Intranasal immunization with recombinant Vaccinia virus encoding trimeric SARS-CoV-2 spike receptor-binding domain induces neutralizing antibody.

Respiratory transmission of SARS-CoV-2 is considered to be the major dissemination route for COVID-19, therefore, mucosal immune responses have great importance in preventing SARS-CoV-2 from infection. In this study, we constructed a recombinant Vaccinia virus (VV) harboring trimeric receptor-binding domain (RBD) of SARS-CoV-2 spike protein (VV_-tRBD), and evaluated the immune responses towards RBD following intranasal immunization against mice and rabbits. In BALB/c mice, intranasal immunization with VV_-tRBD elicited robust humoral and cellular immune responses, with high-level of both neutralizing IgG and IgA in sera against SARS-CoV-2 psudoviruses, and a number of RBD-specific IFN-γ-secreting lymphocytes. Sera from immunized rabbits also exhibited neutralization effects. Notably, RBD-specific secretory IgA (sIgA) in both nasal washes and bronchoalveolar lavage fluids (BALs) were detectable and showed substantial neutralization activities. Collectively, a recombinant VV expressing trimeric RBD confers robust systemic immune response and mucosal neutralizing antibodies, thus warranting further exploration as a mucosal vaccine.

Potential of large "first generation" human-to-human transmission of 2019-nCoV.

To investigate the genetic diversity, time origin, and evolutionary history of the 2019-nCoV outbreak in China and Thailand, a total of 12 genome sequences of the virus with known sampling date (24 December 2019 and 13 January 2020) and geographic location (primarily Wuhan city, Hubei Province, China, but also Bangkok, Thailand) were analyzed. Phylogenetic and likelihood-mapping analyses of these genome sequences were performed. On the basis of our results, the star-like signal and topology of 2019-nCoV may be indicative of potentially large "first generation" human-to-human virus transmission. We estimated that 2019-nCoV likely originated in Wuhan on 9 November 2019 (95% credible interval: 25 September 2019 and 19 December 2019), and that Wuhan is the major hub for the spread of the 2019-nCoV outbreak in China and elsewhere. Our results could be useful for designing effective prevention strategies for 2019-nCoV in China and beyond.

Genomic epidemiology of a densely sampled COVID-19 outbreak in China.

Analysis of genetic sequence data from the SARS-CoV-2 pandemic can provide insights into epidemic origins, worldwide dispersal, and epidemiological history. With few exceptions, genomic epidemiological analysis has focused on geographically distributed data sets with few isolates in any given location. Here, we report an analysis of 20 whole SARS- CoV-2 genomes from a single relatively small and geographically constrained outbreak in Weifang, People's Republic of China. Using Bayesian model-based phylodynamic methods, we estimate a mean basic reproduction number (R 0) of 3.4 (95% highest posterior density interval: 2.1-5.2) in Weifang, and a mean effective reproduction number (Rt) that falls below 1 on 4 February. We further estimate the number of infections through time and compare these estimates to confirmed diagnoses by the Weifang Centers for Disease Control. We find that these estimates are consistent with reported cases and there is unlikely to be a large undiagnosed burden of infection over the period we studied.

Phylogenetic and phylodynamic analyses of SARS-CoV-2.

To investigate the evolutionary and epidemiological dynamics of the current COVID-19 outbreak, a total of 112 genomes of SARS-CoV-2 strains sampled from China and 12 other countries with sampling dates between 24 December 2019 and 9 February 2020 were analyzed. We performed phylogenetic, split network, likelihood-mapping, model comparison, and phylodynamic analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis with the best-fitting combination models, we estimated the time to the most recent common ancestor (TMRCA) and evolutionary rate of SARS-CoV-2 to be 12 November 2019 (95 % BCI: 11 October 2019 and 09 December 2019) and 9.90 × 10-4 substitutions per site per year (95 % BCI: 6.29 × 10-4-1.35 × 10-3), respectively. Notably, the very low Re estimates of SARS-CoV-2 during the recent sampling period may be the result of the successful control of the pandemic in China due to extreme societal lockdown efforts. Our results emphasize the importance of using phylodynamic analyses to provide insights into the roles of various interventions to limit the spread of SARS-CoV-2 in China and beyond.

Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2.

To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.

Transmission dynamics and evolutionary history of 2019-nCoV.

To investigate the time origin, genetic diversity, and transmission dynamics of the recent 2019-nCoV outbreak in China and beyond, a total of 32 genomes of virus strains sampled from China, Thailand, and the USA with sampling dates between 24 December 2019 and 23 January 2020 were analyzed. Phylogenetic, transmission network, and likelihood-mapping analyses of the genome sequences were performed. On the basis of the likelihood-mapping analysis, the increasing tree-like signals (from 0% to 8.2%, 18.2%, and 25.4%) over time may be indicative of increasing genetic diversity of 2019-nCoV in human hosts. We identified three phylogenetic clusters using the Bayesian inference framework and three transmission clusters using transmission network analysis, with only one cluster identified by both methods using the above genome sequences of 2019-nCoV strains. The estimated mean evolutionary rate for 2019-nCoV ranged from 1.7926 × 10-3 to 1.8266 × 10-3 substitutions per site per year. On the basis of our study, undertaking epidemiological investigations and genomic data surveillance could positively impact public health in terms of guiding prevention efforts to reduce 2019-nCOV transmission in real-time.

Cross-species transmission of the newly identified coronavirus 2019-nCoV.

The current outbreak of viral pneumonia in the city of Wuhan, China, was caused by a novel coronavirus designated 2019-nCoV by the World Health Organization, as determined by sequencing the viral RNA genome. Many initial patients were exposed to wildlife animals at the Huanan seafood wholesale market, where poultry, snake, bats, and other farm animals were also sold. To investigate possible virus reservoir, we have carried out comprehensive sequence analysis and comparison in conjunction with relative synonymous codon usage (RSCU) bias among different animal species based on the 2019-nCoV sequence. Results obtained from our analyses suggest that the 2019-nCoV may appear to be a recombinant virus between the bat coronavirus and an origin-unknown coronavirus. The recombination may occurred within the viral spike glycoprotein, which recognizes a cell surface receptor. Additionally, our findings suggest that 2019-nCoV has most similar genetic information with bat coronovirus and most similar codon usage bias with snake. Taken together, our results suggest that homologous recombination may occur and contribute to the 2019-nCoV cross-species transmission.