Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

Background: The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Methods: Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). Results: The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. Conclusions: The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys.

The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak.

The Diamond Princess cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a coronavirus disease 2019 case. We performed whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from PCR+ clinical specimens and conducted a phylogenetic analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the Diamond Princess originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters could be linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network/phylogeny analysis in identifying potential infection routes.