Li-Meng Yan (MD, PhD), Shu Kang (PhD), Jie Guan (PhD), Shanchang Hu (PhD)
Rule of Law Society & Rule of Law Foundation, New York, NY, USA.
Correspondence:
[email protected]
Abstract
The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to over 910,000 deaths worldwide and unprecedented decimation of the global economy. Despite its tremendous impact, the origin of SARS-CoV-2 has remained mysterious and controversial. The natural origin theory, 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. The evidence shows that SARS-CoV-2 should be a laboratory product created by using bat coronaviruses ZC45 and/or ZXC21 as a template and/or backbone. Building upon the evidence, we further postulate a synthetic route for SARS-CoV-2, demonstrating that the laboratory-creation of this coronavirus is convenient and can be accomplished in approximately six months. Our work emphasizes the need for an independent investigation into the relevant research laboratories. It also argues for a critical look into certain recently published data, which, albeit problematic, was used to support and claim a natural origin of SARS-CoV-2. From a public health perspective, these actions are necessary as knowledge of the origin of SARS-CoV-2 and of how the virus entered the human population are of pivotal importance in the fundamental control of the COVID-19 pandemic as well as in preventing similar, future pandemics.
Introduction
COVID-19 has caused a world-wide pandemic, the scale and severity of which are unprecedented. Despite the tremendous efforts taken by the global community, management and control of thispandemic remains difficult and challenging.
As a coronavirus, SARS-CoV-2 differs significantly from other respiratory and/or zoonotic viruses: it attacks multiple organs; it is capable of undergoing a long period of asymptomatic infection; it is highly transmissible and significantly lethal in high-risk populations; it is well-adapted to humans since the very start of its emergence; it is highly efficient in binding the human ACE2 receptor (hACE2), the affinity of which is greater than that associated with the ACE2 of any other potential host.
The origin of SARS-CoV-2 is still the subject of much debate. A widely cited Nature Medicine publication has claimed that SARS-CoV-2 most likely came from nature. However,the article and its central conclusion are now being challenged by scientists from all over the world. In addition, authors of this Nature Medicine article show signs of conflict of interests, raising further concerns on the credibility of this publication.
The existing scientific publications supporting a natural origin theory rely heavily on a single piece of evidence –a previously discovered bat coronavirus named RaTG13, which shares a 96% nucleotide sequence identity with SARS-CoV-2. However,the existence of RaTG13 in nature and the truthfulnessof its reported sequence are being widely questioned. It is noteworthy that scientific journals have clearly censored any dissenting opinions that suggest a non-natural origin of SARS-CoV-2. Because of this censorship, articles questioning either the natural origin of SARS-CoV-2 or the actual existence of RaTG13, although of high quality scientifically, can only exist as preprints or other non-peer-reviewed articles published on various online platforms. Nonetheless, analyses of these reports have repeatedly pointedto severe problems and a probable fraud associated with the reporting of RaTG13. Therefore, the theory that fabricated scientific data has been published to mislead the world’s efforts in tracing the origin of SARS-CoV-2 has become substantially convincing and is interlocked with the notion that SARS-CoV-2 is of a non-natural origin.
Consistent with this notion, genomic, structural, and literature evidence also suggest a non-natural origin of SARS-CoV-2. In addition, abundant literature indicates that gain-of-function research has long advanced to the stage where viral genomes can be precisely engineered and manipulated to enable the creation of novel coronaviruses possessing unique properties. In this report, we present such evidence and the associated analyses. Part 1 of the report describes the genomic and structural features of SARS-CoV-2, the presence of which could be consistent with the theory that the virus is a product of laboratory modification beyond what could be afforded by simple serial viral passage. Part 2 of the report describes a highly probable pathway for the laboratory creation of SARS-CoV-2, key steps of which are supported by evidence present in the viral genome. Importantly, part 2 should be viewed as a demonstration of how SARS-CoV-2 could be conveniently created in a laboratory in a short period of time using available materials and well-documented techniques. This report is produced by a team of experienced scientists using our combined expertise in virology, molecular biology, structural biology, computational biology, vaccine development, and medicine.
1. Has SARS-CoV-2 been subjected to in vitro manipulation?
We present three lines of evidence to support our contention that laboratory manipulation is part of the history of SARS-CoV-2:
i. The genomic sequence of SARS-CoV-2 is suspiciously similar to that of a bat coronavirus discovered by military laboratories in the Third Military Medical University (Chongqing, China) and the Research Institute for Medicine of Nanjing Command (Nanjing, China).
ii. The receptor-binding motif (RBM) within the Spike protein of SARS-CoV-2, which determines the host specificity of the virus,resembles that of SARS-CoV from the 2003 epidemic in a suspicious manner. Genomic evidence suggests that the RBM has been genetically manipulated.
iii. SARS-CoV-2 contains a unique furin-cleavage site in its Spike protein,which is known to greatly enhance viral infectivity and cell tropism. Yet, this cleavage site is completely absent in this particular class of coronaviruses found in nature. 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.
1.1 Genomic sequence analysis reveals that ZC45, or a closely related bat coronavirus, should be the backbone used for the creation of SARS-CoV-2
The structure of the ~30,000 nucleotides-long SARS-CoV-2 genome is shown in Figure 1. Searching the NCBI sequence database reveals that, among all known coronaviruses, there were two related bat coronaviruses, ZC45 and ZXC21, that share the highest sequence identity with SARS-CoV-2 (each bat coronavirus is~89% identical to SARS-CoV-2 on the nucleotide level). Similarity between the genome of SARS-CoV-2 and those of representative β coronaviruses is depicted in Figure 1. ZXC21, which is 97% identical to and shares a very similar profile with ZC45, is not shown. Note that the RaTG13 virus is excluded from this analysis given the strong evidence suggesting that its sequence may have been fabricated and the virus does not exist in nature. (A follow-up report, which summarizes the up-to-date evidence proving the spurious nature of RaTG13, will be submitted soon)
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. 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.
Orf8 is an accessory protein, the function of which is largely unknown in most coronaviruses, although recent data suggests that Orf8 of SARS-CoV-2 mediates the evasion of host adaptive immunity by down regulating MHC-I24. Normally, Orf8 is poorly conserved in coronaviruses. Sequence blast indicates that, while the Orf8 proteins of ZC45/ZXC21 share a 94.2% identity with SARS-CoV-2 Orf8,
no other coronaviruses share more than 58% identity with SARS-CoV-2 on this particular protein. The very high homology here on the normally poorly conserved Orf8 protein is highly unusual.
The coronavirus E protein is a structural protein, which is embedded in and lines the interior of the membrane envelope of the virion. The E protein is tolerant of mutations as evidenced in both SARS (Figure 2A) and related bat coronaviruses (Figure 2B). This tolerance to amino acid mutations of the E protein is further evidenced in the current SARS-CoV-2 pandemic. After only a short two-month spread of the virus since its outbreak in humans, the E proteins in SARS-CoV-2 have already undergone mutational changes. Sequence data obtained during the month of April reveals that mutations have occurred at four different locations in different strains (Figure 2C). Consistent with this finding, 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 (suspicious coronaviruses published after the start of the current pandemic are excluded). 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. Therefore, 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 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.
Importantly, ZC45 and ZXC21 are bat coronaviruses that were discovered (between July 2015 and February 2017), isolated, and characterized by
military research laboratories in the Third Military Medical University (Chongqing, China) and the Research Institute for Medicine of Nanjing Command (Nanjing, China). The data and associated work were published in 2018. Clearly, this backbone/template, which is essential for the creation of SARS-CoV-2, exists in these and other related research laboratories.
What strengthens our contention further is the published RaTG13 virus, the genomic sequence of which is reportedly 96% identical to that of SARS-CoV-2. 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. In fact, a Chinese BSL-3 lab (the Shanghai Public Health Clinical Centre), which published a Nature article reporting a conflicting close phylogenetic relationship between SARS-CoV-2 and ZC45/ZXC21 rather than with RaTG1335, was quickly shut down for “rectification”. It is believed that the researchers of that laboratory were being punished for having disclosed the SARS-CoV-2—ZC45/ZXC21 connection. 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. A very recent publication also indicated that the receptor-binding domain (RBD) of the RaTG13’s Spike protein could not bind ACE2 of two different types of horseshoe bats (they closely relate to the horseshoe bat R. affinis, RaTG13’s alleged natural host), implicating the inability of RaTG13 to infect horseshoe bats. This 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. 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.
1.2 The receptor-binding motif of SARS-CoV-2 Spike cannot be born from nature and should have been created through genetic engineering
The Spike proteins decorate the exterior of the coronavirus particles. They play an important role in infection as they mediate the interaction with host cell receptors and thereby help determine the host range and tissue tropism of the virus. The Spike protein is split into two halves (Figure 3). The front or N-terminal half is named S1, which is fully responsible for binding the host receptor. In both SARS-CoV and SARS-CoV-2 infections, the host cell receptor is hACE2. Within S1, a segment of around 70 amino acids makes direct contacts with hACE2 and is correspondingly named the receptor-binding motif (RBM)(Figure 3C). In SARS-CoV and SARS-CoV-2, the RBM fully determines the interaction with hACE2. The C-terminal half of the Spikeprotein is named S2. The main function of S2 includes maintaining trimer formation and, upon successive protease cleavages at the S1/S2 junction and a downstream S2’ position, mediating membrane fusion to enable cellular entry of the virus.
Similar to what is observed for other viral proteins, S2 of SARS-CoV-2 shares a high sequence identity (95%) with S2 of ZC45/ZXC21. In stark contrast, between SARS-CoV-2 and ZC45/ZXC21, the S1 protein, which dictates which host (human or bat) the virus can infect, is much less conserved with the amino acid sequence identity being only 69%.
Figure 4 shows the sequence alignment of the Spike proteins from six β coronaviruses. Two are viruses isolated from the current pandemic (Wuhan-Hu-1, 2019-nCoV_USA-AZ1); two are the suspected template viruses (Bat_CoV_ZC45, Bat_CoV_ZXC21); two are SARS coronaviruses (SARS_GZ02, SARS). The RBM is highlighted in between two orange lines. Clearly, despite the high sequence identity for the overall genomes, the RBM of SARS-CoV-2 differs significantly from those of ZC45 and ZXC21. Intriguingly, the RBM of SARS-CoV-2 resembles, on a great deal, the RBM of SARS Spike. Although this is not an exact “copy and paste”, careful examination of the Spike-hACE2 structures reveals that all residues essential for either hACE2 binding or protein folding (orange sticks in Figure 3C and what is highlighted by red short lines in Figure 4) are “kept”. Most of these essential residues are precisely preserved, including those involved in disulfide bond formation (C467, C474) and electrostatic interactions (R444, E452, R453, D454), which are pivotal for the structural integrity of the RBM (Figure 3C and 4). The few changes within the group of essential residues are almost exclusively hydrophobic “substitutions” (I428 to L, L443 to F, F460 to Y, L472 to F, Y484 to Q), which should not affect either protein folding or the hACE2-interaction. At the same time, majority of the amino acid residues that are non-essential have “mutated” (Figure 4, RBM residues not labeled with short red lines). Judging from this sequence analysis alone, we were convinced early on that not only would the SARS-CoV-2 Spike protein bind hACE2 but also the binding would resemble, precisely, that between the original SARS Spike protein and hACE223. Recent structural work has confirmed our prediction.
As elaborated below, 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. Collectively, this suggests that portions of the SARS-CoV-2 genome have not been derived from natural quasi-species viral particle evolution.
If SARS-CoV-2 does indeed come from natural evolution, its RBM could have only been acquired in one of the two possible routes: 1) an ancient recombination event followed by convergent evolution or 2) a natural recombination event that occurred fairly recently.
In the first scenario, the ancestor of SARS-CoV-2, a ZC45/ZXC21-like bat coronavirus would have recombined and “swapped” its RBM with a coronavirus carrying a relatively “complete” RBM (in reference to SARS). This recombination would result in a novel ZC45/ZXC21-like coronavirus with all the gaps in its RBM “filled”(Figure 4). Subsequently, the virus would have to adapt extensively in its new host, where the ACE2 protein is highly homologous to hACE2. Random mutations across the genome would have to have occurred to eventually shape the RBM to its current form –resembling SARS-CoV RBM in a highly intelligent manner. However, this convergent evolution process would also result in the accumulation of a large amount of mutations in other parts of the genome, rendering the overall sequence identity relatively low. The high sequence identity between SARS-CoV-2 and ZC45/ZXC21 on various proteins (94-100% identity) do not support this scenario and, therefore, clearly indicates that SARS-CoV-2 carrying such an RBM cannot come from a ZC45/ZXC21-like bat coronavirus through this convergent evolutionary route.
In the second scenario, the ZC45/ZXC21-like coronavirus would have to have recently recombined and swapped its RBM with another coronavirus that had successfully adapted to bind an animal ACE2 highly homologous to hACE2. The likelihood of such an event depends, in part, on the general requirements of natural recombination: 1) that the two different viruses share significant sequence similarity; 2) that they must co-infect and be present in the same cell of the same animal; 3) that the recombinant virus would not be cleared by the host or make the host extinct; 4) that the recombinant virus eventually would have to become stable and transmissible within the host species.
In regard to this recent recombination scenario, the animal reservoir could not be bats because the ACE2 proteins in bats are not homologous enough to hACE2 and therefore the adaption would not be able to yield an RBM sequence as seen in SARS-CoV-2. This animal reservoir also could not be humans as the ZC45/ZXC21-like coronavirus would not be able to infect humans. In addition, there has been no evidence of any SARS-CoV-2 or SARS-CoV-2-like virus circulating in the human population prior to late 2019. Intriguingly, according to a recent bioinformatics study, SARS-CoV-2 was well-adapted for humans since the start of the outbreak.
Only one other possibility of natural evolution remains, which is that the ZC45/ZXC21-like virus and a coronavirus containing a SARS-like RBM could have recombined in an intermediate host where the ACE2 protein is homologous to hACE2. Several laboratories have reported that some of the Sunda pangolins smuggled into China from Malaysia carried coronaviruses, the receptor-binding domain (RBD) of which is almost identical to that of SARS-CoV-2. They then went on to suggest that pangolins are the likely intermediate host for SARS-CoV-2. However, recent independent reports have found significant flaws in this data. Furthermore, contrary to these reports, no coronaviruses have been detected in Sunda pangolin samples collected for over a decade in Malaysia and Sabah between 2009 and 2019. A recent study also showed that the RBD, which is shared between SARS-CoV-2 and the reported pangolin coronaviruses, binds to hACE2 ten times stronger than to the pangolin ACE2, further dismissing pangolins as the possible intermediate host. Finally, an in silico study, while echoing the notion that pangolins are not likely an intermediate host, also indicated that none of the animal ACE2 proteins examined in their study exhibited more favorable binding potential to the SARS-CoV-2 Spike protein than hACE2 did. This last study virtually exempted all animals from their suspected roles as an intermediate host, which is consistent with the observation that SARS-CoV-2 was well-adapted for humans from the start of the outbreak. This is significant because these findings collectively suggest that no intermediate host seems to exist for SARS-CoV-2, which at the very least diminishes the possibility of a recombinant event occurring in an intermediate host.
Even if we ignore the above evidence that no proper host exists for the recombination to take place and instead assume that such a host does exist, it is still highly unlikely that such a recombination event could occur in nature.
As we have described above, if natural recombination event is responsible for the appearance of SARS-CoV-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 (Figure 4). 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. Importantly, this, and the other recombination event described below in section 1.3 (even more impossible to occur in nature), would both have to happen to produce a Spike as seen in SARS-CoV-2.
While the above evidence and analyses together appear to disapprove a natural origin of SARS-CoV-2’s RBM, abundant literature shows that gain-of-function research, where the Spike protein of a coronavirus was specifically engineered, has repeatedly led to the successful generation of human-infecting coronaviruses from coronaviruses of non-human origin.
Record also shows that research laboratories, for example, the Wuhan Institute of Virology (WIV), have successfully carried out such studies working with US researchers and also working alone. In addition, the WIV has engaged in decades-long coronavirus surveillance studies and therefore owns the world’s largest collection of coronaviruses. Evidently, the technical barrier is non-existent for the WIV and other related laboratories to carry out and succeed in such Spike/RBM engineering and gain-of-function research.
Strikingly, consistent with the RBM engineering theory, we have identified two unique restriction sites, EcoRI and BstEII, at either end of the RBM of the SARS-CoV-2 genome, respectively (Figure 5A). These two sites, which are popular choices of everyday molecular cloning, do not exist in the rest of this spike gene. This particular setting makes it extremely convenient to swap the RBM within spike, providing a quick way to test different RBMs and the corresponding Spike proteins.
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. Although ZC45 spike also does not have these two sites (Figure 5B), they can be introduced very easily as described in part 2 of this report.
It is noteworthy that introduction of the EcoRI site here would change the corresponding amino acids from -WNT-to -WNS-(Figure 5AB). As far as we know, all SARS and SARS-like bat coronaviruses exclusively carry a T(threonine) residue at this location. SARS-CoV-2 is the only exception in that this T has mutated to an S (serine), save the suspicious RaTG13 and pangolin coronaviruses published after the outbreak.
Once the restriction sites were successfully introduced, the RBM segment could be swapped conveniently using routine restriction enzyme digestion and ligation. Although alternative cloning techniques may leave no trace of genetic manipulation (Gibson assembly as one example), this old-fashioned approach could be chosen because it offers a great level of convenience in swapping this critical RBM.
Given that RBM fully dictates hACE2-binding and that the SARS RBM-hACE2 binding was fully characterized by high-resolution structures (Figure 3), this RBM-only swap would not be any riskier than the full Spike swap. In fact, the feasibility of this RBM-swap strategy has been proven. In 2008, Dr. Zhengli Shi’s group swapped a SARS RBM into the Spike proteins of several SARS-like bat coronaviruses after introducing a restriction site into a codon-optimized spikegene (Figure 5C). They then validated the binding of the resulted chimeric Spike proteins with hACE2. Furthermore, in a recent publication, the RBM of SARS-CoV-2 was swapped into the receptor-binding domain (RBD) of SARS-CoV, resulting in a chimeric RBD fully functional inbinding hACE2 (Figure 5C).
Strikingly, in both cases, the manipulated RBM segments resemble almost exactly the RBM defined by the positions of the EcoRI and BstEII sites (Figure 5C). Although cloning details are lacking in both publications, it is conceivable that the actual restriction sites may vary depending on the spike gene receiving the RBM insertion as well as the convenience in introducing unique restriction site(s) in regions of interest. It is noteworthy that the corresponding author of this recent publication, Dr. Fang Li, has been an active collaborator of Dr. Zhengli Shi since 2010. Dr. Li was the first person in the world to have structurally elucidated the binding between SARS-CoV RBD and hACE2 and has been the leading expert in the structural understanding of Spike-ACE2 interactions. 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. The more deceiving approach would be to change a few non-essential residues, while preserving the ones critical for binding. This design could be well-guided by the high-resolution structures (Figure 3). This way, when the overall sequence of the RBM would appear to be more distinct from that of the SARS RBM, the hACE2-binding ability would be well-preserved. We believe that all of the crucial residues (residues labeled with red sticks in Figure 4, which are the same residues shown in sticks in Figure 3C) should have been “kept”. As described earlier, while some should be direct preservation, some should have been switched to residues with similar properties, which would not disrupt hACE2-binding and may even strengthen the association further. Importantly, changes might have been made intentionally at non-essential sites, making it less like a “copy and paste” of the SARS RBM.
1.3 An unusual furin-cleavage site is present in the Spike protein of SARS-CoV-2 and is associated with the augmented virulence of the virus
Another unique motif in the Spike protein of SARS-CoV-2 is a polybasic furin-cleavage site located at the S1/S2 junction (Figure 4, segment in between two green lines). Such a site can be recognized and cleaved by the furin protease. Within the lineage B of β coronaviruses and with the exception of SARS-CoV-2, no viruses contain a furin-cleavage site at the S1/S2 junction (Figure 6). In contrast, furin-cleavage site at this location has been observed in other groups of coronaviruses. Certain selective pressure seems to be in place that prevents the lineage B of β coronaviruses from acquiring or maintaining such a site in nature.
As previously described, during the cell entry process, the Spike protein is first cleaved at the S1/S2 junction. This step, and a subsequent cleavage downstream that exposes the fusion peptide, are both mediated by host proteases. The presence or absence of these proteases in different cell types greatly affects the cell tropism and presumably the pathogenicity of the viral infection. Unlike other proteases, furin protease is widely expressed in many types of cells and is present at multiple cellular and extracellular locations. Importantly, the introduction of a furin-cleavage site at the S1/S2 junction could significantly enhance the infectivity of a virus as well as greatly expand its cell tropism —a phenomenon well-documented in both influenza viruses and other coronaviruses.
If we leave aside the fact that no furin-cleavage site is found in any lineage B β coronavirus in nature and instead assume that this site in SARS-CoV-2 is a result of natural evolution, then only one evolutionary pathway is possible, which is that the furin-cleavage site has to be derived from a homologous recombination event. Specifically, an ancestor β coronavirus containing no furin-cleavage site would have to recombine with a closely related coronavirus that does contain a furin-cleavage site.
However, two facts disfavor this possibility. First, although some coronaviruses from other groups or lineages do contain polybasic furin-cleavage sites, none of them contains the exact polybasic sequence present in SARS-CoV-2 (-PRRAR/SVA-). Second, between SARS-CoV-2 and any coronavirus containing a legitimate furin-cleavage site, the sequence identity on Spike is no more than 40%. Such a low level of sequence identity rules out the possibility of a successful homologous recombination ever occurring between the ancestors of these viruses. Therefore, the furin-cleavage site within the SARS-CoV-2 Spike protein is unlikely to be of natural origin and instead should bea result of laboratory modification.
Consistent with this claim, a close examination of the nucleotide sequence of the furin-cleavage site in SARS-CoV-2 spike has revealed that the two consecutive Arg residues within the inserted sequence(-PRRA-) are both coded by the rare codon CGG (least used codon for Arg in SARS-CoV-2) (Figure 7). In fact, this CGGCGG arrangement is the only instance found in the SARS-CoV-2 genome where this rare codon is used in tandem.
This observation strongly suggests that this furin-cleavage site should be a result of genetic engineering. Adding to the suspicion, a FauI restriction site is formulated by the codon choices here, suggesting the possibility that the restriction fragment length polymorphism, a technique that a WIV lab is proficient at, could have been involved. There, the fragmentation pattern resulted from FauI digestion could be used to monitor the preservation of the furin-cleavage site in Spike as this furin-cleavage site is prone to deletions in vitro. Specifically, RT-PCR on the spike gene of the recovered viruses from cell cultures or laboratory animals could be carried out, the product of which would be subjected to FauI digestion. Viruses retaining or losing the furin-cleavage site would then yield distinct patterns, allowing convenient tracking of the virus(es) of interest.
In addition, although no known coronaviruses contain the exact sequence of -PRRAR/SVA-that is present in the SARS-CoV-2 Spike protein, a similar -RRAR/AR-sequence has been observed at the S1/S2 junction of the Spike protein in a rodent coronavirus, AcCoV-JC34, which was published by Dr. Zhengli Shi in 2017. It is evident that the legitimacy of -RRAR- as a functional furin-cleavage site has been known to the WIV experts since 2017.
The evidence collectively suggests that the furin-cleavage site in the SARS-CoV-2 Spike protein may not have come from nature and could be the result of genetic manipulation. The purpose of this manipulation could have been to assess any potential enhancement of the infectivity and pathogenicity of the laboratory-made coronavirus. Indeed, recent studies have confirmed that the furin-cleavage site does confer significant pathogenic advantages to SARS-CoV-2.
1.4 Summary
Evidence presented in this part reveals that certain aspects of the SARS-CoV-2 genome are extremely difficult to reconcile to being a result of natural evolution. The alternative theory we suggest is that the virus may have been created by using ZC45/ZXC21 bat coronavirus(es) as the backbone and/or template. The Spike protein, especially the RBM within it, should have been artificially manipulated, upon which the virus has acquired the ability to bind hACE2 and infect humans. This is supported by the finding of a unique restriction enzyme digestion site at either end of the RBM. An unusual furin-cleavage site may have been introduced and inserted at the S1/S2 junction of the Spike protein, which contributes to the increased virulence and pathogenicity of the virus. These transformations have then staged the SARS-CoV-2 virus to eventually become a highly-transmissible, onset-hidden, lethal, sequelae-unclear, and massively disruptive pathogen.
Evidently, the possibility that SARS-CoV-2 could have been created through gain-of-function manipulations at the WIV (Wuhan Institute of Virology) is significant and should be investigated thoroughly and independently.
