Is Wuhan Institute of Virology Responsible for COVID-19?

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.

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.