links in the Latham–Wilson theory that SARS-CoV-2 evolved within one of the miners.
“First of all, such an extensive genetic evolution needed to go from its closest bat coronavirus relative, RaTG13, to SARS-CoV-2, about 1,200 nucleotides, has never been described to occur in a single patient,” Sellin writes.
“Furthermore, the structure of RaTG13’s receptor binding domain, which binds to the human cell, is sufficiently different from that of SARS-CoV-2, making an initial human infection by RaTG13 to begin a human adaptation process highly unlikely.”
Sellin says that, even if bat-to-human transmission had occurred in the miners and high adaptation for human infection was achieved, there is no record of any human-to-human transmission, despite no extraordinary precautions being implemented during the hospitalisation.
The simplest explanation for the Mojiang miners’ illness in April 2012, Sellin says, is histoplasmosis, a respiratory infection caused by the inhalation of fungal spores found in bat and bird droppings.
The United States directly, and through collaboration with the EcoHealth Alliance, has funded Shi Zhengli’s research since at least 2014 and the EcoHealth Alliance has received further SARS-related funding from the US National Institutes of Health (NIH) dating back to 2008.
Zhengli (pictured left) has been referred to as the ‘bat woman’ because of her extensive work in the field of bat virology. She has publicly denied that SARS-CoV-2 originated in her laboratory. In a WeChat post on February 2 she wrote: “… I promise with my life that the virus has nothing to do with the lab.”
The NIH funded the EcoHealth Alliance to surveil and understand the risks of the transmission to humans of SARS-related coronaviruses. That funding has now been withdrawn.
The director of the NIH’s National Institute of Allergy and Infectious Diseases (NIAID), Anthony Fauci, said the NIH cut off funding for the collaboration between the EcoHealth Alliance, which is based in New York, and researchers in Wuhan after president Donald Trump told it to.
The EcoHealth Alliance is also sponsored by the United States Agency for International Development (USAID), and NIAID. Its president is a British zoologist and an expert on disease ecology and zoonosis in particular who is on the board of the World Health Organisation (WHO).
While researchers from the EcoHealth Alliance, working with scientists from the WIV, identified a sequence of SARS-related coronaviruses found in bats that was 96.11% similar to SARS-CoV-2 in July 2013, the sequence was not published on the NIH website until March this year.
Andre Watson, who is the founder and CEO of the regenerative medicine and pandemic defence biotechnology company Ligandal, which is based in San Francisco, says that a number of other coronaviruses have more than 60% sequence similarity to SARS-CoV-2 and they include viruses that cause neurological, vascular, gastric, and respiratory symptoms. Some of them have been studied for decades, “since the 1970s and earlier”, Watson says.
SARS from 2003 was only 80% similar to SARS-CoV-2 and MERS from 2012 was only 64.3% similar to SARS-CoV-2.
In 2017, the EcoHealth Alliance collaborated with the WIV and the Duke-NUS Medical School in Singapore on research that was funded by the NIH, NIAID, the USAID Emerging Pandemic Threats programme (under the umbrella of the PREDICT project), and by funding sources in China.
The scientists published a paper about research done in bat caves. Researchers drew blood from the bats, or took nasal swabs or fecal samples. They identified 11 new viruses that they called SARS R (SARS-related viruses) in bats, Watson notes. Of these 11 viruses, three of them had spike proteins that had a high affinity for human ACE2.
The authors said: “In this study, we confirmed the use of human ACE2 as receptor of two novel SARSr-CoVs by using chimeric viruses with the WIV1 backbone replaced with the S gene of the newly identified SARSr-CoVs.”
Watson (pictured left) says this means that the researchers admit to doing recombination work with higher-binding ACE2 spike proteins on various SARS-CoV-2-related viral scaffolds.
“Furthermore, overlapping authors published a study in 2015 in which they admitted to discovering ‘SARSr-CoVs’ that had immunological cross-reactivity with SARS-CoV-1 antibodies,” he said.
The 2015 paper is the one Jonathan Latham and Allison Wilson refer to in their June 2 article. The paper’s authors include Zhengli and Baric.
Watson said: “They have still not published the sequences of viruses taken from those infected with the virus.
“The authors directly admit to doing genetic engineering of the viruses, which means that they were taking bits and pieces of one type of virus and mixing it with bits and pieces of other clades and strains of virus.”
Vineet Menachery, Zhengli, Baric et al. said in their article that both wild-type and chimeric viruses were derived from either the epidemic SARS-CoV Urbani strain or the corresponding mouse-adapted (SARS-CoV MA15) infectious clone.
“Plasmids containing spike sequences for SHC014 were extracted by restriction digest and ligated into the E and F plasmid of the MA15 infectious clone,” they wrote.
Menachery et al. go on to explain that plasmids containing wild-type, chimeric SARS-CoV and SHC014-CoV genome fragments were amplified, excised, ligated, and purified.
“In vitro transcription reactions were then preformed to synthesise full-length genomic RNA, which was transfected into Vero E6 cells … The medium from transfected cells was harvested and served as seed stocks for subsequent experiments.”
Synthetic construction of chimeric mutant and full-length SHC014-CoV was approved by the University of North Carolina Institutional Biosafety Committee and the NIH’s Dual Use Research of Concern Committee, the authors said.
The EcoHealth Alliance researchers and their Chinese colleagues took an RNA strand from each of the viruses they identified and converted it to DNA, Watson says. They then put the DNA into a plasmid, where it could replicate.
“When you want to do genetic engineering, you don’t do it on RNA, you do it on DNA,” Watson explained. “A plasmid is stable. You can put it in the fridge or the freezer and put it in some cells and they’ll produce RNA.”
The researchers took spike proteins and combined them with envelope and nucleic acid proteins.
“Basically you’re holding the core of the virus stable, and you’re swapping out the pieces on the outside to get one that binds more strongly to human cells,” Watson said.
“They did not publish any of those sequences, but they admitted that they did that work.
“The nature of studying these viruses in cells is that there are going to be mutation events; evolutions are going to happen – especially if you are optimising the virus for infectivity of ACE2-expressing cells with human ACE2.”
The researchers also admitted to infecting humanised mice that had the human ACE2 receptor, Watson says. “It’s clear that they were doing gain-of-function studies.”
Watson says it is known that SARS-related viruses were infecting people in November 2017, or earlier, in rural China, and that these viruses were subsequently transferred to the WIV.
“The funding trail certainly suggests that the USA has collaborated with China, the WHO, and others and had these projects funded since 2011 or earlier,” he said. “This was a joint, multinational effort.”
Watson added: “In rural China people living near bat caves were catching these ‘SARS-related viruses’ and were getting acute respiratory distress.
“The researchers admitted that they drew the blood of these people, collected the viruses, and studied whether or not those viruses were cross-reactive immunity wise with SARS-Cov-1. They were, but they weren’t SARS-Cov-1.
“Those sequences were never published. So, to this day, they did not show us what the sequences were that were part of that outbreak in 2017.”
The paper trails dating back to November 2017 or earlier show that humans were testing positive for a new SARS-related coronavirus, Watson says.
“The WHO, the United States, and China knew that this had occurred in November 2017, or earlier.
“Circulating bat coronaviruses evolved from SARS-CoV-1 (the 2003 outbreak) were known to have potential for human transmission in 2015, or earlier.”
Watson suspects that, in 2019, a SARS virus escaped from the Wuhan laboratory.
“There’s clear evidence that the first infections were not from people coming from the wet market in Wuhan, but that the virus was brought to the wet market.
“It’s awfully suspicious that Wuhan would be home to the only known biosafety level-4 research facility in China, and they worked on these viruses there.”
In the EcoHealth Alliance grant application for 2017, Daszak said the aim of the project was “to understand what factors increase the risk of the next CoV emerging in people by studying CoV diversity in a critical zoonotic reservoir (bats), at sites of high risk for emergence (wildlife markets) in an emerging disease hotspot (China)”.
He said: “Predictive models of host range (i.e. emergence potential) will be tested experimentally using reverse genetics, pseudovirus and receptor binding assays, and virus infection experiments across a range of cell cultures from different species and humanised mice.”
Writing in Science magazine on April 30, Meredith Wadman and Jon Cohen said that Daszak, with NIH vetting and approval, provided Zhengli with $599,000 out of a total of $3.1 million in grant funding to use lab sequencing techniques to identify bat coronaviruses that were at high risk of jumping to humans.
“The grant has also supported blood testing of people who live near bat caves in southern China to see whether they carried antibodies indicating they had been infected with a bat coronavirus,” Wadman and Cohen reported.
According to Wadman and Cohen, Zhengli and her colleagues collected some 15,000 biological samples in the field from bats. “In January, her team published the genetic sequence of a bat virus that shares 96.2% of its genome with SARS-CoV-2.”
On February 3, Zhengli et al. published an article in Nature entitled ‘A pneumonia outbreak associated with a new coronavirus of probable bat origin’.
The researchers obtained full-length genome sequences from five patients at an early stage of the outbreak in Wuhan.
“The sequences are almost identical and share 79.6% sequence identity to SARS-CoV,” Zhengli et al. said.
The researchers said that a short region of RdRp from BatCoV RaTG13 showed high sequence identity to 2019-nCoV.
“Simplot analysis showed that 2019-nCoV was highly similar throughout the genome to RaTG13, with an overall genome sequence identity of 96.2 percent,” they said.
The SARS-CoV-2 genome and its spike glycoprotein show 96.11% and 92.86% identities to the Rhinolophus affinis bat coronavirus, respectively.
The director of the WIV, Wang Yanyi, told the Beijing-based English-language news channel CGTN, in an interview published on May 25: “Many people might misunderstand that since our institute reported the RaTG13’s genomic similarity to SARS-CoV-2, we must have the RaTG-13 virus in our lab.
“In fact, that’s not the case. When we were sequencing the genes of this bat virus sample, we got the genome sequence of the RaTG13 but we didn’t isolate nor obtain the live virus of RaTG13. Thus, there is no possibility of us leaking RaTG13.”
Asked about speculation that SARS-CoV-2 leaked from the WIV, Wang Yanyi said: “This is pure fabrication. Our institute first received the clinical sample of the unknown pneumonia on December 30 last year. After we checked the pathogen within the sample, we found it contained a new coronavirus, which is now called SARS-CoV-2.
“We didn’t have any knowledge before that, nor had we ever encountered, researched or kept the virus. In fact, like everyone else, we didn’t even know the virus existed. How could it have leaked from our lab when we never had it?”
Watson says that, throughout the progression of the SARS-CoV-2 pandemic, there have been consistent efforts by officials and the mainstream media to repress factual information about the severity, transmission dynamics, origins, and physiological effects of SARS-CoV-2 and Covid-19.
“On January 21, President Xi Jinping asked the director-general of the WHO, Dr Tedros Adhanom, to withhold information about person-to-person transmission of the virus, as well as pandemic classification. Likely as a consequence, pandemic classification of the virus was delayed four to six weeks.”
Luc Montagnier, who was joint winner of the 2008 Nobel Prize for discovery of the human immunodeficiency virus (HIV), has caused controversy with his assertion that some nucleotide sequences of HIV-1 have been found in the SARS-CoV-2 genome. Other scientists have challenged Montagnier’s assertion, saying that each of these sequences also appears in other viruses.
Montagnier (pictured left) also puts forward the theory that SARS-CoV-2 came from the Wuhan laboratory, escaping in an “industrial accident” when Chinese scientists were attempting to develop a vaccine against HIV.
Montagnier says that the sequences he asserts are HIV inserts must have been added to SARS-CoV-2 and that this could not have happened naturally. It is meticulous, professional work, Montagnier says.
Detractors say that a tiny bit of the SARS-CoV-2 genome is about 85% similar to part of the HIV-1 genome, but that the sequence can be found in other viruses.
A paper by Indian scientists who said there were four unique inserts in the 2019-nCoV (SARS-CoV-2) spike glycoprotein that were not present in any other coronavirus reported to date was retracted by the authors.
The paper had been published as a preprint on bioRxiv on January 31. It was stated on bioRxiv that the authors intended to revise the paper “in response to comments received from the research community on their technical approach and their interpretation of the results”.
Prashant Pradhan et al. had said the inserts were either identical or similar to the motifs in the highly variable (V) regions (V1, V4 and V5) in the envelope glycoprotein or in the Gag protein of some unique HIV-1 strains from three different countries (Thailand, Kenya and India).
They speculated that these motif insertions sharing similarity with HIV-1 proteins could provide an enhanced affinity towards host cell receptors and increase the range of host cells of 2019-nCoV. The study implied that 2019-nCoV might be generated by gaining gene fragments from the HIV-1 genome.
In a paper published on February 14 on PubMed Central (PMC), Chuan Xiao et al. discuss the Indian scientists’ assertions and their own examination of the sequences of 2019-nCoV, other CoV viruses and HIV-1 as well as the GenBank database.
Chuan Xiao et al. say their results demonstrate no evidence that the sequences of the four inserts are HIV-1 specific or that 2019-nCoV obtained these insertions from HIV-1.
“First, the results of blast search of these motifs against GenBank shows that the top 100 identical or highly homologous hits are all from host genes of mammalian, insects, bacterial and others,” Chuan Xiao et al. said.
“There are only a few hits on coronaviruses, but none of them are HIV-1 related.”
Chuan Xiao et al. say the insertion sequences in question exist widely in all kinds of viruses.
“While the 100% match between the insertion 1 and 2 sequences and the HIV sequences were found in 19 entries, the matches between the insertion 3 and 4 sequences and HIV-1 sequences were rather poor (from 42% to 88%).
“Sequences that completely match the insertion 3 and 4 sequences were not found in any HIV-1 sequences. This clearly shows that these insertion sequences are widely present in living organisms including viruses, but not HIV-1 specific.”
Evidence of possible manipulation
Andre Watson explains that there is a way that small sequences of HIV may have been inserted into SARS-CoV-2 that would have made the manipulation less noticeable. There could, he says, have been a forensic cover-up.
Watson says it’s very odd that all four of the inserts in question have been found on the surface of the SARS-CoV-2 spike protein.
“One thing that has made this forensically difficult to investigate is that the genomic sequences don’t match. If you line up the RNA letters in those inserts with HIV, they don’t match.
“However, there is something called sense mutation. If I were trying to put a HIV sequence into something, and my job was to cover it up, I wouldn’t use the same genetic sequence, I would get the same protein sequence. I would change the genetic sequence.”
Three DNA or RNA letters create one amino acid, but there are multiple combinations of three that will create a given amino acid, Watson says. “So, you can scramble the genetic letters around and still get the same protein letters to come out.”
Two of the domains that have been found on the surface of the SARS-CoV-2 spike protein are also present in the HIV-1 gp120 surface domain, which, Watson says, is part of HIV’s “viral fusion machinery with CD4+T cells”. (T cells, also called T lymphocytes, are a type of white blood cell. They are an essential part of the body’s immune system.)
Watson says gp120 is known to interfere with dendritic cell function during HIV infections, and also modulates the activity of CD4+ T cells, which interact with dendritic cells to sense and respond to various pathogens.
“While I have yet to see structural modelling data of these domains interacting with known HIV binding motifs and this isn’t damning in and of itself, it does raise several questions, especially when coupled to the known immune-evasive properties of SARS-CoV-2, which includes T cell exhaustion and antibody avoidance in a number of patients,” Watson said.
Watson says he hasn’t a clue what the gp120 domains are doing, “but they are there”.
It’s also very strange, Watson says, to see a malaria sequence on the surface of the SARS-CoV-2 spike protein.
He says the malaria sequences don’t match the known binding domain of malaria to CD147 (malaria’s entry receptor into red blood cell precursor cells and red blood cells).
“However, there is speculation and computational modelling suggesting that SARS-CoV-2 also binds to CD147 and, in our own experiments, we have preliminarily seen some binding responses in the ~100–300 nanomolar range, which need to be further validated.
“What are the odds, if there was not laboratory manipulation, of having four little inserts that match protein sequences from HIV on solvent-accessible domains on the surface of the spike, along with a malaria sequence?”
Watson says he doesn’t want to make claims that cannot be proven, but the presence of the four inserts, and the malaria sequence, need to be investigated – and “certainly line up with the gain-of-function work that is suggested by the plasmid recombination experiments and insertion of higher binding affinity ACE2-binding spike proteins into infectious clones”.
It is important, Watson says, to remember that American and Chinese researchers were doing genetic engineering work in 2015, and admitted that they were doing it.
When a virus is randomly evolving, there is usually a fixed ratio of change if there is mutation, Watson says.
“In the case of SARS-CoV-2, when compared to the 2013 RaTG13 virus that was 96.11% similar to the current outbreak, there is an entire region where the virus has mutated in a way that is statistically impossible,” he said.
“It is virtually impossible for these particular mutations to have happened naturally. And this has happened in the same region of the virus – the spike protein – on which American and Chinese researchers have admitted doing recombination work.”
If SARS-CoV-2 were mutating accidentally, it would have a constant ratio of sense and missense mutations that is typically 5:1, Watson says.
“However, for nearly 2,100 RNA letters, there are virtually no missense mutations, as would be expected naturally between two viruses that are experiencing some form of genetic drift.
“The RNA letters are changing, but most of the protein letters are not – and it’s just in the part of the virus that was known to be spliced around in the SARS-related viral recombination experiments.”
The SARS-CoV-2 research was done in collaboration between the US and China, as well as other global actors, Watson points out.
“To say that we didn’t know what this was when it appeared in December is to outright lie about biological research that we knew was happening. We knew what SARS-CoV-1 did in 2003.”
In a paper published on the pre-print repository ViXr.org in May, independent researcher Murat Seyran from Vienna says that the host tropism (the infection specificity of certain pathogens to particular hosts and host tissues) and the infection pattern of SARS-CoV-2 have three fundamental differences compared to the previous six human pathogenic coronaviruses.
“The unnatural flat pattern of SARS-CoV-2 S protein NTD [N-terminal domain] is conflicting with the evolutionary host tropism strategy of not only the human CoVs but also many different human pathogenic viruses,” Seyran said.
Why have we not seen any pandemic caused by coronaviruses before? Seyran asks. Why did pandemics not emerge in places where people rely on water sources shared with bats or bats are consumed as bushmeat?
Seyran also says that, in the case of SARS-CoV-2, the S Protein RBD is not a high-frequency positive selection site, unlike in other coronaviruses.
The SARS-CoV-2 genome is almost identical to the bat coronavirus, but it is only mutated on the RBD, Seyran says. “Why only the RBD had mutations meanwhile the rest of the genome was almost unaltered?”
It is argued that the presence of a furin cleavage spike in SARS-CoV-2’s spike protein is evidence that the virus did not develop naturally.
A furin cleavage site is a segment of four amino acids that enables a virus to use furin in the human body as an enzyme to dissolve its coating so that it can release its genetic material to infect cells. Furin cleavage sites tend to be more infectious than cleavage sites that use other enzymes.
Seyran is one of 18 scientists who wrote a letter to the editor of the Journal of Medical Virology, which was published on September 3.
The scientists, who are from the US, Austria, Iran, Sudan, India, the UK, New Zealand, Egypt, Switzerland, and Jordan, wrote about the unnatural shape of SARS-CoV-2’s spike protein.
“The SARS-CoV-2 host tropism/adaptation pattern has significant discrepancies compared to other CoVs, raising questions concerning the proximal origin of SARSCoV-2,” Seyran et al. wrote.
“The flat and non-sunken surface of the sialic acid-binding domain of SARS-CoV-2 spike protein (S protein) conflicts with the general adaptation and survival pattern observed for all other CoVs.”
Seyran et al. say that SARS-CoV-2 recombination presumably occurred between the S1/S2 domains of the S protein, enabling host furin protease utilisation.
“Although millions of recorded cases have been recorded globally, SARS-CoV-2 S protein does not have any apparent further recombination, placing it in conflict with the recombination models of other CoVs,” the scientists wrote.
“Similarly, the S protein receptor-binding domain (RBD) of SARS-CoV-2 has not accumulated high-frequency non-synonymous substitutions, differentiating SARS-CoV-2 from other CoVs that have positive selection/adaptation mutations in their RBDs.”
Clinical SARS-CoV-2 isolates to date have only a single high frequency non-synonymous mutation, D614G, in their S protein, Seyran et al. say.
“Based on currently known mutation rates and patterns in clinical isolates of SARS-CoV-2, the S protein does not appear to be a mutational ‘hot spot’ for SARSCoV-2, unlike other human CoVs.”
Seyran et al. say that the furin recognition motif present at the SARS-CoV2 S1/S2 junction has no analogy in other “linage B” beta-coronaviruses, including neither pangolin-CoV nor RaTG13.
They say evidence suggests that the addition of a motif for S1/S2 site furin cleavage constituted a unique recombination occurrence.
“The CoV-unique insertion of 4 amino-acids creating a novel RRAR furin cleavage site introduces two arginine codons CGG-CGG, whose usage is extremely rare in CoVs, further supporting the hypothesis of a unique recombination occurrence.”
Andersen et al. say lab manipulation ‘improbable’
In their article published in Nature Medicine, Kristian G. Andersen et al. say it is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus.
“Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus,” the researchers said.
“It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus.”
Stuart Newman, who is a professor of cell biology and anatomy at New York Medical College, is quoted by GMWatch as saying that a key argument used to deny that SARS-CoV-2 could be a genetically engineered strain that escaped from a laboratory actually points to the exact opposite. In other words, he is quoted as saying, it indicates that SARS-CoV-2 could well be genetically engineered and that it could have escaped from a lab.
Andersen et al. say that, if genetic manipulation had been performed, one of the several reverse-genetic systems available for betacoronaviruses would probably have been used.
The genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone, they say.
Andersen et al. propose two scenarios they say can plausibly explain the origin of SARS-CoV-2: natural selection in an animal host before zoonotic transfer, and natural selection in humans following zoonotic transfer.
The researchers say the high-affinity binding of the SARS-CoV-2 spike protein to ACE2 “is most likely the result of natural selection on a human or human-like ACE2 that permits another optimal binding solution to arise”.
They say that, as many early cases of Covid-19 were linked to the Huanan market in Wuhan, it is possible that an animal source was present at this location.
“Given the similarity of SARS-CoV-2 to bat SARS-CoV-like coronaviruses, it is likely that bats serve as reservoir hosts for its progenitor,” they stated.
They add that Malayan pangolins (Manis javanica) illegally imported into Guangdong province contain coronaviruses similar to SARS-CoV-2.
Although the RaTG13 bat virus remains the closest to SARS-CoV-2 across the genome, some pangolin coronaviruses exhibit strong similarity to SARS-CoV-2 in the receptor-binding domain, the researchers say. This includes all six key RBD residues, they write.
According to Andersen et al., this clearly shows that the SARS-CoV-2 spike protein optimised for binding to human-like ACE2 is the result of natural selection.
It is possible, the researchers say, that a progenitor of SARS-CoV-2 jumped into humans, acquiring the genomic features described in their analysis through adaptation during undetected human-to-human transmission.
The researchers say that, although the evidence shows that SARS-CoV-2 is not a purposefully manipulated virus, it is currently impossible to prove or disprove the other theories of its origin described in their article.
However, they say, since they observed all notable SARS-CoV-2 features in related coronaviruses in nature, they do not believe that any type of laboratory-based scenario is plausible.
In an email interview with GMWatch, Stuart Newman said: “The Nature Medicine paper points to variations in two sites of the spike protein of the new coronavirus that the authors claim must have arisen by natural selection in the wild.
“However, genetic engineering of one of these sites, the ACE2 receptor binding domain, has been proposed since 2005 in order to help generate vaccines against these viruses. It is puzzling that the authors of the Nature Medicine commentary did not cite this paper, which appeared in the prominent journal Science.”
Newman also told GMWatch: “The second site that Andersen et al. assert arose by natural means, a target of enzyme cleavage not usually found in this class of viruses, was in fact introduced by genetic engineering in a similar coronavirus in a paper they do cite. This was done to explore mechanisms of pathogenicity.”
He added that he does not believe that changes were deliberately introduced to increase the pathogenicity of any single strain of SARS-CoV-2, but that the virus may have had genetically engineered components in its history before it was inadvertently introduced into the human population.
GMWatch also quoted the London-based molecular geneticist Michael Antoniou, who has also cast doubt on assertions that SARS-CoV-2 was not genetically engineered. Antoniou said that Andersen et al.’s reasoning was not conclusive because it was based largely on computer modelling, which, Antoniou says, is “not definitive but only predictive”.
Antoniou said that, while Andersen et al. may be correct in how they perceive SARS-CoV-2 to have arisen, the data they present “does not exclude the possibility that this new coronavirus variant could have been created through an in vitro, directed iterative evolutionary selection process”.
He added: “Using this method, a very large library of randomly mutagenised coronavirus spike proteins could be selected for strong binding to the ACE2 receptor and consequently high infectivity of human cells.
“The power of such directed evolution to select for optimal enzymatic and protein-protein interactions was acknowledged by the award of the Nobel Prize in Chemistry in 2018.”
GMWatch states: “Neither Dr Antoniou, nor Prof Newman, nor we ourselves make any suggestion that, in the event that genetic engineering was involved, the intention was to create a bioweapon. Such ‘enhanced infectivity’ research is carried out on viruses all over the world (and not just in China) to investigate their behaviour and to develop vaccines and other therapies, as well as for ‘biodefence’ purposes.
“But the question of whether genetic engineering did play a part in the emergence of SARS-CoV-2 must continue to be investigated so that humanity can place appropriate limits and safeguards on such research.”
‘Escapes’ from laboratories
Concerns about gain-of-function research erupted in 2011 when a team at the University of Wisconsin in the US and researchers at a laboratory at the Erasmus Medical Centre in Rotterdam in the Netherlands announced that they had modified the H5N1 bird flu virus to enable it to spread between ferrets.
The researchers had planned to publish their findings in Science and Nature, but the National Science Advisory Board for Biosecurity in the US asked the journals to refrain from publishing the methods the scientists had used.
The advisory board issued a statement that was published in Science and Nature saying that a pandemic or the deliberate release of a transmissible highly pathogenic influenza A/H5N1 virus “would be an unimaginable catastrophe for which the world is currently inadequately prepared”.
The board added: “Our concern is that publishing these experiments in detail would provide information to some person, organisation or government that would help them to develop similar mammal-adapted influenza A/H5N1 viruses for harmful purposes.”
During the SARS outbreak in 2003 and 2004, there were two separate ‘escapes’ of viruses from the same laboratory in Beijing, China.
The WHO described one of the Beijing incidents, along with two other incidents (one in Singapore and one in Taiwan), as being attributed to “breaches in laboratory biosafety”.
In a report in October 2004, the WHO said that two researchers at the National Institute of Virology (NIV) in Beijing, where experiments using live and inactivated forms of the SARS coronavirus were being carried out, developed SARS in late March and mid-April of that year. The outbreak was reported on April 22 and the institute was closed a day later.
In one of the cases, a nurse who had cared for a researcher at the NIV became ill in April 2004 and was diagnosed as being infected with the SARS virus. The researcher, who had worked at the institute for two weeks in March 2004, developed symptoms on March 25, 2004, and was diagnosed as being infected with the SARS virus. The researcher’s mother also became ill and died on April 19.
Another researcher who also worked at the Beijing virology institute developed SARS symptoms on April 17 and was hospitalised. Health authorities diagnosed him as a suspected SARS case.
The WHO said that many shortcomings in biosecurity were found at the NIV and added: “The specific cause of the outbreak was traced to an inadequately inactivated preparation of SARS virus that was used in general (that is, not biosecure) laboratory areas, including one where the primary cases worked.
“It had not been tested to confirm its safety after inactivation, as it should have been.”
The WHO had said on May 18, 2004, that investigators had serious concerns about biosafety procedures at the Beijing institute – including how and where procedures using the SARS coronavirus were carried out, and how and where SARS coronavirus samples were stored.
The organisation added: “WHO and Chinese authorities view with concern the occurrence of laboratory-associated SARS cases. WHO urges all member states to view this latest outbreak as an opportunity to review the biosafety practices of institutions and laboratories working with SARS coronavirus.”
During and after the SARS outbreak of 2003, a large number of specimens were collected from possible human cases, animals, and the environment, the WHO said.
“These specimens, which may contain live SARS coronavirus, are still kept in various laboratories around the world. Some of them are stored in laboratories at an inappropriate containment level.
“SARS coronavirus has also been propagated in reference and research laboratories, and distributed to other laboratories for research purposes. Research using live and inactivated SARS coronavirus – and other pathogens capable of causing serious illness – is being conducted in many laboratories.”
In Singapore, in August 2003, a microbiology student who had been doing research on the West Nile virus at the country’s national university became ill. He tested positive for the SARS virus. The student has also been doing work on West Nile at Singapore’s Environmental Health Institute (EHI) laboratory, where others researchers were studying the SARS virus.
The Center for Infectious Disease Research and Policy (CIDRAP) at the University of Minnesota in the US reported on the incident in September 2003 and said an international committee concluded that the researcher had probably acquired the virus in the EHI laboratory.
“Inappropriate laboratory procedures and a cross-contamination of West Nile virus samples with SARS coronavirus in the laboratory led to the infection of the doctoral student,” the Singapore Ministry of Health said. “No evidence could be found of any other source of infection.”
The ministry said the committee determined that there was no evidence that the researcher, who recovered from his illness, transmitted the virus to anyone else.
CIDRAP reported that the international committee that investigated the incident also examined Singapore’s four laboratories rated as biosafety level 3 ((BSL-3), the second highest of four risk categories.
“The group found structural problems as well as training and record-keeping deficiencies at the environmental health lab and recommended that the lab not reopen until the problems are corrected,” CIDRAP reported. “Lesser problems were found at the Singapore General Hospital laboratory and the National University of Singapore laboratory.”
In December 2003, a scientist from Taiwan who had been doing research on SARS fell ill on a flight when returning from a meeting in Singapore and was diagnosed as positive for SARS infection. His 74 contacts in Singapore were quarantined, and none developed SARS. It was alleged in one press report that the scientist had handled leaking biohazard waste without gloves, a mask, or a gown.
Calls for augmented security
On December 18, 2003, the WHO called for augmented biosafety in laboratories where SARS-CoV specimens and cultures were being handled.
The WHO said it strongly recommended biosafety level 3 as the appropriate containment level for working with live SARS-CoV material.
“The possibility that a SARS outbreak could occur following a laboratory accident is a risk of considerable importance, given the relatively large number of laboratories currently conducting research using the SARS-CoV or retaining specimens from SARS patients,” the WHO said.
“These laboratories currently represent the greatest threat for renewed SARS-CoV transmission through accidental exposure associated with breaches in laboratory biosafety.
“Given the severity of the threat, WHO strongly recommends that national governments maintain a registry of laboratories that are approved to safely and securely hold and work with specimens of suspected or confirmed SARS patients or cultures containing SARS-CoV.”
Appropriate national authorities should provide guidelines for laboratories to catalogue and control the storage of cultures and specimens of SARS-CoV for periodic inspections, the WHO said.
The WHO also said it encouraged the destruction of unwanted or unneeded clinical and animal specimens that were suspected of, or were confirmed to contain, SARS-CoV, and/or of stocks of SARS-CoV that could not be kept under secure conditions.
In July 2018, the director of the biosafety laboratory at the WIV, Yuan Zhiming, and Shi Zhengli published a paper in ScienceDirect entitled ‘Quality management in a high-containment laboratory’.
The Wuhan Institute of Virology was accredited in 2017 by the China National Accreditation Service for Conformity Assessment, the scientists noted.
Zhiming and Zhengli said, however: “Because no international dedicated standard exists for biosafety level 4 (BSL-4) laboratories, this paper explains the desire of the laboratory’s director to set up a quality management system (QMS) to accredit this first level 4 containment laboratory in China …”
They added: “There are two international standards currently available, the choice of which depends on whether it is a laboratory intended for medical diagnoses … or a laboratory for both medical and environmental purposes …”
Both standards are recognised by the WHO, Zhiming and Zhengli noted. “Nonetheless, none of them has specific requirements for biorisks, i.e. biosafety and biosecurity.
“Although the 2017 version of ISO/IEC 17025:2017 takes into account risks in a general sense (clause 8.5), the management is free to use either another standard or any guidance more appropriate for the laboratory’s activity.”
Zhiming and Zhengli said that accreditation of a biocontainment laboratory in accordance with an international standard was a real challenge.
In the 14 countries hosting one or several BSL-4 laboratories, very few such laboratories had been accredited according to an international standard because there was no suitable international standard for accrediting the activities carried out at these laboratories, they added.
Gain-of-function moratorium lifted
On December 19, 2017, federal officials in the US ended a moratorium that had been imposed three years earlier on the funding of gain-of-function research.
The head of the NIH, Francis S. Collins, said the research could only be done if a scientific panel decided that the benefits justified the risks.
Collins (pictured left) said that researchers would have to show that their studies were scientifically sound and that they would be done in a high-security lab.
The pathogen to be modified must pose a serious health threat, he said, and the work must produce knowledge that would benefit humans. In addition, there would have to be no safer way to do the research.
Collins said that the new regulations applied to any pathogen that could potentially cause a pandemic.
In October 2014, all federal funding had been halted for gain-of-function research on the flu virus and those causing Middle East respiratory syndrome (MERS) and SARS.
The NIH said that certain gain-of-function studies with the potential to enhance the pathogenicity or transmissibility of potential pandemic pathogens (PPPs) had raised biosafety and biosecurity concerns, “including the potential dual use risks associated with the misuse of the information or products resulting from such research”.
The White House Office of Science and Technology Policy launched a gain-of-function deliberative process to re-evaluate the potential risks and benefits associated with certain experiments.
During the process the government halted federal funding for gain-of-function studies that were aimed at enhancing the pathogenicity or transmissibility among mammals by respiratory droplets of influenza, MERS, or SARS viruses.
The National Science Advisory Board for Biosecurity (NSABB) finalised its recommendations on May 24, 2016. In January 2017, the US government released policy guidance for the review and oversight of research “anticipated to create, transfer, or use enhanced PPPs”.
A new framework was developed to guide funding decisions about proposed research involving pathogens with enhanced pandemic potential.
Writing in Lancet Infectious Diseases in February 2018 about the rescinding of the moratorium on gain-of-function experiments, Talha Burki referred to the suppression by the NSABB in 2011 of the two studies involving H5N1 viruses that had been modified to allow airborne transmission from ferret to ferret.
“They worried that malign actors could replicate the work to deliberately cause an outbreak in human beings,” Burki wrote.
The Department of Health and Human Services (HHS) issued guidelines for funding decisions on experiments likely to result in highly pathogenic H5N1 viruses transmissible from mammal to mammal via respiratory droplets, Burki reported. The guidelines were later expanded to include H7N9 viruses.
Burki reported that, in 2014, several breaches of protocol at US government laboratories brought matters to a head.
“The news that dozens of workers at the Centers for Disease Control and Prevention (CDC) might have been exposed to anthrax, that vials of smallpox virus had been left lying around in an NIH storeroom, and that the CDC had unwittingly sent out samples of ordinary influenza virus contaminated with H5N1, shook faith in the country’s biosafety procedures,” she wrote.
In July 2014 more than 200 scientists signed the Cambridge Working Group declaration in which they said that, for any experiment, the expected net benefits should outweigh the risks.
“Experiments involving the creation of potential pandemic pathogens should be curtailed until there has been a quantitative, objective and credible assessment of the risks, potential benefits, and opportunities for risk mitigation, as well as comparison against safer experimental approaches,” the scientists said.
“A modern version of the Asilomar process, which engaged scientists in proposing rules to manage research on recombinant DNA, could be a starting point to identify the best approaches to achieve the global public health goals of defeating pandemic disease and assuring the highest level of safety.
“Whenever possible, safer approaches should be pursued in preference to any approach that risks an accidental pandemic.”
The scientists said that “recent incidents involving smallpox, anthrax and bird flu in some of the top US laboratories remind us of the fallibility of even the most secure laboratories, reinforcing the urgent need for a thorough reassessment of biosafety”.
Such incidents had been accelerating, the scientists said and had been occurring on average more than twice a week with regulated pathogens in academic and government labs across the country.
“An accidental infection with any pathogen is concerning. But accident risks with newly created ‘potential pandemic pathogens’ raise grave new concerns.
“Laboratory creation of highly transmissible, novel strains of dangerous viruses, especially but not limited to influenza, poses substantially increased risks. An accidental infection in such a setting could trigger outbreaks that would be difficult or impossible to control.
“Historically, new strains of influenza, once they establish transmission in the human population, have infected a quarter or more of the world’s population within two years.”
The DRASTIC team has explored, and continues to investigate, numerous hypotheses, ranging from the claim that SARS-CoV-2 is a Chinese or American bioweapon or was leaked from a laboratory to the “miners hypothesis”, the theory that the virus is “a vaccine gone wrong”, and the “serial passage” hypothesis.
“We question everything,” one member of the team, who prefers to remain anonymous, and tweets under the handle @BillyBostickson, said. “We have no fixed agenda.”
The team has put together a list of 260 questions that they say the WHO and specific scientists need to answer and is about to add more.
The questions cover an enormous amount of ground, and many are extremely technical.
They range from “Why did Shi Zhengli alter keywords in the WIV database on December 30 when returning to Wuhan?” and “Why are the majority of Chinese viral pathogen databases now offline?” to “How did Shi Zhengli conclusively establish SARS-CoV-2 didn’t come from her lab?” and “When exactly did the first cases of viral pneumonia that are now known to be Covid-19 occur?”.
Other questions include “What was the increased activity (revealed by satellite images) at Wuhan hospitals in October 2019 due to?” and “Did the Wuhan Institute of Virology team newly extract RNA from the RaBtCoV/4991 physical sample or did they just sequence the stored RNA sample?”
Australia has called for an independent investigation into the origins and spread of SARS-CoV-2 and this has further soured its already tense relations with China.
The French news agency AFP reported that the Chinese ambassador in Canberra retaliated by threatening a widespread consumer boycott of Australian products and this was followed by a Chinese ban on imports from four major Australian beef producers.
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