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.

More efforts are needed for background surveys of zoonotic coronaviruses in animals.

Recently, a novel dog-origin coronavirus has been found in humans. The low similarity between the receptor-binding domain from this novel virus and other human-infecting coronaviruses in genus Alphacoronavirus suggests it might use a novel receptor or mechanism to enter human cells and also might trigger a novel immune response.

Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice.

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Genomic and Epidemiological Features of COVID-19 in the Novosibirsk Region during the Beginning of the Pandemic.

In this retrospective, single-center study, we conducted an analysis of 13,699 samples from different individuals obtained from the Federal Research Center of Fundamental and Translational Medicine, from 1 April to 30 May 2020 in Novosibirsk region (population 2.8 million people). We identified 6.49% positive for SARS-CoV-2 cases out of the total number of diagnostic tests, and 42% of them were from asymptomatic people. We also detected two asymptomatic people, who had no confirmed contact with patients with COVID-19. The highest percentage of positive samples was observed in the 80+ group (16.3%), while among the children and adults it did not exceed 8%. Among all the people tested, 2423 came from a total of 80 different destinations and only 27 of them were positive for SARS-CoV-2. Out of all the positive samples, 15 were taken for SARS-CoV-2 sequencing. According to the analysis of the genome sequences, the SARS-CoV-2 variants isolated in the Novosibirsk region at the beginning of the pandemic belonged to three phylogenetic lineages according to the Pangolin classification: B.1, B.1.1, and B.1.1.129. All Novosibirsk isolates contained the D614G substitution in the Spike protein, two isolates werecharacterized by an additional M153T mutation, and one isolate wascharacterized by the L5F mutation.

Apolipoprotein E mediates cell resistance to influenza virus infection.

Viruses exploit host cell machinery to support their replication. Defining the cellular proteins and processes required for a virus during infection is crucial to understanding the mechanisms of virally induced disease and designing host-directed therapeutics. Here, we perform a genome-wide CRISPR-Cas9-based screening in lung epithelial cells infected with the PR/8/NS1-GFP virus and use GFPhi cell as a unique screening marker to identify host factors that inhibit influenza A virus (IAV) infection. We discovered that APOE affects influenza virus infection both in vitro and in vivo. Cell deficiency in APOE conferred substantially increased susceptibility to IAV; mice deficient in APOE manifested more severe lung pathology, increased virus load, and decreased survival rate. Mechanistically, lack of cell-produced APOE results in impaired cell cholesterol homeostasis, enhancing influenza virus attachment. Thus, we identified a previously unrecognized role of APOE in restraining IAV infection.

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.

Relatively rapid evolution rates of SARS-CoV-2 spike gene at the primary stage of massive vaccination.

A series of stringent non-pharmacological and pharmacological interventions were implemented to contain the pandemic but the pandemic continues. Moreover, vaccination breakthrough infection and reinfection in convalescent coronavirus disease 2019 (COVID-19) cases have been reported. Further, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants emerged with mutations in spike (S) gene, the target of most current vaccines. Importantly, the mutations exhibit a trend of immune escape from the vaccination. Herein the scientific question that if the vaccination drives genetic or antigenic drifts of SARS-CoV-2 remains elusive. We performed correlation analyses to uncover the impacts of wide vaccination on epidemiological characteristics of COVID-19. In addition, we investigated the evolutionary dynamics and genetic diversity of SARS-CoV-2 under immune pressure by utilizing the Bayesian phylodynamic inferences and the lineage entropy calculation respectively. We found that vaccination coverage was negatively related to the infections, severe cases, and deaths of COVID-19 respectively. With the increasing vaccination coverage, the lineage diversity of SARS-CoV-2 dampened, but the rapid mutation rates of the S gene were identified, and the vaccination could be one of the explanations for driving mutations in S gene. Moreover, new epidemics resurged in several countries with high vaccination coverage, questioning their current pandemic control strategies. Hence, integrated vaccination and non-pharmacological interventions are critical to control the pandemic. Furthermore, novel vaccine preparation should enhance its capabilities to curb both disease severity and infection possibility.

A tandem-repeat dimeric RBD protein-based covid-19 vaccine zf2001 protects mice and nonhuman primates.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 µg or 50 µg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

SARS-CoV-2 transmissibility compared between variants of concern and vaccination status.

Since the start of the SARS-CoV-2 pandemic in late 2019, several variants of concern (VOC) have been reported to have increased transmissibility. In addition, despite the progress of vaccination against SARS-CoV-2 worldwide, all vaccines currently in used are known to protect only partially from infection and onward transmission. We combined phylogenetic analysis with Bayesian inference under an epidemiological model to infer the reproduction number (Rt) and also trace person-to-person transmission. We examined the impact of phylogenetic uncertainty and sampling bias on the estimation. Our result indicated that lineage B had a significantly higher transmissibility than lineage A and contributed to the global pandemic to a large extent. In addition, although the transmissibility of VOCs is higher than other exponentially growing lineages, this difference is not very high. The probability of detecting onward transmission from patients infected with SARS-CoV-2 VOCs who had received at least one dose of vaccine was approximate 1.06% (3/284), which was slightly lower but not statistically significantly different from a probability of 1.21% (10/828) for unvaccinated individuals. In addition to VOCs, exponentially growing lineages in each country should also be account for when tailoring prevention and control strategies. One dose of vaccination could not efficiently prevent the onward transmission of SARS-CoV-2 VOCs. Consequently, nonpharmaceutical interventions (such as wearing masks and social distancing) should still be implemented in each country during the vaccination period.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion, and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones 2-3 weeks after the onset of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34, and IGHV4-39 in COVID-19 patients were more abundant than those of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

VarEPS: an evaluation and prewarning system of known and virtual variations of SARS-CoV-2 genomes.

The genomic variations of SARS-CoV-2 continue to emerge and spread worldwide. Some mutant strains show increased transmissibility and virulence, which may cause reduced protection provided by vaccines. Thus, it is necessary to continuously monitor and analyze the genomic variations of SARS-COV-2 genomes. We established an evaluation and prewarning system, SARS-CoV-2 variations evaluation and prewarning system (VarEPS), including known and virtual mutations of SARS-CoV-2 genomes to achieve rapid evaluation of the risks posed by mutant strains. From the perspective of genomics and structural biology, the database comprehensively analyzes the effects of known variations and virtual variations on physicochemical properties, translation efficiency, secondary structure, and binding capacity of ACE2 and neutralizing antibodies. An AI-based algorithm was used to verify the effectiveness of these genomics and structural biology characteristic quantities for risk prediction. This classifier could be further used to group viral strains by their transmissibility and affinity to neutralizing antibodies. This unique resource makes it possible to quickly evaluate the variation risks of key sites, and guide the research and development of vaccines and drugs. The database is freely accessible at www.nmdc.cn/ncovn.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection.

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/µL) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 min. Given our system's simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice.

As COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.