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.

Human-to-human transmission of Chlamydia psittaci in China, 2020: an epidemiological and aetiological investigation.

BACKGROUND: Chlamydia psittaci can infect a wide range of avian species, occasionally causing psittacosis (also known as parrot fever) in humans. Most human psittacosis cases are associated with close contact with pet birds or poultry. In December, 2020, an outbreak of severe community-acquired pneumonia of unknown aetiology was reported in a hospital in Shandong province, China, and some of the patients' close contacts had respiratory symptoms. Our aims were to determine the causative agent of this epidemic and whether there had been human-to-human transmission. METHODS: For this epidemiological and aetiological investigation study, we enrolled patients who had community-acquired pneumonia confirmed by chest CT at two local hospitals in Shandong Province in China. We collected sputum, bronchoalveolar lavage fluid, and nasopharyngeal swab samples from participants and detected pathogens by surveying for 22 target respiratory microbes using a commercial assay, followed by metagenomic next-generation sequencing, specific nested PCR, and qPCR tests. We excluded individuals who were C psittaci-negative on both tests. We recruited close contacts of the C psittaci-positive patients, and tested nasopharyngeal swabs from the close contacts and samples from ducks from the processing plant where these patients worked. We then integrated the epidemiological, clinical, and laboratory data to reveal the potential chain of transmission of C psittaci that characterised this outbreak. FINDINGS: Between Dec 4 and 29, 2020, we used metagenomic next-generation sequencing and different PCR-based approaches to test 12 inpatients with community-acquired pneumonia, of whom six (50%) were workers at a duck-meat processing plant and two (17%) were unemployed people, who were positive for C psittaci and enrolled in this study. We contacted 61 close contacts of the six patients who worked at the duck-meat processing plant, of whom 61 (100%) were enrolled and tested, and we determined that the community-acquired pneumonia outbreak was caused by C psittaci. Within the outbreak cluster, 17 (77%) of 22 participants had confirmed C psittaci infections and five (23%) of 22 participants were asymptomatic C psittaci carriers. The outbreak had begun with avian-to-human transmission, and was followed by secondary and tertiary human-to-human transmission, which included transmission by several asymptomatic carriers and by health-care workers. In addition, some of the participants with confirmed C psittaci infection had no identified source of infection, which suggested cryptic bacterial transmission. INTERPRETATION: Our study data might represent the first documented report of human-to-human transmission of C psittaci in China. Therefore, C psittaci has the potential to evolve human-to-human transmission via various routes, should be considered an elevated biosecurity and emergent risk, and be included as part of the routine diagnosis globally, especially for high-risk populations. FUNDING: Academic Promotion Programme of Shandong First Medical University, National Science and Technology Major Project, ARC Australian Laureate Fellowship.

Seroprevalence of influenza viruses in Shandong, Northern China during the COVID-19 pandemic.

Nonpharmaceutical interventions (NPIs) have been commonly deployed to prevent and control the spread of the coronavirus disease 2019 (COVID-19), resulting in a worldwide decline in influenza prevalence. However, the influenza risk in China warrants cautious assessment. We conducted a cross-sectional, seroepidemiological study in Shandong Province, Northern China in mid-2021. Hemagglutination inhibition was performed to test antibodies against four influenza vaccine strains. A combination of descriptive and meta-analyses was adopted to compare the seroprevalence of influenza antibodies before and during the COVID-19 pandemic. The overall seroprevalence values against A/H1N1pdm09, A/H3N2, B/Victoria, and B/Yamagata were 17.8% (95% CI 16.2%-19.5%), 23.5% (95% CI 21.7%-25.4%), 7.6% (95% CI 6.6%-8.7%), and 15.0 (95% CI 13.5%-16.5%), respectively, in the study period. The overall vaccination rate was extremely low (2.6%). Our results revealed that antibody titers in vaccinated participants were significantly higher than those in unvaccinated individuals (P < 0.001). Notably, the meta-analysis showed that antibodies against A/H1N1pdm09 and A/H3N2 were significantly low in adults after the COVID-19 pandemic (P < 0.01). Increasing vaccination rates and maintaining NPIs are recommended to prevent an elevated influenza risk in China.

Co-existence and co-infection of influenza A viruses and coronaviruses: Public health challenges.

Since the 20th century, humans have lived through five pandemics caused by influenza A viruses (IAVs) (H1N1/1918, H2N2/1957, H3N2/1968, and H1N1/2009) and the coronavirus (CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IAVs and CoVs both have broad host ranges and share multiple hosts. Virus co-circulation and even co-infections facilitate genetic reassortment among IAVs and recombination among CoVs, further altering virus evolution dynamics and generating novel variants with increased cross-species transmission risk. Moreover, SARS-CoV-2 may maintain long-term circulation in humans as seasonal IAVs. Co-existence and co-infection of both viruses in humans could alter disease transmission patterns and aggravate disease burden. Herein, we demonstrate how virus-host ecology correlates with the co-existence and co-infection of IAVs and/or CoVs, further affecting virus evolution and disease dynamics and burden, calling for active virus surveillance and countermeasures for future public health challenges.

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.

Identification of a recombinant equine coronavirus in donkey, China.

Equine coronavirus (ECoV) was first identified in the USA and has been previously described in several countries. In order to test the presence of ECoV in China, we collected 51 small intestinal samples from donkey foals with diarrhoea from a donkey farm in Shandong Province, China between August 2020 and April 2021. Two samples tested positive for ECoV and full-length genome sequences were successfully obtained using next-generation sequencing, one of which was further confirmed by Sanger sequencing. The two strains shared 100% sequence identity at the scale of whole genome. Bioinformatics analyses further showed that the two Chinese strains represent a novel genetic variant of ECoV and shared the highest sequence identity of 97.05% with the first identified ECoV strain - NC99. In addition, it may be a recombinant, with the recombination region around the NS2 gene. To our knowledge, this is the first documented report of ECoV in China, highlighting its risk to horse/donkey breeding. In addition, its potential risk to public health also warrants further investigation.

The emergence, genomic diversity and global spread of SARS-CoV-2.

Since the first cases of COVID-19 were documented in Wuhan, China in 2019, the world has witnessed a devastating global pandemic, with more than 238 million cases, nearly 5 million fatalities and the daily number of people infected increasing rapidly. Here we describe the currently available data on the emergence of the SARS-CoV-2 virus, the causative agent of COVID-19, outline the early viral spread in Wuhan and its transmission patterns in China and across the rest of the world, and highlight how genomic surveillance, together with other data such as those on human mobility, has helped to trace the spread and genetic variation of the virus and has also comprised a key element for the control of the pandemic. We pay particular attention to characterizing and describing the international spread of the major variants of concern of SARS-CoV-2 that were first identified in late 2020 and demonstrate that virus evolution has entered a new phase. More broadly, we highlight our currently limited understanding of coronavirus diversity in nature, the rapid spread of the virus and its variants in such an increasingly connected world, the reduced protection of vaccines, and the urgent need for coordinated global surveillance using genomic techniques. In summary, we provide important information for the prevention and control of both the ongoing COVID-19 pandemic and any new diseases that will inevitably emerge in the human population in future generations.

Bioinformatics resources for SARS-CoV-2 discovery and surveillance.

In early January 2020, the novel coronavirus (SARS-CoV-2) responsible for a pneumonia outbreak in Wuhan, China, was identified using next-generation sequencing (NGS) and readily available bioinformatics pipelines. In addition to virus discovery, these NGS technologies and bioinformatics resources are currently being employed for ongoing genomic surveillance of SARS-CoV-2 worldwide, tracking its spread, evolution and patterns of variation on a global scale. In this review, we summarize the bioinformatics resources used for the discovery and surveillance of SARS-CoV-2. We also discuss the advantages and disadvantages of these bioinformatics resources and highlight areas where additional technical developments are urgently needed. Solutions to these problems will be beneficial not only to the prevention and control of the current COVID-19 pandemic but also to infectious disease outbreaks of the future.

A novel SARS-CoV-2 related coronavirus with complex recombination isolated from bats in Yunnan province, China.

At the end of 2019, A new type of beta-CoV, SARS-CoV-2 emerged and triggered the COVID-19 pandemic, which spread overwhelmingly around the world in less than a year. However, the origin and direct ancestral viruses of SARS-CoV-2 remain unknown. RaTG13, a novel coronavirus found in bats in China's Yunnan Province, is the closest relative virus of the SARS-CoV-2 identified so far. In this study, a new SARS-CoV-2 related virus, provisionally named PrC31, was discovered in Yunnan province by retrospectively analyse bat next generation sequencing (NGS) data of intestinal samples collected in 2018. PrC31 shared 90.7% and 92.0% nucleotide identities to the genomes of SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment of PrC31 showed that several genomic regions, especially orf1a and orf8 had the highest homology with those corresponding genomic regions of SARS-CoV-2 than any other related viruses. Phylogenetic analysis indicated that PrC31 shared a common ancestor with SARS-CoV-2 in evolutionary history. The differences between the PrC31 and SARS-CoV-2 genomes were mainly manifested in the spike genes. The amino acid homology between the receptor binding domains of PrC31 and SARS-CoV-2 was only 64.2%. Importantly, recombination analysis revealed that PrC31 underwent multiple complex recombination events (including three recombination breakpoints) involving the SARS-CoV and SARS-CoV-2 sub-lineages, indicating that PrC31 evolved from yet-to-be-identified intermediate recombination strains. Combined with previous studies, it is revealed that the beta-CoVs may possess a more complex recombination mechanism than we thought.

Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses.

Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2-related and three SARS-CoV-related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2-related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV.

Uncovering two phases of early intercontinental COVID-19 transmission dynamics.

BACKGROUND: The COVID-19 pandemic has posed an ongoing global crisis, but how the virus spread across the world remains poorly understood. This is of vital importance for informing current and future pandemic response strategies. METHODS: We performed two independent analyses, travel network-based epidemiological modelling and Bayesian phylogeographic inference, to investigate the intercontinental spread of COVID-19. RESULTS: Both approaches revealed two distinct phases of COVID-19 spread by the end of March 2020. In the first phase, COVID-19 largely circulated in China during mid-to-late January 2020 and was interrupted by containment measures in China. In the second and predominant phase extending from late February to mid-March, unrestricted movements between countries outside of China facilitated intercontinental spread, with Europe as a major source. Phylogenetic analyses also revealed that the dominant strains circulating in the USA were introduced from Europe. However, stringent restrictions on international travel across the world since late March have substantially reduced intercontinental transmission. CONCLUSIONS: Our analyses highlight that heterogeneities in international travel have shaped the spatiotemporal characteristics of the pandemic. Unrestricted travel caused a large number of COVID-19 exportations from Europe to other continents between late February and mid-March, which facilitated the COVID-19 pandemic. Targeted restrictions on international travel from countries with widespread community transmission, together with improved capacity in testing, genetic sequencing and contact tracing, can inform timely strategies for mitigating and containing ongoing and future waves of COVID-19 pandemic.

Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.

Genomic characterization of SARS-CoV-2 identified in a reemerging COVID-19 outbreak in Beijing's Xinfadi market in 2020.

After 56 days without coronavirus disease 2019 (COVID-19) cases, reemergent cases were reported in Beijing, China on June 11, 2020. Here, we report the genetic characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequenced from the clinical specimens of 4 human cases and 2 environmental samples. The nucleotide similarity among six SARS-CoV-2 genomes ranged from 99.98% to 99.99%. Compared with the reference strain of SARS-CoV-2 (GenBank No. NC_045512), all six genome sequences shared the same substitutions at nt241 (C â†’ T), nt3037 (C â†’ T), nt14408 (C â†’ T), nt23403 (A â†’ G), nt28881 (G â†’ A), nt28882 (G â†’ A), and nt28883 (G â†’ C), which are the characteristic nucleotide substitutions of L-lineage European branch I. This was also proved by the maximum likelihood phylogenetic tree based on the full-length genome of SARS-CoV-2. They also have a unique shared nucleotide substitution, nt6026 (C â†’ T), which is the characteristic nucleotide substitution of SARS-CoV-2 in Beijing's Xinfadi outbreak. It is noteworthy that there is an amino acid D614G mutation caused by nt23403 substitution in all six genomes, which may enhance the virus's infectivity in humans and help it become the leading strain of the virus to spread around the world today. It is necessary to continuously monitor the genetic variation of SARS-CoV-2, focusing on the influence of key mutation sites of SARS-CoV-2 on viral transmission, clinical manifestations, severity, and course of disease.

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.

BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).