NEW YORK – Several new lines of genetic and geospatial evidence have linked early SARS-CoV-2 cases to wild animal trading at Wuhan's Huanan Seafood Wholesale Market.
The results track with past epidemiological studies implicating the market as the source of the pandemic rather than an accidental leak from a nearby laboratory that studied coronaviruses.
"Extensive epidemiological evidence supports wildlife trade at the Huanan market as the most likely conduit for the COVID-19 pandemic origin," co-senior and co-corresponding author Florence Débarre, a researcher at Sorbonne University, and her colleagues wrote in a study published in Cell on Thursday.
Prior research linked roughly one-third of the first 174 COVID-19 cases reported in Wuhan in December 2019 to individuals who worked at or visited the market, the team noted. Likewise, past studies pointed to higher-than-anticipated pneumonia death rates in a geographic region surrounding the market, which also showed higher rates of seropositivity for SARS-CoV-2 exposure in subsequent studies.
"Although some of the early case findings could have preferentially identified market-linked cases," Débarre and coauthors explained, "a geospatial analysis of residences of the early cases with no identified link to the Huanan market showed that they lived unexpectedly close to and centered around the market, even though geographic proximity was not used as a case criterion."
For their new study, Débarre and colleagues from Sorbonne University, the University of Arizona, the Scripps Research Institute, and elsewhere used reverse-transcription quantitative PCR, metagenomic sequencing, SARS-CoV-2 genome analyses, phylogenetics, and geospatial mapping to assess more than 800 environmental samples collected by the Chinese CDC at the Huanan market on Jan. 1 and Jan. 12 of 2020, along with additional samples collected until late March 2020.
In these samples, the researchers identified SARS-CoV-2 from both early viral lineages, known as lineage A and lineage B. They also used four nearly complete SARS-CoV-2 genomes to show that the most recent common ancestor (MRCA) for viral isolates present at the Huanan market matched the MRCA for the vast collection of genetically characterized isolates from the COVID-19 pandemic overall.
"That the MRCA of SARS-CoV-2 linked to the Huanan market is equivalent to the MRCA of the pandemic establishes that the timing of the origin of the market outbreak is genetically indistinguishable from the timing of the origin of the pandemic as a whole," the authors explained.
From geospatial patterns, the team demonstrated that SARS-CoV-2 sequences were enriched in a region in the southwest section of the market, particularly in and around a single stall, dubbed stall A, where wild animals were sold.
"We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and racoon dogs, previously identified as possible intermediate hosts," the authors reported.
The team went on to show that SARS-CoV-2-positive samples were enriched in the region around the suspicious stall, where the virus turned up in samples from the ground, an animal cart, a hair- and feather-removal machine, and a nearby drain. Another stall in the vicinity that also sold wild animals contained SARS-CoV-2-positive samples, though the virus appeared to be less common there.
Consistent with their hypothesis that animals at the Huanan market were capable of shedding viral material, the researchers tracked down a range of other known animal viruses in the market samples, ranging from raccoon dog amdoparvovirus, bamboo rat betacoronavirus, or civet kobuvirus to canine or hedgehog coronaviruses, polyomaviruses, and influenza A.
"These results further add to the evidence for the presence of live animals at the market," the authors noted, "and establish it as a place where potential wildlife hosts of SARS-CoV-2 were actively shedding other viruses."
Based on the phylogenetic relationships between the animal viruses and those described previously, the team saw signs that at least some of the wild animals found at the market originated in southern China.
Likewise, the investigators got a glimpse at the animal species and subspecies found at the market using mitochondrial genome sequencing and haplotyping, leading to a shortlist of potential host intermediates for SARS-CoV-2 — animals suspected of bridging the gap between humans and bat hosts for SARS-CoV-2-related viruses in southern China, northern Laos, and Vietnam.
In particular, the researchers noted that SARS-CoV-2-positive samples from the market also contained DNA from animals previously suspected of transmitting coronaviruses, such as the raccoon dog, masked palm civets, hoary bamboo rats, and Malayan porcupines.
While the intermediate host species remains to be identified, the authors explained, raccoon dog DNA was especially common at the Huanan market, in particular at the stall with the most SARS-CoV-2-containing samples.
"Many of the key animal species had been cleared out before the Chinese CDC teams arrived, so we can't have direct proof that the animals were infected," Débarre said in a statement.
"We are seeing the DNA and RNA ghosts of these animals in the environmental samples, and some are in stalls where SARS-CoV-2 was found, too," she explained. "This is what you would expect under a scenario in which there were infected animals in the market."
Based on these and other findings, the researchers suggested that "genetic and serological sampling of raccoon dogs and the other mammalian species reported here throughout Southeast Asia and southern China can shed light on the animal trade networks that may have led to the emergence of SARS-CoV-2, as previously recommended."
In a statement, co-senior author Michael Worobey, a researcher at the University of Arizona, noted that "there has been an increased focus on lab safety" in the wake of the COVID-19 pandemic, though "not much has been done to decrease the chance of a zoonotic scenario like this happening again."