The following Journal Pre-proof due for publication in September adds further to the substantial body of scientific evidence supporting a zoonotic origin while one still struggles to find any evidence in support of the lab leak theory, perhaps that's because there never was any? There is currently no evidence that SARS-CoV-2 has a laboratory origin. There is no evidence that any early cases had any connection to the Wuhan Institute Of Virology (WIV), in contrast to the clear epidemiological links to animal markets in Wuhan, no evidence that the WIV possessed or worked on a progenitor of SARS-CoV-2 prior to the pandemic. There is no rational experimental reason why a new genetic system would be developed using an unknown and unpublished virus, with no evidence nor mention of a SARS-CoV-2-like virus in any prior publication or study from the WIV, no evidence that the WIV sequenced a virus that is closer to SARS-CoV-2 than RaTG13, and no reason to hide research on a SARS-CoV-2-like virus prior to the COVID-19 pandemic. Under any laboratory escape scenario SARS-CoV-2 would have to have been present in a laboratory prior to the pandemic, yet no evidence exists to support such a notion and no sequence has been identified that could have served as a precursor.
Journal Pre-proof - Sept 2021
The Origins of SARS-CoV-2: A Critical Review
Since the first reports of a novel SARS-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how SARS-CoV-2 emerged in the human population. Recent debate has coalesced around two competing ideas: a “laboratory escape” scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.
Evidence supporting a zoonotic origin of SARS-CoV-2
Coronaviruses have long been known to present a high pandemic risk. SARS-CoV-2 is the ninth documented coronavirus that infects humans and the seventh identified in the last 20 years (Lednicky et al., 2021; Vlasova et al., 2021). All previous human coronaviruses have zoonotic origins, as have the vast majority of human viruses. The emergence of SARS-CoV-2 bear several signatures of these prior zoonotic events. It displays clear similarities to SARS-CoV that spilled over into humans in Foshan, Guangdong province, China in November 2002, and again in Guangzhou, Guangdong province in 2003 (Xu et al., 2004). Both these SARS-CoV emergence events were associated with markets selling live animals and involved species, particularly civets and raccoon dogs (Guan et al., 2003), that were also sold live in Wuhan markets in 2019 (Xiao et al., 2021) and are known to be susceptible to SARS-CoV-2 infection (Freuling et al., 2020). Animal traders working in 2003, without a SARS diagnosis, were documented to have high levels of IgG to SARS-CoV (13% overall and >50% for traders specializing in civets; Centers for Disease Control and Prevention, 2003). Subsequent serological surveys found ~3% positivity rates to SARS-related coronaviruses (SARSr-CoV) in residents of Yunnan province living close to bat caves (Wang et al., 2018), demonstrating regular exposure in rural locations. The closest known relatives to both SARS-CoV and SARS-CoV-2 are viruses from bats in Yunnan, although animals from this province have been preferentially sampled. For both SARS-CoV and SARSCoV-2 there is a considerable geographic gap between Yunnan and the location of the first human cases, highlighting the difficulty in identifying the exact pathway of virus emergence and the importance of sampling beyond Yunnan.
SARS-CoV-2 also shows similarities to the four endemic human coronaviruses: HCoV-OC43,HCoV-HKU1, HCoV-229E, and HCoV-NL63. These viruses have zoonotic origins and the circumstances of their emergence are unclear. In direct parallel to SARS-CoV-2, HCoV-HKU1,which was first described in a large Chinese city (Shenzhen, Guangdong) in the winter of 2004,has an unknown animal origin, contains a furin cleavage site in its spike protein, and was originally identified in a case of human pneumonia (Woo et al., 2005).
Based on epidemiological data, the Huanan market in Wuhan was an early and major epic enter of SARS-CoV-2 infection. Two of the three earliest documented COVID-19 cases were directly linked to this market selling wild animals, as were 28% of all cases reported in December 2019(WHO, 2021). Overall, 55% of cases during December 2019 had an exposure to either the Huanan or other markets in Wuhan, with these cases more prevalent in the first half of that month (WHO, 2021). Examination of the locations of early cases shows that most cluster around the Huanan market, located north of the Yangtze river (Figure 1B-E), although case reporting may be subject to sampling biases reflecting the density and age structure of the population in central Wuhan, and exact location of some early cases is uncertain. These districts were also the first to exhibit excess pneumonia deaths in January 2020 (Figure 1F-H), a metric that is less susceptible to the potential biases associated with case reporting. There is no epidemiological link to any other locality in Wuhan, including the Wuhan Institute of Virology (WIV) located south of the Yangtze and the subject of considerable speculation. Although some early cases do not have a direct epidemiological link to a market (WHO, 2021), this is expected given high rates of asymptomatic transmission and undocumented secondary transmission events, and was similarly observed in early SARS-CoV cases in Foshan (Xu et al., 2004).
During 2019, markets in Wuhan – including the Huanan market – traded many thousands of live wild animals including high-risk species such as civets and raccoon dogs (Xiao et al., 2021).Following its closure, SARS-CoV-2 was detected in environmental samples at the Huanan market, primarily in the western section that traded in wildlife and domestic animal products, as well as in associated drainage areas (WHO, 2021). While animal carcasses retrospectively tested negative for SARS-CoV-2, these were unrepresentative of the live animal species sold, and specifically did not include raccoon dogs and other animals known to be susceptible to SARSCoV-2 (Xiao et al., 2021).
The earliest split in the SARS-CoV-2 phylogeny defines two lineages - denoted A and B(Rambaut et al., 2020) - that likely circulated contemporaneously (Figure 1A). Lineage B, whichbecame dominant globally, was observed in early cases linked to the Huanan market andenvironmental samples taken there, while lineage A contains a case with exposure to othermarkets (Figure 1A-B) as well as with later cases in Wuhan and other parts of China (WHO,2021). This phylogenetic pattern is consistent with the emergence of SARS-CoV-2 involving oneor more contacts with infected animals and/or traders, including multiple spill-over events, aspotentially infected or susceptible animals were moved into or between Wuhan markets viashared supply chains and sold for human consumption (Xiao et al., 2021). The potentialemergence of SARS-CoV-2 across multiple markets again mirrors SARS-CoV in which highlevels of infection, sero prevalence and genetic diversity in animals were documented at both theDongmen market in Shenzhen (Al, 2004; Guan et al., 2003) and the Xinyuan market inGuangzhou (Tu et al., 2004; Wang et al., 2005).
Viruses closely related to SARS-CoV-2 have been documented in bats and pangolins in multiplelocalities in South-East Asia, including in China, Thailand, Cambodia, and Japan (Lytras et al.2021; Zhou et al., 2021), with serological evidence for viral infection in pangolins for more thana decade (Wacharapluesadee et al., 2021). However, a significant evolutionary gap existsbetween SARS-CoV-2 and the closest related animal viruses: for example, the bat virus RaTG13collected by the WIV has a genetic distance of approximately 4% (~1,150 mutations) to theWuhan-Hu-1 reference sequence of SARS-CoV-2, reflecting decades of evolutionary divergence(Boni et al., 2020). Widespread genomic recombination also complicates the assignment ofwhich viruses are closest to SARS-CoV-2. Although RaTG13, sampled from a Rhinolophusaffinis bat in Yunnan (Zhou et al., 2020b), has the highest average genetic similarity to SARSCoV-2, a history of recombination means that three other bat viruses – RmYN02, RpYN06 andPrC31 – are closer in most of the virus genome (particularly ORF1ab) and thus share a morerecent common ancestor with SARS-CoV-2 (Li et al., 2021; Lytras et al. 2021; Zhou et al.,2021). None of these three closer viruses were collected by the WIV and all were sequencedafter the pandemic had begun (Li et al., 2021; Zhou et al., 2020a; Zhou et al., 2021).Collectively, these data demonstrate beyond reasonable doubt that RaTG13 is not the progenitorof SARS-CoV-2, with or without laboratory manipulation or experimental mutagenesis.
No bat reservoir nor intermediate animal host for SARS-CoV-2 has been identified to date. Thisis presumably because the right animal species and/or populations have not yet been sampledand/or any progenitor virus may be at low prevalence. Initial cross-species transmission eventsare also very likely to go undetected. Most SARS-CoV-2 index case infections will not haveresulted in sustained onward transmission (Pekar et al., 2021) and only a very small fraction ofspillovers from animals to humans result in major outbreaks. Indeed, the animal origins of manywell-known human pathogens, including Ebola virus, Hepatitis C virus, poliovirus, and thecoronaviruses HCoV-HKU1 and HCoV-NL63, are yet to be identified, while it took over adecade to discover bat viruses with >95% similarity to SARS-CoV and able to use hACE-2 as areceptor (Hu et al., 2017).
Could SARS-CoV-2 have escaped from a laboratory?
There are precedents for laboratory incidents leading to isolated infections and transienttransmission chains, including SARS-CoV (Parry, 2004). However, with the exception ofMarburg virus (Ristanović et al., 2020), all documented laboratory escapes have been of readilyidentifiable viruses capable of human infection and associated with sustained work in high titercultures (Geddes, 2006; Lim et al., 2004; Senio, 2003). The 1977 A/H1N1 influenza pandemic,that most likely originated from a large-scale vaccine challenge trial (Rozo and Gronvall, 2015),is the only documented example of a human epidemic or pandemic resulting from researchactivity. No epidemic has been caused by the escape of a novel virus and there is no data tosuggest that the WIV—or any other laboratory—was working on SARS-CoV-2, or any virusclose enough to be the progenitor, prior to the COVID-19 pandemic. Viral genomic sequencingwithout cell culture, which was routinely performed at the WIV, represents a negligible risk asviruses are inactivated during RNA extraction (Blow et al., 2004). No case of laboratory escapehas been documented following the sequencing of viral samples.Known laboratory outbreaks have been traced to both workplace and family contacts of indexcases and to the laboratory of origin (Geddes, 2006; Lim et al., 2004; Ristanović et al., 2020;Senio, 2003). Despite extensive contact tracing of early cases during the COVID-19 pandemic,there have been no reported cases related to any laboratory staff at the WIV and all staff in the laboratory of Dr. Shi Zhengli were said to be seronegative for SARS-CoV-2 when tested inMarch 2020 (WHO, 2021), with the laboratory reportedly following the appropriate biosafetyprotocols during their coronavirus work (Cohen, 2020). During a period of high influenzatransmission and other respiratory virus circulation (Liu et al., 2020a) reports of illnesses wouldneed to be confirmed as caused by SARS-CoV-2 to be relevant. Epidemiological modelingsuggests that the number of hypothetical cases needed to result in multiple hospitalized COVID19 patients prior to December 2019 is incompatible with observed clinical, genomic, andepidemiological data (Pekar et al., 2021).
The WIV possesses an extensive catalogue of samples derived from bats (Latinne et al., 2020)and has reportedly successfully cultured three SARSr-CoVs from bats – WIV1, WIV16 andRs4874 (Ge et al., 2013; Hu et al., 2017; Yang et al., 2015). Importantly, all three viruses aremore closely related to SARS-CoV than to SARS-CoV-2 (Ge et al., 2013; Hu et al., 2017; Yanget al., 2015). In contrast, bat virus RaTG13 from the WIV has reportedly never been isolated norcultured and only exists as a nucleotide sequence assembled from short sequencing reads(Cohen, 2020). The three cultured viruses were isolated from fecal samples through serialamplification in Vero E6 cells, a process that consistently results in the loss of the SARS-CoV-2furin cleavage site (Davidson et al., 2020; Klimstra et al., 2020; Liu et al., 2020b; Ogando et al.,2020; Sasaki et al., 2021; Wong et al., 2020; Zhu et al., 2021b). It is therefore highly unlikelythat these techniques would result in the isolation of a SARS-CoV-2 progenitor with an intactfurin cleavage site. No published work indicates that other methods, including the generation ofnovel reverse genetics systems, were used at the WIV to propagate infectious SARSr-CoVsbased on sequence data from bats. Gain-of-function research would be expected to utilize anestablished SARSr-CoV genomic backbone, or at a minimum a virus previously identified viasequencing. However, past experimental research using recombinant coronaviruses at the WIVhas used a genetic backbone (WIV1) unrelated to SARS-CoV-2 (Hu et al., 2017) and SARSCoV-2 carries no evidence of genetic markers one might expect from laboratory experiments(Andersen et al., 2020). There is no rational experimental reason why a new genetic systemwould be developed using an unknown and unpublished virus, with no evidence nor mention of aSARS-CoV-2-like virus in any prior publication or study from the WIV (Ge et al., 2012; Hu etal., 2017; Menachery et al., 2015), no evidence that the WIV sequenced a virus that is closer to SARS-CoV-2 than RaTG13, and no reason to hide research on a SARS-CoV-2-like virus prior tothe COVID-19 pandemic. Under any laboratory escape scenario SARS-CoV-2 would have tohave been present in a laboratory prior to the pandemic, yet no evidence exists to support such anotion and no sequence has been identified that could have served as a precursor.
A specific laboratory escape scenario involves accidental infection in the course of serial passageof a SARSr-CoV in common laboratory animals such as mice. However, early SARS-CoV-2isolates were unable to infect wild-type mice (Wan et al., 2020). While murine models are usefulfor studying infection in vivo and testing vaccines, they often result in mild or atypical disease inhACE2 transgenic mice (Bao et al., 2020; Hassan et al., 2020; Israelow et al., 2020;Rathnasinghe et al., 2020; Sun et al., 2020b). These findings are inconsistent with a virusselected for increased pathogenicity and transmissibility through serial passage throughsusceptible rodents. Although SARS-CoV-2 has since been engineered (Dinnon et al., 2020) andmouse-adapted by serial passage (Gu et al., 2020; Leist et al., 2020; Sun et al., 2020a), specificmutations in the spike protein, including N501Y, are necessary for such adaptation in mice (Guet al., 2020; Sun et al., 2020a). Notably, N501Y has arisen convergently in multiple SARS-CoV2 variants of concern in the human population, presumably being selected to increase ACE2binding affinity (Khan et al., 2021; Kuzmina et al., 2021; Liu et al., 2021; Starr et al., 2020). IfSARS-CoV-2 resulted from attempts to adapt a SARSr-CoV for study in animal models, itwould likely have acquired mutations like N501Y for efficient replication in that model, yetthere is no evidence to suggest such mutations existed early in the pandemic. Both the lowpathogenicity in commonly used laboratory animals and the absence of genomic markersassociated with rodent adaptation indicate that SARS-CoV-2 is highly unlikely to have beenacquired by laboratory workers in the course of viral pathogenesis or gain-of-functionexperiments.
Evidence from genomic structure and ongoing evolution of SARS-CoV-2
Considerable attention has been devoted to claims that SARS-CoV-2 was genetically engineeredor adapted in cell culture or “humanized” animal models to promote human transmission (Zhanet al., 2020). Yet, since its emergence, SARS-CoV-2 has experienced repeated sweeps ofmutations that have increased viral fitness (Deng et al., 2021; Otto et al., 2021; Simmonds, The first clear adaptive mutation, the D614G substitution in the spike protein, occurredearly in the pandemic (Korber et al., 2020; Volz et al., 2021). Recurring mutations in the receptorbinding domain of the spike protein, including N501Y, K417N/T, L452R, and E484K/Q—constituent mutations of the variants of concern—similarly enhance viral infectivity (Cai et al.,2021; Khan et al., 2021; Kuzmina et al., 2021) and ACE2 binding (Liu et al., 2021; Starr et al.,2020; Zhu et al., 2021a), refuting claims that the SARS-CoV-2 spike protein was optimized forbinding to human ACE2 upon its emergence (Piplani et al., 2021). Further, some pangolin derived coronaviruses have receptor binding domains that are near-identical to SARS-CoV-2 atthe amino acid level (Andersen et al., 2020; Xiao et al., 2020) and bind to human ACE2 evenmore strongly than SARS-CoV-2, showing that there is capacity for further human adaptation(Dicken et al., 2021). SARS-CoV-2 is also notable for being a host generalist virus (Conceicao etal., 2020), capable of efficient transmission in multiple mammalian species, including mink,tigers, cats, gorillas, dogs, raccoon dogs, ferrets, and large outbreaks have been documented inmink with spill-back to humans (Oude Munnink et al., 2021) and to other animals (van Aart etal., 2021). Combined, these findings show that no specific human “pre” adaptation was requiredfor the emergence or early spread of SARS-CoV-2, and the claim that the virus was alreadyhighly adapted to the human host (Zhan et al., 2020), or somehow optimized for binding tohuman ACE2, is without validity.
The genesis of the polybasic (furin) cleavage site in the spike protein of SARS-CoV-2 has beensubject to recurrent speculation. Although the furin cleavage site is absent from the closestknown relatives of SARS-CoV-2 (Andersen et al., 2020), this is unsurprising as the lineageleading to this virus is poorly sampled and the closest bat viruses have divergent spike proteinsdue to recombination (Boni et al., 2020; Lytras et al. 2020; Zhou et al., 2021). Furin cleavagesites are commonplace in other coronavirus spike proteins, including some felinealphacoronaviruses, MERS-CoV, most but not all strains of mouse hepatitis virus, as well as inendemic human betacoronaviruses such as HCoV-OC43 and HCoV-HKU1 (Gombold et al.,1993; de Haan et al., 2008; Kirchdoerfer et al., 2016). A near identical nucleotide sequence isfound in the spike gene of the bat coronavirus HKU9-1 (Gallaher, 2020), and both SARS-CoV-2and HKU9-1 contain short palindromic sequences immediately upstream of this sequence thatare indicative of natural recombination break-points via template switching (Gallaher, 2020).
Hence, simple evolutionary mechanisms can readily explain the evolution of an out-of-frameinsertion of a furin cleavage site in SARS-CoV-2 (Figure 2).
The SARS-CoV-2 furin cleavage site (containing the amino acid motif RRAR) does not matchits canonical form (R-X-R/K-R), is suboptimal compared to those of HCoV-HKU1 and HCoVOC43, lacks either a P1 or P2 arginine (depending on the alignment), and was caused by an outof-frame insertion (Figure 2). The RRAR and RRSR S1/S2 cleavage sites in felinecoronaviruses (FCoV) and cell-culture adapted HCoV-OC43, respectively, are not cleaved byfurin (de Haan et al., 2008). There is no logical reason why an engineered virus would utilizesuch a suboptimal furin cleavage site, which would entail such an unusual and needlesslycomplex feat of genetic engineering. The only previous studies of artificial insertion of a furincleavage site at the S1/S2 boundary in the SARS-CoV spike protein utilized an optimal‘RRSRR’ sequence in pseudotype systems (Belouzard et al., 2009; Follis et al., 2006). Further,there is no evidence of prior research at the WIV involving the artificial insertion of completefurin cleavage sites into coronaviruses.
The recurring P681H/R substitution in the proline (P) residue preceding the SARS-CoV-2 furincleavage site improves cleavage of the spike protein and is another signature of ongoing humanadaptation of the virus (Peacock et al., 2021a). The SARS-CoV-2 furin site is also lost understandard cell culture conditions involving Vero E6 cells (Ogando et al., 2020; Peacock et al.,2021b), as is true of HCoV-OC43 (Follis et al., 2006). The presence of two adjacent CGGcodons for arginine in the SARS-CoV-2 furin cleavage site is similarly not indicative of geneticengineering (Maxmen and Mallapaty, 2021). Although the CGG codon is rare in coronaviruses,it is observed in SARS-CoV, SARS-CoV-2 and other human coronaviruses at comparablefrequencies (Maxmen and Mallapaty, 2021). Further, if low-fitness codons had been artificiallyinserted into the virus genome they would have been quickly selected against during SARSCoV-2 evolution, yet both CGG codons are more than 99.8% conserved among the >2,300,000near-complete SARS-CoV-2 genomes sequenced to date, indicative of strong functionalconstraints (Supplementary Information, Table S1).
Conclusions
As for the vast majority of human viruses, the most parsimonious explanation for the origin ofSARS-CoV-2 is a zoonotic event. The documented epidemiological history of the virus iscomparable to previous animal market-associated outbreaks of coronaviruses with a simple routefor human exposure. The contact tracing of SARS-CoV-2 to markets in Wuhan exhibits strikingsimilarities to the early spread of SARS-CoV to markets in Guangdong, where humans infectedearly in the epidemic lived near or worked in animal markets. Zoonotic spillover by definitionselects for viruses able to infect humans. Although strong safeguards should be consistentlyemployed to minimize the likelihood of laboratory accidents in virological research, thoselaboratory escapes documented to date have almost exclusively involved viruses brought intolaboratories specifically because of their known human infectivity.
There is currently no evidence that SARS-CoV-2 has a laboratory origin. There is no evidencethat any early cases had any connection to the WIV, in contrast to the clear epidemiological linksto animal markets in Wuhan, nor evidence that the WIV possessed or worked on a progenitor ofSARS-CoV-2 prior to the pandemic. The suspicion that SARS-CoV-2 might have a laboratoryorigin stems from the coincidence that it was first detected in a city that houses a majorvirological laboratory that studies coronaviruses. Wuhan is the largest city in central China withmultiple animal markets and is a major hub for travel and commerce, well connected to otherareas both within China and internationally. The link to Wuhan therefore more likely reflects thefact that pathogens often require heavily populated areas to become established (Pekar et al.,2021).
We contend that although the animal reservoir for SARS-CoV-2 has not been identified and thekey species may not have been tested, in contrast to other scenarios there is substantial body ofscientific evidence supporting a zoonotic origin. While the possibility of a laboratory accidentcannot be entirely dismissed, and may be near impossible to falsify, this conduit for emergence ishighly unlikely relative to the numerous and repeated human-animal contacts that occur routinelyin the wildlife trade. Failure to comprehensively investigate the zoonotic origin throughcollaborative and carefully coordinated studies would leave the world vulnerable to futurepandemics arising from the same human activities that have repeatedly put us on a collisioncourse with novel viruses.
Holmes, E.C., Goldstein, S.A., Rasmussen, A.L., Robertson, D.L., CritsChristoph, A., Wertheim, J.O., Anthony, S.J., Barclay, W.S., Boni, M.F., Doherty, P.C., Farrar, J.,Geoghegan, J.L., Jiang, X., Leibowitz, J.L., Neil, S.J.D., Skern, T., Weiss, S.R., Worobey, M., Andersen,K.G., Garry, R.F., Rambaut, A., 1. Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life andEnvironmental Sciences and School of Medical Sciences, The University of Sydney, Sydney,NSW 2006, Australia.2. Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.3. Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, S7N5E3, Canada.4. MRC-University of Glasgow Centre for Virus Research, Glasgow, G61 1QH, UK.5. Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA94704, USA.6. Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.7. Department of Pathology, Microbiology, and Immunology, University of California DavisSchool of Veterinary Medicine, Davis, CA 95616, USA.8. Department of Infectious Disease, Imperial College London, W2 1PG, UK.9. Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania StateUniversity, University Park, PA 16802, USA.10. Department of Microbiology and Immunology, The University of Melbourne at the DohertyInstitute, 792 Elizabeth St, Melbourne, VIC 3000, Australia.11. The Wellcome Trust, London, NW1 2BE, UK.12. Department of Microbiology and Immunology, University of Otago, Dunedin 9010, NewZealand. Institute of Environmental Science and Research, Wellington 5022, New Zealand.13. Department of Biological Sciences, Xi'an Jiaotong-Liverpool University (XJTLU), Suzhou,China.14. Department of Microbial Pathogenesis and Immunology, Texas A&M University, CollegeStation, TX 77807, USA.15. Department of Infectious Diseases, King’s College London, Guy’s Hospital, London SE1 9RT,UK.16. Max Perutz Labs, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, A1030 Vienna, Austria.17. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania.Philadelphia, PA 19104, USA.18. Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721,USA.19. Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA92037, USA.20. Department of Microbiology and Immunology, Tulane University School of Medicine, NewOrleans, LA 70112, USA.21. Zalgen Labs, Germantown, MD 20876, USA.22. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3FL, UK.
