Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

One-Year Sustained Cellular and Humoral Immunities in Coronavirus Disease 2019 (COVID-19) Convalescents.

BACKGROUND: The longitudinal antigen-specific immunity in COVID-19 convalescents is crucial for long-term protection upon individual re-exposure to SARS-CoV-2, and even more pivotal for ultimately achieving population-level immunity. We conducted this cohort study to better understand the features of immune memory in individuals with different disease severities at 1 year post-disease onset. METHODS: We conducted a systematic antigen-specific immune evaluation in 101 COVID-19 convalescents, who had asymptomatic, mild, moderate, or severe disease, through 2 visits at months 6 and 12 after disease onset. The SARS-CoV-2-specific antibodies, comprising neutralizing antibody (NAb), immunoglobulin (Ig) G, and IgM, were assessed by mutually corroborated assays (ie, neutralization, enzyme-linked immunosorbent assay [ELISA], and microparticle chemiluminescence immunoassay [MCLIA]). Meanwhile, T-cell memory against SARS-CoV-2 spike, membrane, and nucleocapsid proteins was tested through enzyme-linked immunospot assay (ELISpot), intracellular cytokine staining, and tetramer staining-based flow cytometry, respectively. RESULTS: SARS-CoV-2-specific IgG antibodies, and NAb, can persist among >95% of COVID-19 convalescents from 6 to 12 months after disease onset. At least 19/71 (26%) of COVID-19 convalescents (double positive in ELISA and MCLIA) had detectable circulating IgM antibody against SARS-CoV-2 at 12 months post-disease onset. Notably, numbers of convalescents with positive SARS-CoV-2-specific T-cell responses (≥1 of the SARS-CoV-2 antigen S1, S2, M, and N proteins) were 71/76 (93%) and 67/73 (92%) at 6 and 12 months, respectively. Furthermore, both antibody and T-cell memory levels in the convalescents were positively associated with disease severity. CONCLUSIONS: SARS-CoV-2-specific cellular and humoral immunities are durable at least until 1 year after disease onset.

External Quality Assessment for Severe Acute Respiratory Syndrome Coronavirus 2 RNA Detection in Chongqing, China.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Therefore, clinical laboratories must have the ability to detect SARS-CoV-2 RNA. With the enhanced detection in Chongqing, many laboratories rapidly implemented assays for the molecular detection of SARS-CoV-2 based on real-time reverse transcription polymerase chain reaction (rRT-PCR) assays. This study aimed to improve the detection capabilities of clinical laboratories by evaluating their performance for SARS-CoV-2 RNA detection through the external quality assessment (EQA) programs of 2020 in Chongqing to contribute to the prevention of this epidemic. METHODS: The EQA panels consist of eight positive samples with concentrations within 2.7 - 5.0 log10 copies/mL quantified by digital PCR and two negative samples with other human coronaviruses clinically validated by four commercial assays. All 21 samples from four rounds were distributed to the participating laboratories through cold-chain transportation. Depending on the results from each sample, laboratories were asked to use one or two assays to detect SARS-CoV-2 RNA. Test results and raw data were also required. All data were evaluated, and the testing performance of commercial assays was compared. For the rounds, all laboratories used commercial assays. RESULTS: Four rounds of EQA programs were performed, and the percent agreements of participants were 97.5% (39/40), 97.5% (39/40), 98.9% (88/89), 100.0% (131/131). Only three false negative results and one false positive result were obtained. Statistical significance in the Ct values of the ORF region and N region of SARS-CoV-2-RNA was found by using one-step, one-step concentration, and magnetic bead methods (p < 0.05). The Ct values of the ORF region of SARS-CoV-2-RNA in P5 and P6 were significantly different in the different batches of reagent A (p < 0.05). The ORF region of SARS-CoV-2-RNA was not detected in a batch of reagent B. CONCLUSIONS: The majority of laboratories in Chongqing have reliable diagnostic ability for SARS-CoV-2 detection. Our data emphasized the importance of EQA for monitoring the performance of clinical laboratories. However, clinical laboratories must first effectively evaluate the performance of reagents prior to their use.

A Novel Potentially Recombinant Rodent Coronavirus with a Polybasic Cleavage Site in the Spike Protein.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

Parallel isolation of calicivirus and reovirus from lethal co-infected mink during a potential epidemic of farmed mink infections

Mink has been identified as an animal with susceptibility to SARS-CoV-2 and also as the only animal with evidence to transmit the virus back to humans. Thus, the surveillance of viruses among high-density farmed minks has a significant meaning for the control of zoonotic emerging diseases in humans. Within anal swabs of minks that died of unknown causes in a mink farm, mink calicivirus (MCV) and mammalian reovirus (MRV) were detected and simultaneously observed within MDCK cell culture from the sample of the same lethal mink. The parallel isolation was successfully performed by utilizing cell lines from different host sources with distinct viral sensitivities, i.e. Mv.1.Lu and Vero-E6 and the two viruses were independently separated. The prevalence of the virus among the minks and its genomic characteristics were investigated through deep sequencing technology. Phylogenetic analysis of the viral genome showed a close relationship of the newly isolated MCV-GCCDC8-2020 with MCV strains belonging to the genus Vesivirus, but with unique mutations derived from the major structural protein (VP1). The reovirus MRV-GCCDC9-2020 isolated from the same mink belongs to serotype 3 mammalian reovirus and genome analysis showed a potential reassortment derived from reoviruses in different species. This study provides a beneficial reference on viral co-infection within disease investigation in farmed minks and raises the concern for the virus surveillance among the high-density fed animal farms.

Cold-chain transportation in the frozen food industry may have caused a recurrence of COVID-19 cases in destination: Successful isolation of SARS-CoV-2 virus from the imported frozen cod package surface.

Coronavirus disease 2019 (COVID-19) pandemic has spread in 220 countries/regions to wreak havoc to human beings around the world. At present, the second wave of COVID-19 has begun in many European countries. The complete control of COVID-19 is very urgent. Although China quickly brought the virus under control, there have been eight sporadic outbreaks in China since then. Both in Xinfadi of Beijing and Dalian outbreak of COVID-19, environmental swab samples related to imported cold chain food were tested nucleic acid positive for SARS-CoV-2. In this outbreak in Qingdao, we directly isolated SARS-CoV-2 from the cod outer package's surface swab samples. This is the first time worldwide, SARS-CoV-2 were isolated from the imported frozen cod outer package's surface, which showed that imported frozen food industry could import SARS-CoV-2 virus.