ICTV Virus Taxonomy Profile: Coronaviridae 2023.

The family Coronaviridae includes viruses with positive-sense RNA genomes of 22-36 kb that are expressed through a nested set of 3' co-terminal subgenomic mRNAs. Members of the subfamily Orthocoronavirinae are characterized by 80-160 nm diameter, enveloped virions with spike projections. The orthocoronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome-related coronavirus are extremely pathogenic for humans and in the last two decades have been responsible for the SARS and MERS epidemics. Another orthocoronavirus, severe acute respiratory syndrome coronavirus 2, was responsible for the recent global COVID-19 pandemic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Coronaviridae which is available at www.ictv.global/report/coronaviridae.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Unique Features of Hepatitis B Virus-Related Hepatocellular Carcinoma in Pathogenesis and Clinical Significance.

Hepatitis B virus (HBV) infection is one of the important risk factors for hepatocellular carcinoma (HCC) worldwide, accounting for around 50% of cases. Chronic hepatitis B infection generates an inflammatory microenvironment, in which hepatocytes undergoing repeated cycles of damage and regeneration accumulate genetic mutations predisposing them to cancer. A striking male dominance in HBV-related HCC highlights the influence of sex hormones which interact with viral factors to influence carcinogenesis. HBV is also considered an oncogenic virus since its X and surface mutant proteins showed tumorigenic activity in mouse models. The other unique mechanism is the insertional mutagenesis by integration of HBV genome into hepatocyte chromosomes to activate oncogenes. HCC survival largely depends on tumor stages at diagnosis and effective treatment. However, early diagnosis by the conventional protein biomarkers achieves limited success. A new biomarker, the circulating virus-host chimera DNA from HBV integration sites in HCC, provides a liquid biopsy approach for monitoring the tumor load in the majority of HBV-HCC patients. To maximize the efficacy of new immunotherapies or molecular target therapies, it requires better classification of HCC based on the tumor microenvironment and specific carcinogenic pathways. An in-depth study may benefit both the diagnosis and treatment of HBV-related HCC.

D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Virus Titer via Enhanced Furin-Mediated Spike Cleavage.

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.

Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects.

Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.

The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.