Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic
Alexander Crits‐ChristophiD, Joshua I. LevyiD, Jonathan E. PekariD, Stephen A. GoldsteiniD, Reema SinghiD, Zach HenseliD, Karthik GangavarapuiD, Matthew Brian RogersiD, Niema MoshiriiD, Robert F. GarryiD, Edward Charles HolmesiD, Marion KoopmansiD, Philippe LemeyiD, Thomas P. PeacockiD, Saskia V. PopescuiD, Andrew RambautiD, David L. RobertsoniD, Marc A. SuchardiD, Joel O. WertheimiD, Angela L. RasmusseniD, Kristian G. AnderseniD, Michael WorobeyiD, Florence DébarreiD
Abstract
Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Wholesale Seafood Market, the site with the most reported wildlife vendors in the city of Wuhan, China. Here, we analyze publicly available qPCR and sequencing data from environmental samples collected in the Huanan market in early 2020. We demonstrate that the SARS-CoV-2 genetic diversity linked to this market is consistent with market emergence, and find increased SARS-CoV-2 positivity near and within a particular wildlife stall. We identify wildlife DNA in all SARS-CoV-2 positive samples from this stall. This includes species such as civets, bamboo rats, porcupines, hedgehogs, and one species, raccoon dogs, known to be capable of SARS-CoV-2 transmission. We also detect other animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them to those from other markets. This analysis provides the genetic basis for a short list of potential intermediate hosts of SARS-CoV-2 to prioritize for retrospective serological testing and viral sampling.
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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (14 Sept 2023) · CC BY 4.0
Reanalysis of public environmental metatranscriptomes from the Huanan market links SARS-CoV-2 positivity to one wildlife stall containing raccoon dog, civet and bamboo rat genetic material.
Earlier work on these market samples qPCR-tested only a narrow set of animals (none from raccoon dogs, civets or porcupines) and reported negative correlations between animal reads and SARS-CoV-2. This analysis adds species-level mitochondrial, viral and phylodynamic evidence to shortlist candidate intermediate hosts for follow-up serology.
Key results
ShownSARS-CoV-2 genomes tied to the market share a common ancestor date with the global pandemic, and both lineage A and lineage B occur in market environmental samples.
BEAST tMRCA from market-linked genomes (n=17), Wuhan genomes (n=93) and global genomes (n=789) overlapped; 95% HPD intervals spanned Nov 13 to Dec 23. Sample A20 was lineage A; F54, F13 and B5 were lineage B. · SARS-CoV-2 genetic diversity linked to the…; SARS-CoV-2 phylogenetics
ShownSARS-CoV-2 positivity concentrates in the southwest of the market and in and around one wildlife stall (stall A), supported by three independent spatial signals: qPCR, metatranscriptomics and drain sampling.
Stall A had 3 of 10 samples qPCR-positive (30%). Six of 70 sequenced Jan 12 samples had SARS-CoV-2 reads. Drains: 4 of 60 positive (Jan 27 and 29), including the one in front of stall A; 3 of 17 positive later. · High SARS-CoV-2 positivity in and near a…; Spatial relative risk analyses of environmental…
ShownAll SARS-CoV-2-positive samples from stall A contained wildlife mitochondrial DNA, including raccoon dog, hoary bamboo rat, hedgehog, porcupine and civet. No bat or pangolin DNA was found.
In the stall A positive samples, raccoon dog, hoary bamboo rat and European rabbit mtDNA occurred in all of them; Amur hedgehog and Malayan porcupine in four; civet in one. Only 8 reads mapped to Myotis, all non-specific by BLAST. · Mammalian wildlife species detected in five…; Mitochondrial mapping
ShownPooling reads across samples yielded mostly complete genomes for three wildlife-associated viruses (raccoon dog amdoparvovirus, bamboo rat betacoronavirus, civet kobuvirus); each grouped with viruses from one host species, and some relatives came from southern China.
Genomes were reconstructed by pooling reads mapped across samples. The bamboo rat betacoronavirus was a close, recent relative of a virus from a 2019 Guangxi bamboo rat farm. Five influenza A segments, closest to H9N2, were found together in one sample from a SARS-CoV-2 negative wildlife stall. · Wildlife stalls and SARS-CoV-2 positive samples…; Viral mapping and assembly; Viral phylogenetics
SuggestedRaccoon dog mitochondrial SNVs from the Huanan market resemble wild Hubei populations rather than northern fur-market and farm lineages, pointing to a central or southern China origin.
Across seven wildlife species, 33 consensus mitochondrial haplotypes were >50% complete (ten >90%), diverging on average 0.57% from references. For raccoon dogs, market SNVs matched two wild Hubei populations from 2020; SNVs on branches to N. p. ussuriensis and N. p. procyonoides seen in northern markets were absent. · Reconstruction of mitochondrial genotypes of…; Cytochrome B phylogenetics
Methods, data and reproducibility
Public market qPCR and metatranscriptomic reads were mapped to SARS-CoV-2, mitochondrial and viral reference databases, then combined with spatial relative risk analysis, BEAST phylodynamics, Spearman correlation, and maximum likelihood phylogenies of viral and raccoon dog mitochondrial sequences.
- Huanan market environmental swab metatranscriptomes originally generated by Liu et al. (early 2020), downloaded as sequencing runs · environmental samples (human, wildlife, viral) · n = 184 sequencing runs; 783 of 819 samples precisely located · PRJNA948658
- Jan 1 and Jan 12 2020 sampling with qPCR results; Jan 12 wildlife stall samples (10 per stall, seven stalls) all sequenced · n = 515 samples on Jan 1 (27 qPCR-positive); 70 sequenced on Jan 12
- Early SARS-CoV-2 genomes (collected by Feb 14, 2020) for phylodynamics, plus four environmental genomes from the market · SARS-CoV-2 (human cases and market environment) · n = 17 market-linked, 93 Wuhan, 789 global genomes · Supplementary Table S19
- Published raccoon dog mitochondrial sequences used for cytB and whole-mitogenome phylogenies; reference mitogenome NC_013700.1 · Nyctereutes procyonoides (raccoon dog) · n = 44 published sequences · NC_013700.1
- Independent pooled raccoon dog samples from markets in five cities north of Hubei province, 2020, used for SNV comparison · Nyctereutes procyonoides (raccoon dog) · n = five cities
- GenBank viral genomes used for phylogenies of market viruses · amdoparvoviruses, kobuviruses, embecoviruses · n = 299 amdoparvovirus, 283 kobuvirus, 54 Embecovirus genomes
- Methods & tools
- BBDuk, Bowtie2, ViralConsensus v0.0.3, iVar, BEAST v1.10.5, sparr (R), MAFFT v.7.490, IQ-TREE 2 v2.0.7, TreeTime v0.8.1, rnaSPAdes v3.15.5, BLAST v2.14.0+, Mash
- Data & accessions
- https://github.com/sars-cov-2-origins/huanan-market-environment
- Software & environment
- ViralConsensus v0.0.3; BEAST v1.10.5; Tracer v1.7.1; MAFFT v.7.490; IQ-TREE 2 v2.0.7; TreeTime v0.8.1; rnaSPAdes v3.15.5; BLAST v2.14.0+; pysam 0.21.0; Seaborn 0.12.2; baltic 0.2.2.
- Assay conditions & compute
- BBDuk run with 60 threads. BEAST chains ran 100 million generations (market and Wuhan analyses) and three chains of 400 million generations (global analysis). No GPU or wall-clock details given.
- Reproducibility gaps
- Genome accession IDs for the phylodynamic dataset are only in Supplementary Table S19; the accession for the northern-market raccoon dog data is not printed in the text.
Limitations (6)
- Mitochondrial DNA co-detection cannot show which species were infected or introduced the virus; the data fit either animal shedding or unreported human cases at the same stall. · stated by the authors
- SARS-CoV-2 read counts were low, and wildlife stalls were sampled 11 days after the first sampling round, so RNA decay probably reduced detection there. · stated by the authors
- Raccoon dog geographic origin is uncertain because genetic data for the species in southern China are lacking, so the extent of the matched populations is unknown. · stated by the authors
- The authors caution that correlating species abundance with SARS-CoV-2 reads is unreliable for deciding which species were infected, given unbalanced sequencing of samples, temporal trends and compositional effects. · stated by the authors
- No serology from market animals has been described and no raccoon dog, civet or porcupine samples were qPCR-tested, so host infection is inferred only from environmental co-detection of animal DNA and viral RNA. · noted in this summary
- The Spearman analysis rests on 70 samples from a single sampling day, with relative abundances that are compositional, limiting power to detect weaker host associations. · noted in this summary
Who should read:Read this if you want a computational reanalysis of Huanan market environmental metagenomics linking wildlife DNA, animal viruses and SARS-CoV-2 positivity.
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