A response to “The Origins of SARS-CoV-2: A Critical Review”

By Alina Chan, on Twitter @ayjchan

Declaration of competing interests:

The author of this article is co-authoring a book, VIRAL: The Search for the Origin of Covid-19, with Matt Ridley. It will be published in late 2021 by HarperCollins. The book explores both natural and lab Covid-19 origin hypotheses.

Introduction

In this work, I summarize and assess the arguments for a natural origin and against a laboratory-based origin of Covid-19 presented in the Holmes et al. preprint [1]. The preprint review by Holmes et al. represents one of the most extensive compilations of arguments for a zoonotic origin and was authored by numerous experts in relevant fields. Although Holmes et al. “contend that there is substantial body of scientific evidence supporting a zoonotic origin for SARS-CoV-2” [1], I argue that all publicly available evidence and information are consistent with both natural and laboratory origin scenarios. In the absence of dispositive evidence in support of either a natural spillover or a research-related incident, it is necessary to rigorously investigate both hypotheses [2,3]. Only with more data and information can scientists confidently evaluate the likelihood of each origin hypothesis. A credible, transparent, evidence-based, and international investigation of the origin of Covid-19 is not only vital but also feasible [3–5].

A comparison of the origin stories of SARS-CoV and SARS-CoV-2

The review points out parallels between the emergence of SARS-CoV and SARS-CoV-2, but does not clarify critical differences between the two outbreaks. In short, in the first SARS epidemic, Chinese investigators more rapidly tracked down early cases in the province, likely animal sources, and a well-substantiated path for SARS-CoV to have been introduced into human beings via the trade of infected animals — and all of this had been achieved despite less sophisticated technology in the early 2000s.

The first SARS-CoV broke out twice in Guangdong province (the first time in late 2002 and the second time in late 2003). Based on the genetic data, each SARS-CoV outbreak was determined to have emerged from separate spillover events [6]. In both outbreaks, a considerable portion of the index patients were restaurant employees or other food handlers [7]. This spurred investigators to sample animals at live animal markets or restaurants, which successfully identified a variety of SARS-CoV-positive animals [8–11]. In the first outbreak, animals carrying SARS-CoV were detected via RT-PCR, virus isolation, and neutralizing antibody tests, and known to scientists by May 2003 [8]. This tracking of animal hosts of the virus was rapid considering both the relatively immature surveillance technology in 2003 and that the first SARS-CoV isolate from human patients was obtained only two months prior in March (and determined to be a coronavirus in late March) and genome sequenced in April [8,12].

Similarly, in the second outbreak, palm civets carrying SARS-CoV were even more swiftly detected at the workplace of an index patient and at an animal market; within only a few days of diagnosis of the index patient, samples were collected from civets and people at the patient’s workplace, a restaurant, and confirmed to be positive for SARS-CoV RNA and/or antibodies [9–11]. In addition, a May 2003 survey of 508 market traders in Guangdong exhibited higher seroprevalence (13%) against SARS-CoV compared to control populations (1.2% in healthy adults at a clinic for routine physical examinations, 2.9% in healthcare workers involved with SARS control), despite none of these traders being among the detected SARS epidemic cases [7,13]. These findings collectively pointed to an animal origin for SARS-CoV and indicated that SARS-related CoVs (SARSr-CoVs) were prevalent in the Guangdong animal trading community [6,14,15]. By mid-2005, scientists had traced SARSr-CoVs to bats, which were determined to be the natural reservoir [6,16].

Investigators now face the reverse situation for tracing the origins of SARS-CoV-2: a paucity of evidence and a cold trail. Closely related bat SARSr-CoVs have been identified, but there is still no sign of an intermediate host 1.5 years into the pandemic. The closest bat virus relatives have been found in Yunnan province about a thousand miles away from Wuhan in Hubei province [17–19]. Specifically, when SARS-CoV-2 was detected in Wuhan in 2019, the Wuhan Institute of Virology (WIV) had already been characterizing nine of its closest virus relatives at the time (one of the nine, RaTG13 is still the closest genomic match to SARS-CoV-2) [20,21]. Other closely related viruses have since been described [17,22–25].

Scientists widely agree that an ancestral form of SARS-CoV-2 has its origin in bats so it is unsurprising that related bat coronaviruses continue to be found in nature. The question is what was the journey that a bat virus ancestor of SARS-CoV-2 took before becoming SARS-CoV-2 and starting an outbreak in Wuhan? Wuhan is not located proximally to where close virus relatives to SARS-CoV-2 have been found, and is not a place where some leading coronavirus experts would have predicted human pathogenic SARSr-CoVs to emerge [26–29]. In fact, the Wuhan human population was used as a negative control in a 2015 serological survey to look for SARSr-CoV spillover: of 240 blood donors in Wuhan, none (0.0%) had antibodies against SARSr-CoVs; in comparison to the 2.7% seropositivity observed in 218 Yunnan residents living close to bat colonies [30].

Despite today’s advanced technologies and extensive searches for an intermediate host of SARS-CoV-2, none of the tested market or Wuhan animals carried SARS-CoV-2 according to the China-WHO joint study report [31]. What exactly was sampled? According to the China-WHO joint study, “more than 80 000 wildlife, livestock and poultry samples were collected from 31 provinces in China and no positive result was identified for SARS-CoV-2 antibody or nucleic acid before and after the SARS-CoV-2 outbreak in China”; of these, 38,515 were livestock and poultry samples and 41,696 were wild animal samples, all collected during 2018 to 2020 [31]. Furthermore, in the case of SARS-CoV-2, market workers were highly represented among symptomatic, hospitalized index cases [18]. This contrasts what was observed in the 2003 SARS epidemic in which animal sellers had been determined to be frequently exposed to SARSr-CoVs and were underrepresented among the SARS epidemic clinically confirmed cases [7,13,32]. Due to the large 2019 outbreak in Wuhan including market workers among index cases, it is no longer straightforward to test whether Wuhan market workers had a high level of exposure to SARSr-CoVs prior to the Covid-19 outbreak. They could have been infected during the outbreak and many infected individuals develop only mild symptoms.

In other words, there is zero evidence that animal sellers at the Huanan seafood market had been frequently exposed to animals carrying SARSr-CoVs or that natural spillovers of SARSr-CoVs would be expected in Wuhan prior to the emergence of SARS-CoV-2.

The furin cleavage site

Next, the review highlights the presence of furin cleavage sites (FCS) in other coronaviruses that infect human beings. Briefly, the particular FCS of interest in the spike of SARS-CoV-2 plays a critical role in the virus’ ability to grow in different types of host cells and cause severe disease [33,34]. Similar FCSs can indeed be found in other human and animal pathogenic coronaviruses, but among all known SARSr-CoVs (lineage B betacoronaviruses or sarbecoviruses), SARS-CoV-2 is the only member with an inserted FCS at the spike’s S1/S2 junction. Despite the discovery of a growing number of viruses closely related to SARS-CoV-2, not a single one has been found with such an FCS insertion. Holmes et al. rightly point out later in the review that the lineage leading to SARS-CoV-2 is poorly sampled and the spikes in closely related SARSr-CoVs are divergent and prone to recombination. So it is possible that as more SARSr-CoVs are collected in the wild, we will find another rare strain that has a similar S1/S2 FCS insertion as SARS-CoV-2. Or, we may continue to sample dozens or even hundreds more close relatives of SARS-CoV-2 without ever finding one with an S1/S2 FCS insertion.

In addition, Holmes et al. assert that it is highly unlikely that growing viruses in VeroE6 cells would retain the FCS, which has been observed to be lost after serial passaging in Vero cells [35–40]. Although Vero cells (green monkey kidney cells) are commonly used for growing lab strains of viruses, they are not the only cell type used by the WIV which has previously described an array of cell lines derived from different organs of different animal species for isolating novel viruses found in nature [41]. For example, to isolate a novel swine coronavirus of bat origin, they tried Vero cells, five bat cell lines, and six swine cell lines. We do not know what other cell lines are available in Wuhan labs for isolating and studying novel coronaviruses. We know that the FCS confers an advantage to SARS-CoV-2 and would not be lost during serial passage in some cell lines such as a human respiratory cell line, Calu-3 (a human lung adenocarcinoma cell line), which has also been used at the WIV to study SARSr-CoVs [34,42].

Finally, Holmes et al. speculate that this is not how a scientist would logically engineer a novel S1/S2 FCS into a SARSr-CoV and they contend that there is no evidence of research at the WIV that artificially inserted complete FCSs into coronaviruses. They also push back on the argument that the codon usage in the FCS insertion suggests human intervention [43,44]. These arguments for or against the artificial insertion of an FCS are difficult to substantiate on either side because we have little insight to the viruses and virus sequences available to scientists and the experiments being conducted in labs prior to the detected Covid-19 outbreak — these would have a great influence on the types of insertions being made [45]. Ultimately, the FCS of SARS-CoV-2 may have evolved naturally or it may have been inserted by scientists. The genomic sequence alone cannot definitively support or rule out either hypothesis [46,47]. Importantly, a laboratory-based origin of SARS-CoV-2 is not dependent on an artificial insertion of the FCS.

The geographic distribution of early Covid-19 cases

The Holmes et al. review emphasizes the density of early cases (mapped by patient home address) and excess pneumonia deaths in January 2020 north of the Yangtze river, i.e., the part of the city where the Huanan market is located (see Figure 1 of Holmes et al.) [1,31].

Figure 1 | Phylogenetic and epidemiological data on the early COVID-19 pandemic in Wuhan. (a) Phylogenetic tree of early SARS-CoV-2 genomes sampled from Wuhan during December 2019-January 2020. The split between lineages A and B is labelled with the coordinates and base of the two differentiating nucleotide mutations. Cases with a known association to the Huanan or other markets are denoted by symbols (reported in ref. 10). (b) Map of districts of Wuhan showing the location of markets, the BSL-4 campus of the Wuhan Institute of Virology (where the coronavirus work of Dr. Shi Zhengli is performed) and the earliest known cases. (c-e) Location of recorded COVID-19 cases in Wuhan from 8th December to 31st December 2019. Cases with a home address outside of Wuhan city are not shown. (f-h) Map of districts of Wuhan indicating the first record of excess deaths due to pneumonia (shaded green) from 15th January 2020. Case and excess death data were extracted and redrawn from figures provided in ref 10. For more details see supplementary information.

Figure and caption from Holmes et al. [1]: Holmes, Edward C, Goldstein, Stephen A, Rasmussen, Angela L, Robertson, David L, Crits-Christoph, Alexander, Wertheim, Joel O, … Rambaut, Andrew. (2021, July 7). The Origins of SARS-CoV-2: A Critical Review (Version 1.0). Zenodo. http://doi.org/10.5281/zenodo.5075888. These are copyright of the authors, made available under a CC-BY-NC-ND 4.0 International license. The material has not been adapted. It is important to note that the Holmes et al. map data was extracted from figures in the China-WHO joint study report using Adobe Illustrator, presumably because there is no public access to the map data used by the China-WHO team.

This region of Wuhan is where the Huanan seafood market is located, but it is also characterized by higher population density that happens to be elderly; both the higher density of the general population and the elderly are factors that could be expected to concentrate the detected Covid-19 case load. In February 2020, Wuhan experts from the Wuhan Land Use and Urban Spatial Planning Research Center and Huaqiao University published a map of Wuhan showing the population density of all age groups (see Figure 1 of Jia et al.) [48]. The density of the general population and especially the elderly population in Wuhan city closely resemble the distribution of early Covid-19 cases by home address [49].

Figure 1. Location of Wuhan metropolitan area and population density of all age groups. (a) The location of Wuhan city and the red border is Wuhan metropolitan area; (b) The density of the total population; © The density of the children population; (d) The density of the adult population; (e) The density of the elderly population. Source: Authors.

Figure and caption from Jia et al. [48]: Jia, Y.; Zheng, Z.; Zhang, Q.; Li, M.; Liu, X. Associations of Spatial Aggregation between Neighborhood Facilities and the Population of Age Groups Based on Points-of-Interest Data. Sustainability 2020, 12, 1692. https://doi.org/10.3390/su12041692 These are copyright of the authors, made available under a CC BY 4.0 International License. The material has not been adapted.

It is also worth bearing in mind that the criteria by which early cases had been identified suffered from ascertainment biases (see Annex E3 of the China-WHO joint report) [31]. Specifically, exposure to the Huanan market was one of the key factors in determining whether a patient was a suspected case of Covid-19. Cases were marked as suspect if they were linked to “related markets in Wuhan” and presented at least two clinical manifestations, i.e., fever, (pulmonary) imaging characteristic of pneumonia, reduction in white blood cell count or lymphocyte count, or no significant improvement in response to three days of antibiotic treatment. Eventually, these criteria was expanded to evaluate cases with links to other fever or respiratory disease patients or Covid-19 clusters. Based on this information, it is plausible that the initial approach of identifying Covid-19 cases may have been biased towards cases with links to the Huanan market and its cluster of cases, as well as the elderly who are concentrated in that area North of the Yangtze and are more likely to exhibit multiple clinical manifestations when infected by SARS-CoV-2.

The Holmes et al. review points out that the Wuhan Institute of Virology (WIV) BSL-4 campus (one of two WIV campuses) is located south of the Yangtze in Jiangxia district. The second WIV campus (not indicated in Holmes et al.’s Figure 1 map) is located in Wuchang district where early Covid-19 cases had also been detected; this campus is ~14 km from the Huanan Seafood Market and one can get from the WIV campus to the market on Line 2 of the Wuhan Metro with ~30-min train ride. The Jiangxia WIV campus was serviced by a regular shuttle bus from the Chinese Academy of Sciences nearby the Wuchang WIV campus [50]. Information on which campus the SARSr-CoV research and sample storage had been located is not publicly available, although we now know that the live virus SARSr-CoV work at the WIV had been performed in its BSL-2 lab [51]. In any case, readers have insightfully commented that the home addresses of WIV staff may not be proximal to either campus [52] so a map of early cases plotted by their home addresses would not reveal their occupations or where the initial introduction of the virus into humans occurred. It is worth noting that Wuhan is the capital of Hubei province with a population of ~11 million and a modern rapid transit Metro system. Workers can get from home to work each day, traversing long distances in reasonable amounts of time.

Previous examples of natural spillovers and lab-acquired infections illustrate the importance of tracking down the earliest patients and identifying their exposures to various possible sources of the pathogen. For example, when a Taiwanese researcher was accidentally infected with SARS-CoV while working in a BSL-4 laboratory in 2003, he only developed symptoms after taking an international flight to attend a conference in Singapore and taking another international flight back to Taiwan [53]. If the patient had become infectious during his travels, the location of a resulting cluster could have been in Singapore or traced back to one of the international flights, as opposed to where the lab was located in Taipei. In another example, a Beijing researcher who was accidentally infected with SARS-CoV in 2004 was not actually the first case in the cluster to be diagnosed; a nurse that had treated the researcher was the first diagnosed with SARS and several other people had been infected by then [54]. Furthermore, the researcher had traveled long distances via train across China while symptomatic. Another three researchers at the same institute were later found to have been separately infected with SARS-CoV and an estimated eleven people were infected by SARS-CoV from the Beijing institute [55,56]. In this saga, close to 1000 people were quarantined or sent for medical supervision.

The missing intermediate host

A key point is understanding what live animals were sold in Wuhan and whether these may have been the proximal animal source of SARS-CoV-2, leading to the 2019 outbreak. On their trip, the independent experts who were part of the China-WHO joint study on the origins of SARS-CoV-2 met with witnesses who had shopped at Huanan for decades. These witnesses said that they had never seen live animals being sold at the market [31]. Yet, the Huanan seafood market was recently revealed to have sold live wild animals including those that might be susceptible to infection by SARS-CoV-2, albeit no bats or pangolins were detected at any Wuhan market during the study period of May 2017 through November 2019 [57]. Nonetheless, winter (including November 2019) could be described as the off-season for wildlife trade in Wuhan markets; few animals, including those susceptible to Covid-19, were on sale in Wuhan markets at the time.

Despite extensive sampling, no animals found at the Huanan seafood market have tested positive for SARS-CoV-2 (see tables 3 and 4 on page 99 of the China-WHO report). According to the China-WHO joint study report, a total of 457 Huanan market samples from animals were collected and tested between 1 January and 2 March 2020 [31]. These were from 188 individual animals spanning 18 species such as rabbit, snake, badger, cat, bamboo rat, rat, chicken, salamander etc.

Sampling of Huanan market suppliers has also not uncovered any animal source of SARS-CoV-2. Animals raised by Huanan market suppliers in Hubei province were also sampled and tested (616 samples, 10 species); wild animals in the Southern Chinese provinces of Yunnan, Guangdong, and Guangxi were also tested (1287 samples, 27 species including pangolins, civets, bats, bamboo rats, macaques, bear monkeys, porcupines, foxes etc.) — none were positive for the virus (see tables 5.1 and 5.2 on page 100 of the China-WHO report) [31].

A further 1914 serum samples were collected from 35 species between November 2019 and March 2020 in Wuhan and its surrounding areas; a total of 27,000 samples of wild animals spanning 208 species were collected across China between May and September 2020; 6,811 animal samples collected across China between 2015 to 2019 were retrospectively tested — no SARS-CoV-2-specific antibodies were detected (see tables 8, 9 and 10 on pages 103–106 of the China-WHO report) [31].

In response to this absence of an intermediate host for SARS-CoV-2, the Holmes et al. preprint writes “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 SARS-CoV-2.” It is unclear what data supports this statement. Animals susceptible to SARS-CoV-2 were represented among the animal samples collected from the Huanan seafood market, its suppliers, and other animal populations across China (please see Annex F of the China-WHO joint study report) [31]. There is no publicly available data to show which species of live animals had been on sale at the Huanan seafood market in the winter of 2019, and whether these included racoon dogs in particular. However, the China-WHO joint study reports that racoon dogs were sampled in December 2019, among 2328 samples (69 species), from breeding sites, tourist areas, and zoos in Hubei — none were positive for the virus [31]. Unfortunately, farms that had supplied wild animals to Wuhan had been ordered by Chinese officials to shut down without testing for the virus [58].

These questions remain:

  • What animals were on sale in Wuhan markets in the winter of 2019? Were the 457 animal samples from the Huanan seafood market representative of these animals on sale in the winter of 2019?
  • Why were farms that supplied wild animals to Wuhan shut down without testing for the virus to definitively track down the source?
  • In the absence of any animal samples positive for the virus, what is the evidence of an intermediate host?

The multi-market hypothesis

There were two early lineages of SARS-CoV-2: lineage A and lineage B. Importantly, although classified as different lineages, the earliest genomes in each lineage only differ from each other by two letters (nucleotides) out of a total of >29.9K genomic letters. This is a very minute difference that would not, by itself, suggest multiple spillovers of the virus into the human population. Lineage B, but not lineage A, was found in cases linked to the Huanan seafood market and the samples collected from its environment (surfaces such as doors, floors, trash cans, sewage and drains, toilets and public areas).

Holmes et al. speculate that SARS-CoV-2 may have included multiple spillover events across multiple markets, pointing to the market exposure of lineage A Covid-19 cases and the possible transfer of infected animals between markets in Wuhan via shared supply chains. As we already covered above, although the proximal animal sources of SARS-CoV were readily and robustly detected, this is not the case for SARS-CoV-2. Moreover, the definition of a “market exposure” appears to be broad in the China-WHO report cited by Holmes et al. [1,31]. For example, according to the China-WHO joint study, the market that the alleged first known Covid-19 case (symptom onset on 8 December) was exposed to was an RT-Mart in the Jiangxia district (see page 178 in the Annexes of the China-WHO report); RT-mart is a chain of hypermarkets comparable to a Walmart or Costco where it would have been very unlikely for live wild animals to be sold in Wuhan, a high-tech metropolitan city. This first patient had no links to the Huanan market and none of their family members or other contacts had any history of exposure to the Huanan market. The China-WHO report further noted that “After the first case with history of exposure to Huanan Market appeared on December 11, Huanan Market-related cases increased rapidly, and reached a peak (nine cases) on 25 December 2019” [31].

On the basis of the available genetic and epidemiological information, experts have stated that it is more probable that the progenitor of all detected SARS-CoV-2 genomes to date was introduced into human beings a single time, as opposed to multiple spillovers at multiple locations [59,60].

Peer-reviewed research articles on the topic have also considered the relationship among early sequences to be consistent with a single introduction of the virus into the human population [61,62]. In my opinion, Jesse Bloom explained this best in a recent preprint [60]:

Another explanation that I consider less plausible is offered by Garry (2021): that there were multiple zoonoses from distinct markets, with the Huanan Seafood Market being the source of viruses in clade B, and some other market being the source of viruses that lack T8782C and C28144T (Figure 3). However, this explanation requires positing zoonoses in two markets by two progenitors differing by just two mutations, which seems non-parsimonious in the absence of direct evidence for zoonosis in any market.

Discussion of lab origin hypotheses

Holmes et al. (in my view, correctly) reject one of the more speculative lab origin hypotheses that RaTG13 (96% genome match, the highest genomic similarity to SARS-CoV-2) may have been the progenitor of SARS-CoV-2. The review does this by describing viruses closely related to SARS-CoV-2 found in bats and pangolins in China, Japan, Thailand, and Cambodia. Holmes et al. note the significant evolutionary gap between SARS-CoV-2 and all these viruses, equating to “decades of evolutionary divergence” [1,63]. They also point out that some of the bat viruses characterized after the emergence of SARS-CoV-2 could share a more recent common ancestor with SARS-CoV-2 in comparison to RaTG13. However, Holmes et al. do not address the troubling sample history of RaTG13, which was collected in 2013 by the WIV from a Yunnan mine where people had sickened with a SARS-like respiratory illness [44,64]. Out of 6 infected, 3 died — a 50% mortality rate — although, fortunately, none of the patients transmitted their illness to their families or the healthcare workers attending to them. According to Dr. Shi Zhengli of the WIV, they had suspected that the miners had been infected with an unknown virus so multiple groups of scientists had sampled animals in and around the mine; between 2012 and 2015, Dr. Shi’s group made one or two visits a year and found at least nine SARSr-CoVs including RaTG13, which was full genome sequenced in 2018 [20].

Further, Holmes et al. argue that previously documented laboratory escapes have largely (excepting Marburg virus) involved known human pathogens and that past recombinant virus work at the WIV used a specific WIV1 genetic backbone. They press the point that there is no available evidence pointing to the presence of SARS-CoV-2 in a laboratory prior to the pandemic. However, Holmes et al. do not acknowledge that not all research and newly discovered viruses are published in a timely manner or, in some cases, at all. For instance, the other eight SARSr-CoVs collected by the WIV in 2015 from the Yunnan mine with the sick miners were only revealed to the public in 2020 [19]; their origin was only clarified in November 2020 [20]; and the full sequence of only one of the eight viruses was released in May 2021 (the authors reported that the eight viruses shared 99.7% genome sequence identity) [21].

To compound these challenges, some of the work at the WIV is classified. The Washington Post recently obtained documents revealing that WIV employees signed pledges in May 2019 to protect confidential information; in November 2019, students working on classified topics were told that their papers may not be published publicly, but they should submit their reports internally to a review team and confidentiality committee with a copy of a contract indicating the confidentiality of the project; and the WIV had discussions, protocols and training sessions to manage classified projects, lab responsibilities under China’s state secrets law, disclosing information to foreigners, confidentiality management when hosting foreigners, and the sealing of some research reports for up to two decades [65].

In November 2019, students working on classified topics were told that their papers may not be published publicly, but they should submit their reports internally to a review team and confidentiality committee with a copy of a contract indicating the confidentiality of the project.

The Holmes et al. review does not address the more plausible lab origin hypotheses. These include: (1) research personnel being infected while sampling or handling samples from thousands of animals and even humans in the known SARSr-CoV spillover zone [19,30,66]; (2) WIV research or non-research personnel being accidentally exposed to live viruses, natural or recombinant, being experimented with at biosafety level 2 [51]. To rule out these plausible lab origin hypotheses, it will be at minimum necessary to know what samples the WIV had collected from animals and humans (where and when) and obtain access to the currently missing WIV pathogen database that was taken offline in September 2019 [67]. Gaining access to comprehensive Wuhan research and personnel records and being able to confidentially interview relevant individuals through a safe whistleblower mechanism would boost confidence in an investigation of possible lab origins. For more suggestions of what a rigorous investigation should include, please see the open letter published by Butler et al., of which I am a co-author [68]. Without these data and information, it would be challenging to understand what viruses and genetic backbones were in Wuhan lab collections or studied in Wuhan labs, what experiments were being conducted in Wuhan or with research collaborators, and whether any Wuhan research institute personnel and/or their contacts were among the early Covid-19 cases.

On the last point about the tracking of early cases, the Chinese authorities have remained firm that none of the detected early cases were linked to the WIV, and Dr. Shi had reported that none of the WIV laboratory staff were seropositive for SARS-CoV-2 when tested in March 2020 [1,31]. However, there is contentious debate on intelligence that could point to multiple WIV researchers exhibiting Covid-19-like symptoms and going to the hospital in late 2019 (some reports have specified November 2019) [69]. It is unclear whether these WIV researchers had Covid-19 and, if so, how severe their illness was. It is also unknown what the protocol is when a Wuhan researcher working at BSL-2 falls ill with symptoms that could easily be attributed to a common cold or flu. A recent analysis suggests that human-to-human transmission of SARS-CoV-2 likely began between mid-October to mid-November 2019 in Hubei [70]. Nonetheless, if new data on early sequences were to emerge, this could substantially revise estimates of when the virus might have been introduced into the human population [71].

In their review, Holmes et al. also discuss a specific laboratory escape scenario involving laboratory animals. Holmes et al. then narrow in on an even more specific scenario of a virus having been selected for increased pathogenicity and transmissibility through serial passage through rodents; and speculate that if such attempts had been made we would observe some of the more recently detected mutations in mouse adaptation studies and in the general human population infected with SARS-CoV-2. In this discussion, it is unclear why the authors focused on SARS-CoV-2’s inability to infect wild-type mice and the types of mutations seen in mouse adaptation studies, when the animal models used by the WIV are humanized mice (expressing the human ACE2 receptor utilized by SARS-CoV, SARS-CoV-2, and other SARSr-CoVs in their collection) as well as civets (the intermediate host of SARS-CoV) as reported by Dr. Shi in a July 2020 interview with Science magazine [27,72]. Surprisingly, these arguments relying on an unlikely wild-type mouse adaptation laboratory origin scenario are used to claim that “SARS-CoV-2 is highly unlikely to have been acquired by laboratory workers in the course of viral pathogenesis or gain-of-function experiments” [1].

Was the virus pre-adapted to human beings?

In response to claims that the virus had been adapted in the lab to better utilize human ACE2 and infect human cells, Holmes et al. point to the singular first clear adaptive mutation, D614G, and some recurring mutations in the ongoing pandemic of more than 187 million confirmed cases. They also emphasize that SARS-CoV-2 is a “generalist” virus that can infect numerous mammalian species; therefore, no human-specific pre-adaptation was required for the emergence of the virus [50].

Before critiquing this argument, it is important to first note that the possible pre-adaptation of the virus is consistent with both natural and lab-based origins. We know too little about the earliest stages of the outbreak to determine how SARS-CoV-2 evolved its abilities to effectively infect and transmit among people. Some experts have ventured that SARS-CoV-2, after its spillover or first introduction into human beings, may have been transmitting at a low level among people and picking up adaptive mutations before its first detected outbreak in Wuhan. Unfortunately, banked blood samples have not been tested to determine when SARS-CoV-2 might have first emerged in human beings [73,74]. Whether SARS-CoV-2 is pre-adapted or not does not support or rule out either natural or lab origin hypotheses.

The first SARS-CoV was also a generalist virus that could infect numerous mammalian species; indeed, several animal hosts infected by SARS-CoV, including civet, badger, and racoon dog, were rapidly found in markets and restaurants in Guangdong after index cases were identified (see earlier discussion at beginning of article) [8]. SARS-CoV had only infected upwards of 8,000 people in the entirety of the 2003 epidemic [75]. Yet, adaptive mutations had been rapidly acquired in the early months of its transmission among humans. If SARS-CoV had been allowed to infect close to 200 million human beings, even after having rapidly adapted to its new host species in the first few months, it is very probable that recurrent mutations leading to greater human-to-human transmissibility would have been detected.

The key difference in the origin story of SARS-CoV versus that of SARS-CoV-2 is that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had rapidly developed not only one but several adaptive mutations for human transmission [76]. In comparison to the single D614G early adaptive mutation in SARS-CoV-2, a dozen of these adaptive mutations were observed and experimentally validated in the small region of the spike receptor binding-domain of SARS-CoV [76–79].

Conclusion

Holmes et al. have written an extensive argument for a natural origin of SARS-CoV-2 and importantly urge a comprehensive, collaborative, and careful investigation of possible zoonotic origins of the virus. However, their review does not address plausible lab origin scenarios where a virus may have infected a researcher during fieldwork or in the lab. It is too confident in dismissing the possibility that the virus may have been cultured, manipulated, and even engineered or recombined in the laboratory without leaving obvious signs of human interference. Techniques to synthesize entire virus genomes without leaving traces have existed for years and have been used by labs around the world; these can easily evade detection strategies devised by scientists, especially when not all viruses and sequences being studied in labs are shared publicly in a timely manner [80,81].

While the review elaborates on what the authors consider to be compelling (all circumstantial) evidence for a natural origin of SARS-CoV-2, it does not adequately discuss the substantial body of (all circumstantial) evidence for a laboratory-based origin of SARS-CoV-2. If the argument for the lack of direct evidence for a natural origin is that not enough efforts have been made to sample animals, then one should apply the same standards to evaluating lab origin scenarios and advocate for access to relevant samples, data, and information. As numerous scientists have already stated, we must rigorously investigate both natural and laboratory origin hypotheses to prevent future pandemics [2,45,68,82–88].

We must rigorously investigate both natural and laboratory origin hypotheses to prevent future pandemics.

Summary:

  • The 2003 epidemic SARS-CoV was quickly traced to proximal animal sources (intermediate hosts) of the virus. Yet, despite greatly improved surveillance technologies and capabilities, an intermediate host for SARS-CoV-2 has still not been found more than 1.5 years since the virus was detected. Tens of thousands of animal samples including those from susceptible host species in Wuhan, Hubei, and across China have all tested negative for the virus.
  • The geographic distribution of early Covid-19 cases appears to overlap with densely populated Wuhan districts with higher proportions of the elderly individuals, which is a plausible alternative explanation to the location of the Huanan seafood market. Furthermore, sampling of cases around the Huanan seafood market may suffer from ascertainment bias because one of the criteria used initially to determine cases was exposure to the Huanan seafood market and related markets.
  • The fact that the home addresses of early cases are not concentrated close to WIV campuses is a weak argument. Previous natural spillovers and lab escapes illustrate the importance of tracking down early patients and identifying their exposures to various possible sources.
  • Furin cleavage sites exist in other coronaviruses, but have not been found in sarbecoviruses. Despite the discovery of a growing number of close virus relatives of SARS-CoV-2, an S1/S2 FCS insertion remains unique to SARS-CoV-2.
  • We have little insight into the viruses and virus sequences available to scientists and the experiments being conducted in labs prior to the Covid-19 outbreak. Varied cell types were used to culture viruses. It is premature to predict what sequences may have been engineered or that the cells used would have selected against furin cleavage sites.
  • The existing genetic and epidemiological data point to a single introduction of SARS-CoV-2 into humans. An origin hypothesis involving multiple spillovers from animals to humans at multiple locations is not parsimonious, especially given that there is still no direct evidence for spillover at even a single market.
  • Further mutation of SARS-CoV-2 during the pandemic does not contradict the hypothesis that the virus was pre-adapted to human infection when detected in December 2019. Both SARS-CoV and SARS-CoV-2 are generalist viruses. If the 2003 SARS epidemic had been allowed to expand, recurrent mutations would likely have been observed.
  • Plausible lab origin scenarios were not addressed by Holmes et al., who focused on furin cleavage site insertions and unlikely experiments with wild-type mice. The available data and information remain consistent with both natural and lab origin hypotheses.

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Alina Chan is a postdoctoral researcher with a background in medical genetics, synthetic biology, and vector engineering. On twitter @ayjchan

Alina Chan is a postdoctoral researcher with a background in medical genetics, synthetic biology, and vector engineering. On twitter @ayjchan