New and hotly debated genetic evidence from a curious doglike animal is adding some crucial pieces in the puzzle of how and where the virus that causes COVID first infected humans. The pieces don’t solve the puzzle—and haven’t entirely quelled the controversy over speculations about a “lab leak”—but they do help clarify the bigger picture.
In mid-March an international team of researchers released a report based on genetic material from positive COVID samples at a food market in Wuhan, China, where many of the earliest cases of the disease were reported. Scientists from the Chinese Center for Disease Control and Prevention (CCDC) and their colleagues had uploaded the data set used in the report to a scientific database called GISAID in early March. They later took it down but have since made it available again. The data analysis in the international team’s report revealed evidence of DNA and RNA from nonhuman animals—including foxlike creatures called raccoon dogs—in samples that had been swabbed from market stalls and other surfaces and had tested positive for the COVID-causing virus SARS-CoV-2. Last week, Chinese researchers published their own findings on the data in Nature. They confirmed the presence of genetic material from raccoon dogs and other animal species at the market but stated that the data do not prove the animals were infected with the virus.
Raccoon dogs (Nyctereutes procyonoides), members of the dog family with raccoon-like facial marking, are native to eastern Asia where they’re sometimes sold illegally for their fur and meat. Scientists focused on the animals’ presence at the market because they are known to be susceptible to and capable of spreading SARS-CoV-2.
The swab data provide concrete evidence that wild animals were being sold at the Huanan Seafood Wholesale Market in Wuhan and confirm earlier reports and photographs. The international report’s authors and other researchers say that finding animal genetic material in such close proximity to SARS-CoV-2 offers further evidence favoring a natural animal-to-human transmission of the virus at the market.
The findings are “not a ‘smoking raccoon dog,’ but it is pretty indicative that in exactly the same part of the market that our other analyses … suggested we would find the animals, now we found them in that exact spot—with the virus and without, importantly, much human [DNA present],” says Angela Rasmussen, a virologist at the Vaccine and Infectious Disease Organization–International Vaccine Center in Saskatchewan and one of the collaborators on the international report.
The findings don’t confirm the animals were infected or that they first spread the virus to people. And while there is no known evidence to support alternative scenarios in which the virus leaked from one of several virology labs in Wuhan that conduct research on coronaviruses, the new data cannot rule out such scenarios. (Tracing the origin of a new viral disease can take decades—the original SARS virus was traced to bats 15 years after it caused a deadly outbreak in 2002–2003, and the origin of many pandemic viruses has never been found.)
The CCDC released the involved data after the World Health Organization (WHO) urged Chinese researchers to make it public so scientists around the world could analyze it. “These data could have and should have been shared three years ago,” said WHO director general Tedros Adhanom Ghebreyesus in a news briefing on March 17.
Scientific American spoke with members of the international team who wrote the initial report on the market genetic sequences, as well as some scientists who were not involved, about what the findings do and don’t tell us about the origins of SARS-CoV-2.
What do the animal genetic sequences from the market tell us?
The mitochondrial DNA and RNA samples are direct evidence that animals—including raccoon dogs—were indeed being sold at the market near humans who were infected at some point. It’s not clear whether the genetic material was from live animals or animal products such as meat, but others have previously reported the sale of live animals at that market, and evolutionary biologist Edward Holmes—a co-author of the international team’s report—had photographed live raccoon dogs there several years earlier.
Many of the virus-positive samples were clustered in the market’s southwest corner, in the same place where stalls selling live animals were previously reported. Half a dozen virus-positive samples were also positive for raccoon dog DNA or RNA, often at higher amounts than human DNA. One sample contained no human DNA at all. Additionally, the report’s authors found genetic material from Amur hedgehogs, Malayan porcupines, masked palm civets, Siberian weasels, hoary bamboo rats and other animals. These animals could have also possibly acted as an intermediate host of the virus, which scientists believe likely originated in wild bats, but they have not yet been shown to be susceptible to SARS-CoV-2. Masked palm civets were found to be an intermediate host of the SARS virus that caused an epidemic in 2002–2003.
“The report finds genetic evidence of a set of animals that were in wildlife stalls,” says lead author Alex Crits-Christoph, a senior scientist in computational biology at Cultivarium, a nonprofit microbiology research organization. This provides circumstantial evidence in support of the virus spreading to humans from animals—a type of infection known as zoonosis—at the market. “This is not conclusive evidence that an animal was infected, but it’s very consistent with that,” Crits-Christoph says. In science, he adds, “there’s no such thing really as proof. There are only degrees of confidence … that become certain enough that we should then use that science to enact policy change and make decisions.”
The subsequent Nature study by former CCDC head George Gao and his colleagues, who originally collected and shared the swab data, confirms some of the international team’s findings but doesn’t draw the same conclusions. “Our study confirmed the existence of raccoon dogs, and other hypothesized/potential SARS-CoV-2 susceptible animals, at the market, prior to its closure,” the authors write. “However, these environmental samples cannot prove that the animals were infected. Furthermore, even if the animals were infected, our study does not rule out that human-to-animal transmission occurred, considering the sampling time was after the human infection within the market as reported retrospectively. Thus, the possibility of potential introduction of the virus to the market through infected humans, or [frozen] products, cannot be ruled out yet.”
The new findings build on previous studies supporting the market as an early epicenter of SARS-CoV-2 and suggesting multiple zoonotic origins linked to the market.
If the market was not the original origin of the SARS-CoV-2 outbreak but rather just the site of a superspreader event caused by people who were already infected, “you’d have to ask, Why there?” Crits-Christoph says. “If humans brought it there, why did they bring it to the place in Wuhan with the most stalls selling wild animals?”
A previous study led by Jonathan Pekar, a doctoral student in biomedical informatics at the University of California, San Diego, and a co-author of the international team’s report, suggested that there were two lineages of the virus—A and B—circulating in Wuhan in the earliest days of the pandemic and that both were connected to the market. The B lineage is the first one believed to have infected humans. This might mean the virus was introduced there twice, Rasmussen says. “Is it possible that somebody working in the [Wuhan Institute of Virology] could have gotten infected with lineage B [the first lineage believed to infect humans], showed up [at] the market and didn’t infect anybody else on their way there, even though it’s [about 10 miles away]—and then the next week the exact same thing happened with lineage A virus?” Rasmussen says. “It’s possible, but I don’t think it’s very plausible, compared to the alternative: that lineage A and lineage B came from the animals, and then there were two separate spillovers.”
But critics of the two-lineage interpretation have pointed out that these lineages only differed by two genetic mutations. And given how rapidly SARS-CoV-2 evolves, it is possible that one lineage evolved into the other after arriving at the market. “I don’t think that the fact that, among the early viruses…, they can be split into these two groups that differ by just two mutations really means that there had to be two introductions,” says Jesse Bloom, a computational biologist at the Fred Hutchinson Cancer Center. “It’s also possible that one could have evolved into the other in humans.”
Still, the findings offer some of the most compelling evidence to date that COVID-susceptible animals were at the market at roughly the same time and place where COVID was infecting humans. And they give scientists a better idea where to look next for animals closer to the origin of the virus: they can now focus their efforts upstream of the market, in the wildlife trade or on farms where these animals may have been bred. The raccoon dog DNA doesn’t match any of the currently known farmed animals, suggesting the ones sold at the market may have been wild.
The next step, Crits-Christoph and his colleagues say, would be searching for the virus in wild raccoon dogs and some of the other animals that were being sold when the pandemic began—as well as wild bat populations, which are known to harbor related coronaviruses. But finding an infected animal remains a difficult task. Even if one were found, it wouldn’t be clear that the animal hadn’t been infected by a human. Still, by looking at that virus’s genetic sequence, it may be possible to tell whether a progenitor of the pandemic virus had been evolving in an animal host, Crits-Christoph says.
What are some of the limitations of the new genetic evidence?
One of the main limitations of these findings is the fact that these samples were taken more than a month after the first reported COVID case emerged on or around November 17, 2019 (as reported by a Chinese newspaper and supported by evolutionary genetic analyses). It’s impossible to know if the same animals were at the market then or whether they had been infected prior to the first human cases. “I think the major limitation is that, unfortunately, the sampling was being done in January 2020—not the beginning” of December 2019, Bloom says. “It’s difficult to interpret what the correspondence between the animal and human content of these samples and the SARS-CoV-2 content means.” The CCDC reported that none of the live Wuhan market animals it sampled in early January 2020 were infected with the virus—but that doesn’t rule out the possibility that they had been several weeks earlier.
Another possible limitation critics have raised is that the clustering of positive samples in the market’s southwest corner may have merely been the result of investigators sampling more heavily near the animal stalls. The full data set indicates this cluster was not merely a sampling bias, however, according to Crits-Christoph.
Alina Chan, a scientific adviser at the Broad Institute of the Massachusetts Institute of Technology and Harvard University who has been an outspoken advocate of the lab leak hypothesis, doesn’t think the animal genetic data add much that wasn’t already known or suspected but rather simply confirm there were animals at the Wuhan market. “To me, it’s not shocking that you would find raccoon dog material on these surfaces,” Chan says. She notes that SARS-CoV-2 was found all over the market, not just at the animal stalls.
Is there any evidence for the lab leak hypothesis for the origin of SARS-CoV-2?
There is no known evidence that the pandemic started in a laboratory. “The main argument that has been made for research-related scenarios is a proximity-based argument: that the outbreak started in Wuhan, where there are labs that study SARS-like coronaviruses,” Bloom says, adding that this could be a coincidence. “There’s definitely no direct evidence that any of the labs were studying a virus identical to SARS-CoV-2.”
Bloom thinks there are four plausible scenarios by which the pandemic could have started, two of which relate to a laboratory or researcher: a raccoon dog or other intermediate animal host directly infected a human in Wuhan or elsewhere; a bat directly infected a person outside Wuhan and brought the virus back to the city (the bats that carry similar viruses aren’t found in Wuhan); a scientist from one of the Wuhan virology labs got infected by a bat while doing fieldwork; or a scientist at one of the labs collected a virus sample from a bat or other animal, brought the sample back to Wuhan and became infected while working with it in the lab. “In my mind, honestly, all these things sort of remain possible,” Bloom says. “Without knowing a lot more details, including about what was happening with the first infections in Wuhan, I think it’s really hard to rule any of those in or out with high confidence.”
Chan agrees—and adds what she claims is another possible scenario for a lab-related origin: that the virus had been brought to a lab and, in attempt to learn about how it mutates, was engineered to better infect human cells—and then somehow got out into the world. This is the most controversial idea, and a majority of scientists note that there is absolutely no evidence for it. Chan and others have pointed to an unusual feature of the virus called a furin cleavage site as evidence it was engineered, but such sites have also been found in viruses in nature.
At least eight U.S. intelligence agencies have conducted their own investigations of the virus’s origins. Four agencies concluded a natural spillover from animals is most likely, two favor a lab leak, and two are undecided. U.S. president Joe Biden recently signed a bill requiring U.S. government information related to COVID origins to be declassified.
In the meantime scientists are left with imperfect but suggestive evidence that animals susceptible to the virus were being sold at a market where some of the earliest human COVID patients worked or visited. On the other hand, there is the possibility—but zero evidence—that the virus could have jumped into humans working at a Wuhan virology lab that studies coronaviruses. Without more evidence and transparency from authorities in China, finding the truth will be difficult. But may not be impossible.
“People keep betting that no new information will come out, and new information keeps coming out,” Crits-Christoph says. “You see this all the time. People say, ‘I guess we’ll never know more than we know now.’ I will never say that. I would never make that bet. We’re going to know more.”
Editor’s Note (4/12/23): This article was edited after posting to correct the spelling of Alex Crits-Christoph’s last name and to correct Jesse Bloom’s description of when the sampling of the market should have begun.
