Is Wuhan Institute of Virology Responsible for COVID-19?

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Is Wuhan Institute of Virology Responsible for COVID-19? Mar 3, 2020 6:26:24 GMT

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Post by Admin on Mar 3, 2020 6:26:24 GMT

Where are the original sources from?
The evolutionary network suggests that the hypothesized haplotype mv1 may be from an intermediate host or the first infected humans. From those connections, both H13 and H38 would be suggested as ancestral haplotypes. The SH-like approximate likelihood ratio test showed both haplotype H13’s group and H38 (with H45) could be the most basal clades in phylogenies of the 58 haplotypes (Figure S4), but phylogenetic information of the alignment was informative (Figure S5). Two main evolutionary paths of available haplotypes can be from H13 through H3 to H1, or from H38 through H3 to H1 (Figure 3C). Both scenarios demonstrate H3 was the key connection from an ancestral haplotype to H1. Neither H13 nor H38 has samples from Wuhan (Hubei) (Figure 3). H13 was only recovered from five Shenzhen (Guangdong) samples, including the father (patient 2) of the familial cluster, who was one of the first identified infected patients in Guangdong.13 Two derived haplotypes were also only found in Shenzhen (H14 from the grandson of patient 2), and the other three haplotypes were found in three samples from Japan and one sample from Arizona in the United States (Figure 3). According to an epidemiological study, the Shenzhen family traveled to Wuhan after the outbreak was announced, and they could have been infected during their visit in Wuhan from a hospital or an unknown common source13. This suggests that H13 should have originated from Wuhan, but none of the available samples from Wuhan encode haplotypes in Group A. Genetically, haplotypes of Group A have links to only Wuhan haplotype H3 (only one sample EPI_ISL_406801, with no link to the Hua Nan market).

It is possible that H13 was newly derived from H3 in the family from Shenzhen (Figure 3C) and did not spread in Wuhan, or that no samples have been sequenced yet. However, this scenario is not supported by the evolutionary network. H38 has three genomes from the same patient (Table S1), who was the first identified infected patient in the United States.14 He should have been infected while visiting his family in China. The original source of H38 can be explained as that of H13, which is also derived from H3, and the derived H45 was from a Chongqing patient who was working in Wuhan.

The H3 haplotype has only one sample from Wuhan, which was not linked to the Hua Nan market,9 and the other samples in this group were from other countries and regions (Figure 3). Noteworthily, all the samples from the Hua Nan market had the H1 haplotype or its derived haplotypes (H2, H8-H12, see Figure 2 and Table S1), indicating that there were circulated infections within the market in the short term. It is possible that SARS-CoV-2 in the Hua Nan market had been transmitted from other places (Figure 3D), or at least, that Hua Nan market did not host the original source of SARS-CoV-2. As the first identified infected patients had no link to the market,11 it is possible that infected humans transmitted SARS-CoV-2 to workers or sellers in the market, after which it rapidly circulated there. The crowded market boosted SARS-CoV-2 transmissions to buyers and spread it to the whole city in early December 2019, corresponding to the estimated population expansion time.

Regional and worldwide circulation and spread
Of the 54 genomes from patients in China, Chongqing (3 samples), Guangdong (18), Hubei (22), Taiwan (2), and Zhejiang (4) have more than two samples, and the other five provinces sequenced one sample. Hubei (Wuhan) samples from 24 December 2019 to 5 January 2020 encoded 13 haplotypes, belonging to Groups C (H1 and 11 satellite haplotypes) and B (only H3). These relationships indicate a rapid transmission and circulations of SARS-CoV-2 in Wuhan at an early stage of transmission. H1 (no satellite haplotypes) and H3 haplotypes are the ancestors of haplotypes out of Wuhan/Hubei. Eighteen Guangdong samples, collected from 10 to 23 January 2020, encoded 15 haplotypes, belonging to Groups A, C and E, showing that there were multiple sources imported into Guangdong. Three haplotypes (H14, H15 and H17) may have evolved locally, indicating that human-to-human transmissions happened when SARS-CoV-2 initially spread to Shenzhen of Guangdong.13 Two samples from Taiwan encoded H3 and H24 in Groups B and D, respectively, and three samples from Chongqing encoded H1, H40, and H45 in Groups B and C, respectively. There were two sources imported into these two provinces/regions. Four Zhejiang samples encoded H1 and H24 in Group C, which was only imported from the source of the H1 haplotype.

Phylogenetic approaches provide insight into the epidemiology of SARS-CoV-2
Epidemiological study of SARS-CoV-2 using traditional approaches is very difficult, because it was not identified as a new coronavirus until 29 December, and some infected people with mild symptoms or without symptoms16,40 may have been overlooked in late November and early December. Moreover, the Hua Nan market, which was considered as the birthplace of SARS-CoV-2, has been closed since 1 January 2020. In this study, we have used genomic data for SARS-CoV-2 to infer evolutionary relationships of the 58 haplotypes, and to suggest that H1 and its descendant haplotypes from the Hua Nan market should be derived from the H3 haplotype, which was not linked to the market (Figure 3C). This suggests that the source of the coronavirus in the Hua Nan market was imported from elsewhere, as also suggested by other researchers.12 The phylogenetic network indicated that H13 and H38 should be ancestral haplotypes that connected to the outgroup bat-RaTG13-CoV through a hypothesized intermediate haplotype. Because the currently available samples do not include the first identified infected patient and other patients from early December, the most common ancestral haplotype might be missed. If there are any frozen samples from those patients, it would be worth doing genomic sequencing for phyloepidemiologic study to help to locate the birthplace of SARS-CoV-2 in Wuhan. Meanwhile, we expect that the H13 and H38 haplotypes will be found in some samples of infected patients in Wuhan if more samples are sequenced in future. This will be very important in the search for the original sources of SARS-CoV-2, because both H13 and H38 tend to be ancestral haplotypes.

The evolutionary network of haplotypes can be used to recover the directions of human-to-human transmissions at the local scale and spread at the larger scale. The central haplotype can be considered as the super-spreader haplotype, and the tip haplotype is the most recent descendant haplotype. The transmission direction can be identified using the connection information of tips and branches. For example, the confirmed patients from the Hua Nan market shared the common ancestral haplotype H1, indicating they were infected from a common source, who may have been a super-spreader in the market. This transmission phenomenon may also have happened in Shenzhen with the patients of Group A. This approach has recovered specific directions of human-to-human transmission in the Shenzhen family (H13 → H14), the Queensland tour group (H25 → H26), the England family (H28 → H29), and the repatriated Japanese from Wuhan (H53 → H52). Most international infections link to Wuhan directly or indirectly, but for some of them it is not clear exactly where they were infected. As discussed above, we have found that some patients in Japan and United States might have been infected in Guangzhou, and one patient in France might have been infected in Chongqing or Singapore. We suspect that super-spreaders mediate the spreading from China to worldwide. At least, the infected people with H56 and mv2, as well as H54, contributed at least three haplotypes (Figure 3A).

The samples outside of China encoded 31 haplotypes belonging to Groups A-E. Of these, 27 haplotypes are private by regional samplings, only two Thailand samples were the H1 haplotype, one each from Australia and Belgium were the H3 haplotype, one sample from the United States was the H19 haplotype, and one sample from Singapore was the H40 haplotype. Twelve samples, encoding 10 haplotypes, were from patients in five countries in Asia. Seven haplotypes linked to Wuhan and three haplotypes linked to Guangdong (Shenzhen). Human-to-human transmission may have happened from patients with H53 to H52 haplotypes in Tokyo, Japan, who were repatriated Japanese from Wuhan.35 Five Oceanian samples, encoding six haplotypes in Groups B, C, and D, were from patients from three states in Australia, all with links to Wuhan. Patients with H3, with H25 and H26, and with H55 (linked to H1) were directly from Wuhan, and human-to-human transmission was from patients of H25 to H26, who were in a same tour group in Queensland.36 The connection between patients with H56 and H27 is not clear, because the patient with H56 flew to Sydney from Wuhan on 25 January 2020, and the patient with H27 flew to Melbourne from Wuhan on 15 January 2020. One possibility is that there was an intermediary spreader, who also transmitted SARS-CoV-2 to other patients in France, the United States, and Taiwan. Eight European samples, encoding seven haplotypes, were from patients in four countries. Patients in Belgium37 and Germany16 traveled to or stayed in Wuhan. Patients in England did not report a link to Wuhan,38 but a familial transmission was recovered from H28 to H29. Patients in France may have been infected by three different sources, i.e. H44 linked to Wuhan, H43 may link to Chongqing/Singapore, and H30 may link to an intermediary spreader.

Of the 13 genomes from the United States, three were from the same patient in Washington encoding the same haplotype H38, while the other three samples encoded eight haplotypes, covering all five groups (Figure 3A), so the sources of imported infections are complicated. Three haplotypes (H1 (California), H19 (Wisconsin), and H38 (Washington)) were linked to Wuhan, and three (H19 (Wisconsin), H35 (Arizona), H42 (California)) to five (H41 (California) and H58 (Illinois)) haplotypes may link to Guangdong. The remaining haplotypes (H36 (California), H37 (California), and H57 (Massachusetts)) linked to patients out of China (H54 (Vietnam) and H56 (Australia)), who were from Wuhan.15,39 It is not clear where they were infected. There is no human-to-human transmission evidence in the United States from the 11 cases.

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Is Wuhan Institute of Virology Responsible for COVID-19? Mar 17, 2020 23:25:33 GMT

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Post by Admin on Mar 17, 2020 23:25:33 GMT

President Trump drew backlash Monday night after posting a tweet using the phrase “Chinese virus.”

After giving an address Monday afternoon in which he said that the country “may be” headed toward recession and urged social distancing, he later tweeted his confidence in and support for various sectors, while including the offensive remark.

“The United States will be powerfully supporting those industries, like Airlines and others, that are particularly affected by the Chinese Virus. We will be stronger than ever before!,” he wrote.

Many officials, including the Center for Disease Control and Protection chief, have criticized the phrase as both inaccurate and potentially harmful in tying racist associations between the virus and those from China.

The comments prompted massive backlash from many social media users, including New York City mayor Bill de Blasio, who pointed out how the tweet is misplacing blame and could put more Asian Americans in danger.

Trump has previously referred to COVID-19 as a “foreign virus” and has also retweeted a supporter who used the term “China Virus.” His newest reference comes days after CDC director Robert Redfield agreed when questioned at a House hearing that it was "absolutely wrong and inappropriate" to use labels like “Chinese coronavirus” as the virus had now expanded beyond China to other parts of the world. The are currently roughly 3500 confirmed cases of the illness in the U.S. alone as of Monday night.

Last Edit: Mar 17, 2020 23:26:01 GMT by Admin

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Is Wuhan Institute of Virology Responsible for COVID-19? Mar 21, 2020 23:36:35 GMT

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Post by Admin on Mar 21, 2020 23:36:35 GMT

As the novel coronavirus causing COVID-19 spreads across the globe, with cases surpassing 284,000 worldwide today (March 20), misinformation is spreading almost as fast.

One persistent myth is that this virus, called SARS-CoV-2, was made by scientists and escaped from a lab in Wuhan, China, where the outbreak began.

A new analysis of SARS-CoV-2 may finally put that latter idea to bed. A group of researchers compared the genome of this novel coronavirus with the seven other coronaviruses known to infect humans: SARS, MERS and SARS-CoV-2, which can cause severe disease; along with HKU1, NL63, OC43 and 229E, which typically cause just mild symptoms, the researchers wrote March 17 in the journal Nature Medicine.

"Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus," they write in the journal article.

Kristian Andersen, an associate professor of immunology and microbiology at Scripps Research, and his colleagues looked at the genetic template for the spike proteins that protrude from the surface of the virus. The coronavirus uses these spikes to grab the outer walls of its host's cells and then enter those cells. They specifically looked at the gene sequences responsible for two key features of these spike proteins: the grabber, called the receptor-binding domain, that hooks onto host cells; and the so-called cleavage site that allows the virus to open and enter those cells.

That analysis showed that the "hook" part of the spike had evolved to target a receptor on the outside of human cells called ACE2, which is involved in blood pressure regulation. It is so effective at attaching to human cells that the researchers said the spike proteins were the result of natural selection and not genetic engineering.

Here's why: SARS-CoV-2 is very closely related to the virus that causes severe acute respiratory syndrome (SARS), which fanned across the globe nearly 20 years ago. Scientists have studied how SARS-CoV differs from SARS-CoV-2 — with several key letter changes in the genetic code. Yet in computer simulations, the mutations in SARS-CoV-2 don't seem to work very well at helping the virus bind to human cells. If scientists had deliberately engineered this virus, they wouldn't have chosen mutations that computer models suggest won't work. But it turns out, nature is smarter than scientists, and the novel coronavirus found a way to mutate that was better — and completely different— from anything scientists could have created, the study found.

Another nail in the "escaped from evil lab" theory? The overall molecular structure of this virus is distinct from the known coronaviruses and instead most closely resembles viruses found in bats and pangolins that had been little studied and never known to cause humans any harm.

"If someone were seeking to engineer a new coronavirus as a pathogen, they would have constructed it from the backbone of a virus known to cause illness," according to a statement from Scripps.

Where did the virus come from? The research group came up with two possible scenarios for the origin of SARS-CoV-2 in humans. One scenario follows the origin stories for a few other recent coronaviruses that have wreaked havoc in human populations. In that scenario, we contracted the virus directly from an animal — civets in the case of SARS and camels in the case of Middle East respiratory syndrome (MERS). In the case of SARS-CoV-2, the researchers suggest that animal was a bat, which transmitted the virus to another intermediate animal (possibly a pangolin, some scientists have said) that brought the virus to humans.

In that possible scenario, the genetic features that make the new coronavirus so effective at infecting human cells (its pathogenic powers) would have been in place before hopping to humans.

In the other scenario, those pathogenic features would have evolved only after the virus jumped from its animal host to humans. Some coronaviruses that originated in pangolins have a "hook structure" (that receptor binding domain) similar to that of SARS-CoV-2. In that way, a pangolin either directly or indirectly passed its virus onto a human host. Then, once inside a human host, the virus could have evolved to have its other stealth feature — the cleavage site that lets it easily break into human cells. Once it developed that capacity, the researchers said, the coronavirus would be even more capable of spreading between people.

All of this technical detail could help scientists forecast the future of this pandemic. If the virus did enter human cells in a pathogenic form, that raises the probability of future outbreaks. The virus could still be circulating in the animal population and might again jump to humans, ready to cause an outbreak. But the chances of such future outbreaks are lower if the virus must first enter the human population and then evolve the pathogenic properties, the researchers said.

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Is Wuhan Institute of Virology Responsible for COVID-19? Mar 23, 2020 0:44:39 GMT

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Post by Admin on Mar 23, 2020 0:44:39 GMT

The proximal origin of SARS-CoV-2
Kristian G. Andersen, Andrew Rambaut, W. Ian Lipkin, Edward C. Holmes & Robert F. Garry
Nature Medicine (2020)

To the Editor — Since the first reports of novel pneumonia (COVID-19) in Wuhan, Hubei province, China1,2, there has been considerable discussion on the origin of the causative virus, SARS-CoV-23 (also referred to as HCoV-19)4. Infections with SARS-CoV-2 are now widespread, and as of 11 March 2020, 121,564 cases have been confirmed in more than 110 countries, with 4,373 deaths5.

SARS-CoV-2 is the seventh coronavirus known to infect humans; SARS-CoV, MERS-CoV and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms6. Here we review what can be deduced about the origin of SARS-CoV-2 from comparative analysis of genomic data. We offer a perspective on the notable features of the SARS-CoV-2 genome and discuss scenarios by which they could have arisen. Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus.

Notable features of the SARS-CoV-2 genome
Our comparison of alpha- and betacoronaviruses identifies two notable genomic features of SARS-CoV-2: (i) on the basis of structural studies7,8,9 and biochemical experiments1,9,10, SARS-CoV-2 appears to be optimized for binding to the human receptor ACE2; and (ii) the spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site at the S1–S2 boundary through the insertion of 12 nucleotides8, which additionally led to the predicted acquisition of three O-linked glycans around the site.

1. Mutations in the receptor-binding domain of SARS-CoV-2
The receptor-binding domain (RBD) in the spike protein is the most variable part of the coronavirus genome1,2. Six RBD amino acids have been shown to be critical for binding to ACE2 receptors and for determining the host range of SARS-CoV-like viruses7. With coordinates based on SARS-CoV, they are Y442, L472, N479, D480, T487 and Y4911, which correspond to L455, F486, Q493, S494, N501 and Y505 in SARS-CoV-27. Five of these six residues differ between SARS-CoV-2 and SARS-CoV (Fig. 1a). On the basis of structural studies7,8,9 and biochemical experiments1,9,10, SARS-CoV-2 seems to have an RBD that binds with high affinity to ACE2 from humans, ferrets, cats and other species with high receptor homology7.

Fig. 1: Features of the spike protein in human SARS-CoV-2 and related coronaviruses.

a, Mutations in contact residues of the SARS-CoV-2 spike protein. The spike protein of SARS-CoV-2 (red bar at top) was aligned against the most closely related SARS-CoV-like coronaviruses and SARS-CoV itself. Key residues in the spike protein that make contact to the ACE2 receptor are marked with blue boxes in both SARS-CoV-2 and related viruses, including SARS-CoV (Urbani strain). b, Acquisition of polybasic cleavage site and O-linked glycans. Both the polybasic cleavage site and the three adjacent predicted O-linked glycans are unique to SARS-CoV-2 and were not previously seen in lineage B betacoronaviruses. Sequences shown are from NCBI GenBank, accession codes MN908947, MN996532, AY278741, KY417146 and MK211376. The pangolin coronavirus sequences are a consensus generated from SRR10168377 and SRR10168378 (NCBI BioProject PRJNA573298)29,30.

While the analyses above suggest that SARS-CoV-2 may bind human ACE2 with high affinity, computational analyses predict that the interaction is not ideal7 and that the RBD sequence is different from those shown in SARS-CoV to be optimal for receptor binding7,11. Thus, the high-affinity binding of the SARS-CoV-2 spike protein to human ACE2 is most likely the result of natural selection on a human or human-like ACE2 that permits another optimal binding solution to arise. This is strong evidence that SARS-CoV-2 is not the product of purposeful manipulation.

2. Polybasic furin cleavage site and O-linked glycans
The second notable feature of SARS-CoV-2 is a polybasic cleavage site (RRAR) at the junction of S1 and S2, the two subunits of the spike8 (Fig. 1b). This allows effective cleavage by furin and other proteases and has a role in determining viral infectivity and host range12. In addition, a leading proline is also inserted at this site in SARS-CoV-2; thus, the inserted sequence is PRRA (Fig. 1b). The turn created by the proline is predicted to result in the addition of O-linked glycans to S673, T678 and S686, which flank the cleavage site and are unique to SARS-CoV-2 (Fig. 1b). Polybasic cleavage sites have not been observed in related ‘lineage B’ betacoronaviruses, although other human betacoronaviruses, including HKU1 (lineage A), have those sites and predicted O-linked glycans13. Given the level of genetic variation in the spike, it is likely that SARS-CoV-2-like viruses with partial or full polybasic cleavage sites will be discovered in other species.

The functional consequence of the polybasic cleavage site in SARS-CoV-2 is unknown, and it will be important to determine its impact on transmissibility and pathogenesis in animal models. Experiments with SARS-CoV have shown that insertion of a furin cleavage site at the S1–S2 junction enhances cell–cell fusion without affecting viral entry14. In addition, efficient cleavage of the MERS-CoV spike enables MERS-like coronaviruses from bats to infect human cells15. In avian influenza viruses, rapid replication and transmission in highly dense chicken populations selects for the acquisition of polybasic cleavage sites in the hemagglutinin (HA) protein16, which serves a function similar to that of the coronavirus spike protein. Acquisition of polybasic cleavage sites in HA, by insertion or recombination, converts low-pathogenicity avian influenza viruses into highly pathogenic forms16. The acquisition of polybasic cleavage sites by HA has also been observed after repeated passage in cell culture or through animals17.

The function of the predicted O-linked glycans is unclear, but they could create a ‘mucin-like domain’ that shields epitopes or key residues on the SARS-CoV-2 spike protein18. Several viruses utilize mucin-like domains as glycan shields involved immunoevasion18. Although prediction of O-linked glycosylation is robust, experimental studies are needed to determine if these sites are used in SARS-CoV-2.

Last Edit: Mar 23, 2020 0:44:59 GMT by Admin

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Is Wuhan Institute of Virology Responsible for COVID-19? Mar 23, 2020 18:52:10 GMT

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Post by Admin on Mar 23, 2020 18:52:10 GMT

Theories of SARS-CoV-2 origins
It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus. As noted above, the RBD of SARS-CoV-2 is optimized for binding to human ACE2 with an efficient solution different from those previously predicted7,11. Furthermore, if genetic manipulation had been performed, one of the several reverse-genetic systems available for betacoronaviruses would probably have been used19. However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone20. Instead, we propose two scenarios that can plausibly explain the origin of SARS-CoV-2: (i) natural selection in an animal host before zoonotic transfer; and (ii) natural selection in humans following zoonotic transfer. We also discuss whether selection during passage could have given rise to SARS-CoV-2.

1. Natural selection in an animal host before zoonotic transfer
As many early cases of COVID-19 were linked to the Huanan market in Wuhan1,2, it is possible that an animal source was present at this location. Given the similarity of SARS-CoV-2 to bat SARS-CoV-like coronaviruses2, it is likely that bats serve as reservoir hosts for its progenitor. Although RaTG13, sampled from a Rhinolophus affinis bat1, is ~96% identical overall to SARS-CoV-2, its spike diverges in the RBD, which suggests that it may not bind efficiently to human ACE27 (Fig. 1a).

Malayan pangolins (Manis javanica) illegally imported into Guangdong province contain coronaviruses similar to SARS-CoV-221. Although the RaTG13 bat virus remains the closest to SARS-CoV-2 across the genome1, some pangolin coronaviruses exhibit strong similarity to SARS-CoV-2 in the RBD, including all six key RBD residues21 (Fig. 1). This clearly shows that the SARS-CoV-2 spike protein optimized for binding to human-like ACE2 is the result of natural selection.

Neither the bat betacoronaviruses nor the pangolin betacoronaviruses sampled thus far have polybasic cleavage sites. Although no animal coronavirus has been identified that is sufficiently similar to have served as the direct progenitor of SARS-CoV-2, the diversity of coronaviruses in bats and other species is massively undersampled. Mutations, insertions and deletions can occur near the S1–S2 junction of coronaviruses22, which shows that the polybasic cleavage site can arise by a natural evolutionary process. For a precursor virus to acquire both the polybasic cleavage site and mutations in the spike protein suitable for binding to human ACE2, an animal host would probably have to have a high population density (to allow natural selection to proceed efficiently) and an ACE2-encoding gene that is similar to the human ortholog.

2. Natural selection in humans following zoonotic transfer
It is possible that a progenitor of SARS-CoV-2 jumped into humans, acquiring the genomic features described above through adaptation during undetected human-to-human transmission. Once acquired, these adaptations would enable the pandemic to take off and produce a sufficiently large cluster of cases to trigger the surveillance system that detected it1,2.

All SARS-CoV-2 genomes sequenced so far have the genomic features described above and are thus derived from a common ancestor that had them too. The presence in pangolins of an RBD very similar to that of SARS-CoV-2 means that we can infer this was also probably in the virus that jumped to humans. This leaves the insertion of polybasic cleavage site to occur during human-to-human transmission.

Estimates of the timing of the most recent common ancestor of SARS-CoV-2 made with current sequence data point to emergence of the virus in late November 2019 to early December 201923, compatible with the earliest retrospectively confirmed cases24. Hence, this scenario presumes a period of unrecognized transmission in humans between the initial zoonotic event and the acquisition of the polybasic cleavage site. Sufficient opportunity could have arisen if there had been many prior zoonotic events that produced short chains of human-to-human transmission over an extended period. This is essentially the situation for MERS-CoV, for which all human cases are the result of repeated jumps of the virus from dromedary camels, producing single infections or short transmission chains that eventually resolve, with no adaptation to sustained transmission25.

Studies of banked human samples could provide information on whether such cryptic spread has occurred. Retrospective serological studies could also be informative, and a few such studies have been conducted showing low-level exposures to SARS-CoV-like coronaviruses in certain areas of China26. Critically, however, these studies could not have distinguished whether exposures were due to prior infections with SARS-CoV, SARS-CoV-2 or other SARS-CoV-like coronaviruses. Further serological studies should be conducted to determine the extent of prior human exposure to SARS-CoV-2.

3. Selection during passage
Basic research involving passage of bat SARS-CoV-like coronaviruses in cell culture and/or animal models has been ongoing for many years in biosafety level 2 laboratories across the world27, and there are documented instances of laboratory escapes of SARS-CoV28. We must therefore examine the possibility of an inadvertent laboratory release of SARS-CoV-2.

In theory, it is possible that SARS-CoV-2 acquired RBD mutations (Fig. 1a) during adaptation to passage in cell culture, as has been observed in studies of SARS-CoV11. The finding of SARS-CoV-like coronaviruses from pangolins with nearly identical RBDs, however, provides a much stronger and more parsimonious explanation of how SARS-CoV-2 acquired these via recombination or mutation19.

The acquisition of both the polybasic cleavage site and predicted O-linked glycans also argues against culture-based scenarios. New polybasic cleavage sites have been observed only after prolonged passage of low-pathogenicity avian influenza virus in vitro or in vivo17. Furthermore, a hypothetical generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with very high genetic similarity, which has not been described. Subsequent generation of a polybasic cleavage site would have then required repeated passage in cell culture or animals with ACE2 receptors similar to those of humans, but such work has also not previously been described. Finally, the generation of the predicted O-linked glycans is also unlikely to have occurred due to cell-culture passage, as such features suggest the involvement of an immune system18.

Conclusions
In the midst of the global COVID-19 public-health emergency, it is reasonable to wonder why the origins of the pandemic matter. Detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. For example, if SARS-CoV-2 pre-adapted in another animal species, then there is the risk of future re-emergence events. In contrast, if the adaptive process occurred in humans, then even if repeated zoonotic transfers occur, they are unlikely to take off without the same series of mutations. In addition, identifying the closest viral relatives of SARS-CoV-2 circulating in animals will greatly assist studies of viral function. Indeed, the availability of the RaTG13 bat sequence helped reveal key RBD mutations and the polybasic cleavage site.

The genomic features described here may explain in part the infectiousness and transmissibility of SARS-CoV-2 in humans. Although the evidence shows that SARS-CoV-2 is not a purposefully manipulated virus, it is currently impossible to prove or disprove the other theories of its origin described here. However, since we observed all notable SARS-CoV-2 features, including the optimized RBD and polybasic cleavage site, in related coronaviruses in nature, we do not believe that any type of laboratory-based scenario is plausible.

More scientific data could swing the balance of evidence to favor one hypothesis over another. Obtaining related viral sequences from animal sources would be the most definitive way of revealing viral origins. For example, a future observation of an intermediate or fully formed polybasic cleavage site in a SARS-CoV-2-like virus from animals would lend even further support to the natural-selection hypotheses. It would also be helpful to obtain more genetic and functional data about SARS-CoV-2, including animal studies. The identification of a potential intermediate host of SARS-CoV-2, as well as sequencing of the virus from very early cases, would similarly be highly informative. Irrespective of the exact mechanisms by which SARS-CoV-2 originated via natural selection, the ongoing surveillance of pneumonia in humans and other animals is clearly of utmost importance.