Molecular evolutionary analysis of novel NSP4 mono-reassortant G1P[8]-E2 rotavirus strains that caused a discontinuous epidemic in Japan in 2015 and 2018.

In the 2010s, several unusual rotavirus strains emerged, causing epidemics worldwide. This study reports a comprehensive molecular epidemiological study of rotaviruses in Japan based on full-genome analysis. From 2014 to 2019, a total of 489 rotavirus-positive stool specimens were identified, and the associated viral genomes were analyzed by next-generation sequencing. The genotype constellations of those strains were classified into nine patterns (G1P[8] (Wa), G1P[8]-E2, G1P[8] (DS-1), G2P[4] (DS-1), G3P[8] (Wa), G3P[8] (DS-1), G8P[8] (DS-1), G9P[8] (Wa), and G9P[8]-E2). The major prevalent genotype differed by year, comprising G8P[8] (DS-1) (37% of that year's isolates) in 2014, G1P[8] (DS-1) (65%) in 2015, G9P[8] (Wa) (72%) in 2016, G3P[8] (DS-1) (66%) in 2017, G1P[8]-E2 (53%) in 2018, and G9P[8] (Wa) (26%) in 2019. The G1P[8]-E2 strains (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1) isolated from a total of 42 specimens in discontinuous years (2015 and 2018), which were the newly-emerged NSP4 mono-reassortant strains. Based on the results of the Bayesian evolutionary analyses, G1P[8]-E2 and G9P[8]-E2 were hypothesized to have been generated from distinct independent inter-genogroup reassortment events. The G1 strains detected in this study were classified into multiple clusters, depending on the year of detection. A comparison of the predicted amino acid sequences of the VP7 epitopes revealed that the G1 strains detected in different years encoded VP7 epitopes harboring distinct mutations. These mutations may be responsible for immune escape and annual changes in the prevalent strains.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

Chimeric provirus of bovine leukemia virus/SMAD family member 3 in cattle with enzootic bovine leukosis.

Bovine leukemia virus (BLV) is a member of the family Retroviridae that causes enzootic bovine leukemia (EBL). However, the association between BLV infection and EBL development remains unclear. In this study, we identified a BLV/SMAD3 chimeric provirus within CC2D2A intron 30 in monoclonal expanded malignant cells from a cow with EBL. The chimeric provirus harbored a spliced SMAD3 sequence composed of exons 3-9, encoding the short isoform protein, and the BLV-SMAD3 chimeric transcript was detectable in cattle with EBL. This is the first report of a BLV chimeric provirus that might be involved in EBL tumorigenesis.

Generation of JC Polyoma Pseudovirus for High-Throughput Measurement of Neutralizing Antibodies.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) caused by reactivation of dormant JC polyomavirus (JCPyV). PML was mainly observed in immunocompromised individuals, such as HIV-positive patients, autoimmune disease patients, and cancer patients. Given that the presence of anti-JCPyV antibodies in serum is a risk indicator for PML development, it is essential to monitor anti-JCPyV antibody levels. In the present study, we established reporter-based single-infection neutralization assays for JCPyV and the genetically similar BK polyoma virus (BKPyV). We then confirmed the lack of cross-reactivity between the two viruses using test sera obtained from mice immunized with plasmids encoding the JCPyV or BKPyV capsid. Next, we compared neutralization antibody titers in sera from healthy donors, patients with multiple sclerosis (MS), and HIV-positive patients using an in-house enzyme-linked immunosorbent assay (ELISA) with JCPyV-like particles (virus-like particles; VLPs). A positive correlation was demonstrated between the neutralization titer (75% infectious concentration; IC75) against JCPyV and the antibody titer obtained by VLP-based JCPyV ELISA. This assay system may be applied to detect antibodies against other PyVs by generation of pseudoviruses using the respective capsid expression plasmids, and is expected to contribute to the surveillance of PyV as well as basic research on these viruses.

Antiviral effect of peptoids on hepatitis B virus infection in cell culture.

Although antimicrobial peptides have been shown to inactivate viruses through disruption of their viral envelopes, clinical use of such peptides has been hampered by a number of factors, especially their enzymatically unstable structures. To overcome the shortcomings of antimicrobial peptides, peptoids (sequence-specific N-substituted glycine oligomers) mimicking antimicrobial peptides have been developed. We aimed to demonstrate the antiviral effects of antimicrobial peptoids against hepatitis B virus (HBV) in cell culture. The anti-HBV activity of antimicrobial peptoids was screened and evaluated in an infection system involving the HBV reporter virus and HepG2.2.15-derived HBV. By screening with the HBV reporter virus infection system, three (TM1, TM4, and TM19) of 12 peptoids were identified as reducing the infectivity of HBV, though they did not alter the production levels of HBs antigen in cell culture. These peptoids were not cytotoxic at the evaluated concentrations. Among these peptoids, TM19 was confirmed to reduce HBV infection most potently in a HepG2.2.15-derived HBV infection system that closely demonstrates authentic HBV infection. In cell culture, the most effective administration of TM19 was virus treatment at the infection step, but the reduction in HBV infectivity by pre-treatment or post-treatment of cells with TM19 was minimal. The disrupting effect of TM19 targeting infectious viral particles was clarified in iodixanol density gradient analysis. In conclusion, the peptoid TM19 was identified as a potent inhibitor of HBV. This peptoid prevents HBV infection by disrupting viral particles and is a candidate for a new class of anti-HBV reagents.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.