T492I mutation alters SARS-CoV-2 properties via modulating viral non-structural proteins (preprint)

The historically dominant SARS-CoV-2 Delta variants and the currently dominant Omicron variants carry a T492I substitution within the non-structural protein 4 (NSP4). Based on a combination of in silico analyses, we predicted that the T492I mutation increases the transmissibility and adaptability of the virus. We confirmed this hypothesis by performing competition experiments in hamsters and in human airway tissue culture models. Furthermore, we show that the T492I mutation also increases the replication capacity and infectiveness of the virus, and improves its ability to evade antibody neutralization induced by previous variants. Mechanistically, the T492I mutation increases cleavage efficiency of the viral main protease NSP5 by enhancing enzyme-substrate binding, resulting in increased production of nearly all non-structural proteins processed by NSP5. Importantly, T492I mutation suppresses the viral RNA associated chemokines in monocytic macrophages, which may contribute to the attenuated pathogenicity of Omicron variants. Our results highlight the importance of the NSP4 mutation in the evolutionary dynamics of SARS-CoV-2 and identify a novel target for the development of broad-spectrum antiviral agents.

A local COVID - 19 outbreak in Dalian caused by imported cold - chain products contaminated by SARS-CoV-2

On December 15, 2020, four dock workers tested positive for severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2) nucleic acids and were reported by Dalian. Up until then, Dalian City had not reported local cases for 136 consecutive days. In this coronavirus disease 2019 (COVID-19) outbreak (referred to as the "Dalian COVID-19 outbreak"), samples from all infected persons (83) and part from the ship cargoes in contact With them during December 15, 2020 to January 8, 2021 were collected. Confirmed cases accounted for 61.45% (51/83) and asymptomatic infections accounted for 38.55% (32/83). Through high-throughput sequencing, 76 SARS-CoV-2 whole-genome sequences were obtained, of which 72 (86.75%) were from clinical samples, and 4 from cold-chain food packaging surface samples on cargo ship A of country R. Refer to Wuhan reference strain (NC_045512), genome analysis revealed 12-16 nucleotide mutations in 76 whole genomes sharing 12 nucleotide mutations and belong to the SARS-CoV-2 branch of B.1.1. Viral genomics and field epidemiological investigations showed that the Dalian COVID-19 outbreak was a local epidemic caused by dock workers infected with imported cold - chain products contaminated with SARS - CoV - 2. During transmission, 3 Virus generations and three relatively independent transmission chains were formed.

A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants (preprint)

Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion (preprint)

Previous work indicated that the nucleocapsid 203 mutation increase the virulence and transmission of the SARS-CoV-2 Alpha variant. However, Delta later outcompeted Alpha and other lineages, promoting a new wave of infections. Delta also possesses a nucleocapsid 203 mutation, R203M. Large-scale epidemiological analyses suggest a synergistic effect of the 203 mutation and the spike L452R mutation, associated with Delta expansion. Viral competition experiments demonstrate the synergistic effect in fitness and infectivity. More importantly, we found that the combination of R203M and L452R brings in a 3.2-fold decrease in neutralizing titers to the neutralizing serum relative to L452R-only virus. R203M/L452R show an increased fitness after the initiation of global vaccination programmes, possibly associated with the enhanced immune evasion. Another rapidly emerging variant Omicron also bears the 203 mutation. Thus, we proposed that nucleocapsid mutations play an essential role for the rise and predominance of variants in concern.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection (preprint)

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

Nucleocapsid Mutations R203K/G204R Increase the Infectivity, Fitness and Virulence of SARS-CoV-2 (preprint)

In this study, cooccurring mutations R203K/G204R in the nucleocapsid protein are demonstrated adaptive and associated with the emergence of a high-transmissibility SARS-CoV-2 lineage B.1.1.7. Through comparing a R203K/G204R mutant virus, created based on the USA-WA1/2020 SARS-CoV-2 strain, with the native virus in competition experiments, we found that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly during virus replication. Moreover, the 203K/204R virus showed increased infectivity in a human lung cell line and induced increased damage to blood vessels in infected hamster lungs. Accordingly, we observed a positive association between increased COVID-19 severity and the incidence frequency of 203K/204R. Our work suggested a contribution of the 203K/204R mutations to the increased transmission and virulence of SARS-CoV-2. In addition to mutations in the spike protein, the mutations in the nucleocapsid protein are important for viral spreading during the pandemic.Funding Information: This work was supported by grants from the National Natural Science Foundation of China, SGC's Rapid Response Funding for COVID-19 (C-0002), the National Key Research and Development Program (2019YFC1604600), the National Natural Science Foundation of China (81970008 and 31200941), the Fundamental Research Funds for the Central Universities (2021CDJYGRH-009), the Youth Innovative Talents Training Project of Chongqing (CY210102) and the National Natural Science Foundation of HeBei province (19226631D).Declaration of Interests: The authors declare no competing interests.

Nucleocapsid mutation R203K/G204R increases the infectivity, fitness and virulence of SARS-CoV-2 (preprint)

In addition to the mutations on the spike protein (S), co-occurring mutations on nucleocapsid (N) protein are also emerging in SARS-CoV-2 world widely. Mutations R203K/G204R on N, carried by high transmissibility SARS-CoV-2 lineages including B.1.1.7 and P.1, has a rapid spread in the pandemic during the past year. In this study, we performed comprehensive population genomic analyses and virology experiment concerning on the evolution, causation and virology consequence of R203K/G204R mutations. The global incidence frequency (IF) of 203K/204R has rose up from nearly zero to 76% to date with a shrinking from August to November in 2020 but bounced later. Our results show that the emergence of B.1.1.7 is associated with the second growth of R203K/G204R mutants. We identified positive selection evidences that support the adaptiveness of 203K/204R variants. The R203K/G204R mutant virus was created and compared with the native virus. The virus competition experiments show that 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly in relation to the ribonucleocapsid (RNP) assemble during the virus replication. Moreover, the 203K/204R virus increased the infectivity in a human lung cell line and induced an enhanced damage to blood vessel of infected hamsters' lungs. In consistence, we observed a positive association between the increased severity of COVID-19 and the IF of 203K/204R from in silicon analysis of global clinical and epidemic data. In combination with the informatics and virology experiment, our work suggested the contribution of 203K/204R to the increased transmission and virulence of the SARS-CoV-2. In addition to mutations on the S protein, the mutations on the N protein are also important to virus spread during the pandemic.

Patient-derived mutations impact pathogenicity of SARS-CoV-2 (preprint)

The sudden outbreak of the severe acute respiratory syndrome-coronavirus (SARS-CoV-2) has spread globally with more than 1,300,000 patients diagnosed and a death toll of 70,000. Current genomic survey data suggest that single nucleotide variants (SNVs) are abundant. However, no mutation has been directly linked with functional changes in viral pathogenicity. Here we report functional characterizations of 11 patient-derived viral isolates, all of which have at least one mutation. Importantly, these viral isolates show significant variation in cytopathic effects and viral load, up to 270-fold differences, when infecting Vero-E6 cells. We observed intrapersonal variation and 6 different mutations in the spike glycoprotein (S protein), including 2 different SNVs that led to the same missense mutation. Therefore, we provide direct evidence that the SARS-CoV-2 has acquired mutations capable of substantially changing its pathogenicity.