Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

Cytokine and Chemokine Production in Mice Inoculated with NVX-CoV2373 (Nuvaxovid®) in Comparison with Omicron BA.4/5 Bivalent BNT162b2 (Comirnaty®).

A recombinant SARS-CoV-2 spike protein vaccine (NVX-CoV2373) has been licensed and has a lesser incidence of adverse events. To know the immunological mechanisms of adverse events, the production of cytokines and chemokines was investigated in mice inoculated with NVX-CoV2373. Serum IL-6 was detected on Day 1 of the first and second doses and the IFN-γ, IL-4, IL-10, TNF-α, and IL-6 levels increased on Day 1 of the second dose at the inoculation site. The enhanced production of the inflammatory chemokines (CCL2), homeostatic chemokine (CXCL13), and Th2 chemokine (CCL17) was observed at the inoculation site on Day 1 of the second dose. These findings were compared with data obtained following inoculation with BNT162b2 bivalent vaccine containing omicron BA.4/5. Significantly lower levels of inflammatory chemokines were detected on Day 1 after the first dose of NVX-CoV2373 in sera and inoculation site than those following inoculation with bivalent BNT162b2 (p < 0.01), reflecting a lower incidence of adverse events after immunization with NVX-CoV2373 in humans. NVX-CoV2373 induced significantly higher concentrations of IFN-γ, TNF-α, and IL-10 at the inoculation site obtained on Day 1 of the second dose (p < 0.05). Significant higher levels of Th2 chemokines, CCL11 and CCL17, were induced at the inoculation site on Day 1 of the second dose (p < 0.01) and they explain the booster IgG EIA antibody response after the second dose of NVX-CoV2373.

Experimental Adaptation of Murine Norovirus to Calcium Hydroxide.

Slaked lime (calcium hydroxide) is a commonly used disinfectant for fecal sludge. Although viruses are inactivated by lime treatment, whether RNA viruses adapt to lime treatment has not yet been determined. Here, we show that murine norovirus developed higher tolerance during serial passages with lime treatment. We compared synonymous and non-synonymous nucleotide diversities of the three open reading frames of viral genome and revealed that virus populations were subjected to enhanced purifying selection over the course of serial passages with lime treatment. Virus adaptation to lime treatment was coincident with amino acid substitution of lysine to arginine at position 345 (K345R) on the major capsid protein VP1, which accounted for more than 90% of the population. The infectious clones with the K345R produced using a plasmid-based reverse genetics system exhibited greater tolerance in a lime solution, which indicated that the specific amino acid substitution was solely involved in the viral tolerance in lime treatment.

Dynamic rotation of the protruding domain enhances the infectivity of norovirus.

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract.