Infections with novel human coronavirus 2019 (HCoV-19) (1, 2), named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses (ICTV) (3), can result in coronavirus disease 2019 , characterized by various clinical outcomes from asymptomatic infections to severe pneumonia and even death (4)(5)(6). Globally, as of September 10th 2021, there have been 223,022,538 confirmed cases of COVID-19, including 4,602,882 deaths. No sign hints the pandemic has declined. (covid19.who.int).

Early human cases with COVID-19 were first reported in late December 2019 from Wuhan, China with pneumonia of unknown etiology (PUE), and a majority of them were found to be linked to Huanan Seafood Market (HSM) in Wuhan (6,7), where various animal meats and exotic seafoods were available for purchase. Therefore, HSM has been suspected to be the source of the COVID-19 pandemic (8). However, the possibility that some individuals outside the market had brought the virus into the market through human-to-human transmission or cold chain could not be ruled out, considering some of the early cases without epidemiological link to the market (7,9).

In addition, recent studies from different countries suggest that SARS-CoV-2 circulation preceded the initial detection of cases by weeks. Some of the suspected positive samples were detected even earlier than the first case in Wuhan, suggesting that potential circulation of the virus in other regions may have been missed (10)(11)(12)(13)(14).

Considering the findings that SARS-CoV-2 had high similarities with a few coronaviruses derived from bats in Asian countries including China, Japan, Cambodia, Thailand and Laos, scientists have proposed that bats might be the original source of SARS-CoV-2 (1,(15)(16)(17)(18)(19)(20). However, it is yet a mystery whether another animal might act as an intermediate host to have facilitated the virus spillover between bats and humans (21). One of such important findings was the discovery of SARS-CoV-2 related coronaviruses from pangolins that harbored highly similar receptor binding domain with that of SARS-CoV-2 (22)(23)(24). Despite that pangolins might be involved in the ecology of coronaviruses, whether the pangolins are the intermediate hosts is still fairly controversial. A recent study documented the animal species in HSM between May 2017 and November 2019 and noted that no pangolins or bats were traded (25). Thus far, the origins of SARS-CoV-2 (26,27) and the role of HSM in the origins and spread of SARS-CoV-2 remain poorly understood.

The HSM is located in the Jianghan District, a downtown of Wuhan, and is approximately 800 m far from Hankou Railway Station, a major railway travel hub. It occupies >50,000 m 2 , with 678 stalls located close to each other in an extremely crowded condition (Fig. 1A). The market is separated into two zones, eastern and western zone, with seafood and animals mainly sold in the western zone and livestock meat in the eastern zone. Among the 678 stalls of the market, 10 domesticated wildlife animal-selling stalls (1.5%) were identified, located in the south-western corner of western zone (8/10) and the north-western corner of eastern zone (2/10), respectively (Fig. 1A). According to sales records, during late December 2019, animals or animal products were sold in these 10 animal stalls and animals included snakes, avian species (chickens, ducks, gooses, pheasants and doves), Sika deer, badgers, rabbits, bamboo rats, porcupines, hedgehogs, salamanders, giant salamanders, bay crocodiles and Siamese crocodiles, etc., among which snakes, salamanders and crocodiles were traded as live animals.

The market was closed in the morning of January 1 st , 2020 shortly after the identification of the PUE. At the same time, in order to investigate the potential introduction of SARS-CoV-2 into the market, Chinese Center for Disease Control and Prevention (China CDC) dispatched an epidemiological team, together with experts from Hubei Provincial CDC and Wuhan Municipal CDC, to the HSM to collect environmental samples in the early morning of January 1 st , 2020 (Fig. 1B). From January 1 st , 2020 to March 2 nd , 2020, a total of 923 environmental samples from different locations within and around this market and 457 animal samples including animal bodies, stray animals and their feces were collected, with some stray animals sampled until March 30 th (Extended Data Table 1), which may reflect the profile of SARS-CoV-2 contamination in the market during the early phase of the outbreak.

After the closure of the stalls in the market, the outside surface of the rolling shutter doors of the stalls and the corridors was disinfected (with 1% bleach mixed with water) throughout January and February 2020. The goods inside the stalls were completely cleared and disinfected until early March 2020.

Out of the 923 environmental samples collected in and around the market, 73 were found to be positive for SARS-CoV-2 with positive rate of 7.9% through the nucleic acid test (NAT), with Cycle threshold (Ct) values of real-time polymerase chain reaction (PCR) ranging from 23.9 to 41.7 (Table 1). Among the 828 samples inside the HSM, 64 samples (7.7%) were positive. For the 14 samples from warehouses related to the HSM, five tested positive. Among the 51 sewerage wells in the surrounding areas, three tested positive (Table 1). Notably, one out of the 30 environmental samples from other markets in Wuhan tested positive.

For these 64 SARS-CoV-2 positive samples inside HSM, 87.5% (56/64) were collected in the western zone of the market, in particular streets from no. 1 to 8, with 71.4% (40/56) positive samples identified herein (Fig. 1A). Of the 110 samples collected from sewers or sewerage wells in the market, 24 samples were positive for SARS-CoV-2 nucleic acid. All the four sewerage wells in the market tested positive. This suggested that either contaminated sewage may have played a role in the cluster of cases in the market or that the infected people in the market contaminated the sewage.

The merchants' activities were assessed against the NAT results of the environmental samples. The sampling covered 19.8% (134/678) of the vendors in the market (95% confidence interval (CI): 16.8-23.0%). Of the positive samples, 44 were distributed among 21 vendors in the market, 19 of whom were located in the west zone and the remaining two located in the east area (Fig. 1A). Some vendors sold more than one product type, leading to differences in the denominators. While the results provide some indication of association of cases with different products, but no significant differences were observed between different vendors, including cold-chain products (18.4%, 16/87, 95% CI: 10.9-28.1%), aquatic products (17.8%, 13/73, 95% CI: 9.8-28.5%), seafood products (11%, 6/56: 95% CI: 4-21.9%), poultry (22%, 8/37: 95% CI: 9.8-38.2%), livestock (14%, 5/36: 95% CI: 4.7-29.5%), wildlife products (11%, 1/9: 95% CI: 0.3-48.2%) and vegetables (25%, 2/8: 95% CI: 3.2-65%) (Extended Data Fig. 1). These results suggested that SARS-CoV-2 might have been circulating in the market, especially the western zone, for a period of time in December 2019, leading to an extensive distribution of the virus within the market, which might have been facilitated by the crowded buyers and the contaminated environment.

The 457 animal samples mainly collected between January 1 st and March 2 nd , 2020 included 188 individuals belonging to 18 species (with some stray animals sampled until March 30 th ) (Table 2). The sources of the samples include unsold goods kept in refrigerators and freezers in the stalls of HSM, and goods kept in warehouses and refrigerators related to the HSM. Samples from stray animals in the market were also collected, i.e. swab samples from 10 stray cats, 27 cat feces, one dog, one weasel, and 10 rats. All the 457 animal samples tested negative for SARS-CoV-2 nucleic acid, suggesting that the animal infections with SARS-CoV-2 might be rare in the market.

To determine whether there was live virus in the HSM, we inoculated 27 SARS-CoV-2 positive environmental samples collected on January 1 st , 2020 into cell lines, including Vero E6 and Huh7.5 cells. Cytopathic effects (CPE) were observed 3 days post inoculation with sample F13 on Vero E6 cells. CPE was also observed 5 days post inoculation on Huh7.5 cells. We performed RT-qPCR on the supernatant of the inoculated Vero E6 and Huh7.5 cells. The supernatants were found to be SARS-CoV-2 positive, with Ct values of 15.11 (Vero E6) and 14.44 (Huh7.5) for the ORF1ab gene, and 13.98 (Vero E6) and 13.59 (Huh7.5) for the N gene, respectively.

We further used transmission electron microscopy to observe the culture supernatant and ultra-thin section of Vero E6 cells 5 days post inoculation. The electron micrographs showed that virus particles were present in both the supernatant and the cells. Negative-stained virus particles were generally spherical, pleomorphic and 60-140 nm in diameter. Spike protrusions were observed around the particles in a crown (corona) shape. In the ultra-thin cultured cell sections, a group of virus particles could be seen outside the cells, and sheets of virus particles could also be observed inside the cells (Fig. 2). Totally, live viruses were isolated from samples F13, F54, and B5, which were the only three samples with Ct values <30 in the NAT. F54 and B5 were swab samples of the ground and F13 were swab samples of the wall.

Notably, samples F13 and F54 were from the stalls with confirmed patients. All the results of successful virus isolation and the Ct values of the original samples revealed the existence of live SARS-CoV-2 with high titers in the environment of HSM.

We further performed high-throughput sequencing on the Miseq platform and successfully obtained seven complete SARS-CoV-2 genome sequences, including three sequences from three environmental samples (F13, F54, and A20), and four sequences from cell supernatants of F13, F54, and B5 (Fig. 3). The genome sequences of two environmental samples, F13 and F54, were found to be highly identical to the reference strain HCoV/Wuhan/IVDC-HB-01 (WH01, sequence identity of 99.993%) and completely identical to the human stain Wuhan-Hu-1 (GenBank: NC_045512) (Fig. 3A). The genome sequence of the isolated virus from environmental sample F54 had two more synonymous mutations compared to HCoV/Wuhan/IVDC-HB-01, with sequence identity of 99.987% (Fig. 3A). Therefore, the SARS-CoV-2 sequences from environmental samples were highly similar to the clinical strains obtained during the early stage of the COVID-19 outbreak. Previously, SARS-CoV-2 has been proposed to be classified into two major genotypes based on the two highly linked single nucleotide polymorphisms (SNPs): S (8782T and 28144C) and L (8782C and 28144T), with S most likely to be the ancestral lineage, because all of the SARS-CoV-2 related coronaviruses from bats and pangolins possessed 8782T and 28144C (28). Phylogenetic analysis revealed that most of the environmental strains belong to the L lineage and they fall into the basal positions of the L lineage (Fig. 3B). However, the environmental sample A20 fell within the S lineage in the tree (Fig. 3B, Extended Data Table 2).

To explore the potential origins of the SARS-COV-2, we conducted RNA-seq analysis using 27 SARS-CoV-2 positive environmental samples collected on January 1 st , 2020 from the HSM. We analyzed the correlation of SARS-CoV-2 and the abundance of other species. The abundance of Homo sapiens showed the correlation to SARS-CoV-2 (Figure 4), which highly suggests the SARS-CoV-2 might have derived from Homo sapiens in the HSM. No animals were concluded, implying that no animal host of SARS-CoV-2 can be deduced.

In summary, SARS-CoV-2 RNA was detected in stalls in the western zone of HSM, suggesting the prevalence of SARS-CoV-2 in the market. Thus, the market might have acted as an amplifier due to the high number of visitors every day, causing many initially identified infection clusters in the early stage of the outbreak as indicated in the Report of WHO-convened global study of origins of SARS-CoV-2 (10). In addition, live SARS-CoV-2 viruses also existed in the environmental samples.

However, no SARS-CoV-2 was detected in the animal samples from the market.

Definitely, more work involving international coordination is needed to investigate the real origins of SARS-CoV-2(10), especially considering the SARS-CoV-2 positive results of samples collected in 2019 in retrospective studies of different countries (29,30). Surveillance of wild animals using a viromic approach should be enhanced to explore the natural and intermediate hosts for SARS-CoV-2 (8, 31), which would help to prevent future potential pandemics caused by animal-derived coronaviruses or alike. A. As the place of the early cluster of COVID-19 patients, the Huanan Seafood Market is separated into eastern and western zones with the Xinhua Rd. between them.

To detect for the presence of SARS-CoV-2 RNA, reverse transcription, quantitative polymerase chain reaction (RT-qPCR) was performed. The locations of the positive samples were marked in the map of the market within orange, while the location of the samples that the live viruses were isolated from were labeled with red. The map also shows locations of stalls where domesticated wildlife products were sold. B. Timeline of environmental and animal samples collected within and around Huanan Seafood Market. Clustering of environmental samples in Huanan Seafood Market. A, Correlation of SARS-COV-2 with other species in SARS-COV-2 positive cases. The abundance of SARS-COV-2 and other species was used for analysis. Both Pearson's and Spearman's correlation values were plotted. B, Correlation of the abundance of SARS-COV-2 and Homo sapiens.

Huanan Seafood Market (HSM) was closed on early morning of January 1st 2020 and at the same time, China CDC began collecting environmental and animal samples.

Staff from China CDC entered the market about 30 times before the market's final clean-up on 2 March 2020, with some stray animals sampled outside the market until March 30 th . Environmental samples in the HSM were collected to represent exhaustively as possible, from a wide diversity of surfaces, animals and products (Table 1 and For animal samples, depending on the type of animal and whether it was alive or frozen, pharyngeal, anal, body surface and body cavity swabs or tissue samples were collected for nucleic acid testing (NAT). Generally, for alive animal and frozen full bodies, three samples, including pharyngeal, anal, and body surface swabs were collected for each animal individuals. And for animal bodies after "bai tiao" disposing (remaining parts of poultry or livestock after removal of hair and viscera), the body cavity swabs were collected.

Sewage (silt) samples were collected by the use of virus sampling swabs to probe into the silt at the bottom of drainage channels in the market. Sewage and silt samples were preserved in virus preservation solution. For the sewage well, a container was used to take a silt-water mixture from a location near the bottom of the well, and an appropriate amount of sample was collected by using virus sampling swabs and then preserved in virus preservation solution.

A virus nucleic acid extraction kit (Xi'an Tianlong) was used to extract viral nucleic acid from samples using an automated nucleic acid extraction instrument according to

Samples positive for SARS-CoV-2 were cultured in Vero E6 and Huh7.5 cells. The cell lines were inoculated with positive samples and three blind passages were performed for each sample. The culture supernatant and cell pellet of each passage were harvested for RT PCR. The morphology of viral particles in the cell sections and the supernatant were observed by transmission electron microscope (TEM).

Metagenomic sequencing was conducted at Wuhan BGI. Nucleic acid was extracted using Qiagen's viral RNA microextraction kit and human nucleic acid was removed Figures Figure 1 The distribution of the positive environmental samples in Huanan Seafood Market. A. As the place of the early cluster of COVID-19 patients, the Huanan Seafood Market is separated into eastern and western zones with the Xinhua Rd. between them. To detect for the presence of SARS-CoV-2 RNA, reverse transcription, quantitative polymerase chain reaction (RT-qPCR) was performed. The locations of the positive samples were marked in the map of the market within orange, while the locationof the samples that the live viruses were isolated from were labeled with red. The map also shows locations of stalls where domesticated wildlife products were sold. B. Timeline of environmental and animal samples collected within and around Huanan Seafood Market

The SARS-CoV-2 virus isolation from environmental samples of Huanan Seafood Market. The electron micrographs of the SARS-CoV-2 viruses isolated fromthe environmental samples in Huanan Seafood Market. To determine whether SARS-CoV-2 particles could be visualized from the cell supernatant and lysate, we used transmission electron microscopy to observe the culture supernatant and ultra-thin section cells based from both VeroE6 and Huh7.5 cells. The electron micrographs showed that virus particles were present in both the supernatant (A, B) and the cells (C, D). Negative-stained virus particles were generally spherical, pleomorphic and 60-140 nm in diameter. Spike protrusions were observed around the particles in a crown (corona) shape (A, B). In ultra-thin cultured cell sections, a group of virus particles can be seen outside the cell (C), and sheets of virus particles canalso be observed inside the cells (D).

Figure 3 The genomic analysis of SARS-CoV-2 sequenced fromHuanan Seafood Market.