福田 佐織

Live-attenuated rotavirus (RV) vaccines are the most effective interventions for preventing RV gastroenteritis (RVGE) in young children. However, the molecular basis of attenuation remains not well understood. Here, we describe a compact but comprehensive strategy to identify RV virulence determinants by combining low-passage bidirectional selection, sequence analysis, and segment-level phenotype testing via a reverse genetics infectious system. Using the virulent murine RV strain EW, virulence was quantified by diarrhea severity/duration and body-weight gain. Serial passaging in cell culture selected an attenuated population, which regained virulence after passaging in suckling mice. Sequence comparison of the virulent and attenuated EW populations revealed only seven amino acid differences. We summarized literature describing attenuation/virulence-associated mutations in various RV group A (RVA) strains and found previous findings identical or similar to four of the seven mutations: NSP4-T45M, VP4-S470L, VP4-T612A, and VP7-T75P. Virulent- and attenuated-type EW variants of VP2, VP4, VP7, and NSP4 were introduced individually, or as NSP4/VP7 or VP4/VP7 pairs, into a simian SA11-L2 backbone using an 11-plasmid reverse genetics system. Phenotyping of rescued viruses consistently linked cell-culture-adapted VP4 to enhanced replication in vitro and reduced virulence in suckling mice. In vivo passaging strongly favored VP4 residue S470 over cell-culture-selected L470. More generally, our findings (i) underscore VP4 and VP7 as key determinants of EW virulence, (ii) provide a practical framework for identifying driver mutations underlying RVA attenuation, and (iii) highlight attenuation-associated substitutions shared across diverse RVAs.

BACKGROUND: Biliary atresia (BA) is a severe infantile hepatobiliary disorder of unknown etiology. Perinatal rotavirus (RV) infection has been implicated in animal models of BA; however, supporting human data remains limited. The study investigated the serological evidence of recent RV infection in infants with BA using RV-specific immunoglobulin (Ig)-A, a marker of primary infection unaffected by maternal antibodies. METHODS: Serum samples from 17 infants with BA and 30 age-matched controls without gastrointestinal symptoms or prior RV vaccination were retrospectively analyzed. Anti-RV-IgA titers were measured by enzyme-linked immunosorbent assay using purified WA-strain virions. Cytomegalovirus (CMV)-IgM and Epstein-Barr virus (EBV)-viral capsid antigen (VCA)-IgM levels were assessed using commercial enzyme immunoassays. RESULTS: RV-IgA was detected in 70.6% (12/17) of the patients with BA versus 3.4% (1/29) of the controls (p < 0.001). RV-IgA titers were significantly higher in the BA group (median: interquartile range 28.0:26.0-210.0) than in the control group (23.5:22.0-24.8) (p = 0.004). Among patients diagnosed with BA after 14 days of age, 84.6% (11/13) were RV-IgA-positive. CMV-IgM was detected in three patients in the BA group and one individual in the control group, while EBV-VCA-IgM was negative in BA patients and positive in two controls; neither difference was statistically significant. CONCLUSIONS: The study findings support the potential association between RV infection and BA pathogenesis. However, the lack of an epidemiological reduction in BA following the introduction of the RV vaccine warrants caution in other studies. Further prospective multicenter studies are required to elucidate the causal role of RV infection in BA development.

An unusual rotavirus strain with the G3P[10] genotype, designated RVA/Human-wt/THA/PK2016-1-0120/2016/G3P[10] (short name "PK2016-1-0120"), was detected in a stool specimen from a hospitalized 10-year-old boy with acute gastroenteritis in Thailand. In this study, we sequenced and characterized its whole genome. The strain possesses the genotype constellation G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, similar to several previously reported bat and bat-like rotavirus strains (MYAS33-like). In line with this, phylogenetic analyses and sequence comparisons indicate that 10 of the 11 genomic segments (VP4, VP6, VP1-VP3, and NSP1-NSP5) are most similar to those found in MYAS33-like strains. In contrast, the VP7 genomic segment of PK2016-1-0120-which defines the G genotype-is most closely related to those of the Indian equine rotavirus strain Erv105 and DS-1-like G3P[8] human strains. These findings imply a history of segment reassortment involving independent acquisition of the VP7 segment. Given that bats are likely donors in interspecies RVA transmission chains, we speculate that this VP7 lineage originated in bats. In summary, our characterization of the novel bat-like human strain PK2016-1-0120 suggests that the VP7 lineage as found in human DS-1-like G3P[8] strains may have originated from a bat-associated rotavirus, offering an alternative model to the previously proposed equine origin.

Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that establishes lifelong latency in sensory ganglionic neurons. Subsequent viral reactivation causes herpes zoster, a morbid disease often complicated by neuropathic pain. Mechanisms underlying VZV latency and reactivation are not understood, mostly due to the lack of permissive animal models and challenges of current in vitro latency modelling. Here, we evaluated HD10.6 cells, a simplified and easily expandable human sensory neuron line to model VZV latency and reactivation. Mature HD10.6 (mHD10.6) differentiated neurons supported productive VZV infection, viral DNA replication, production of infectious progeny, and viral spread in cultures. VZV infection was associated with limited cytopathic effects and ultrastructural changes. Infecting mHD10.6 neurons with cell-free VZV in the presence of antivirals resulted in a quiescent-persistent state, characterized by persistent VZV genomes with restricted VZV gene expression and absence of infectious virus. Importantly, VZV could be reactivated by treatment with capsaicin, as evidenced by increased lytic viral transcription and virus spread. In conclusion, this study establishes human HD10.6 neurons as a novel and scalable in vitro model for studying VZV latency and reactivation to identify virus and host factors governing latency that may serve as therapeutic targets to restrict VZV reactivation.

Abstract Background Rotavirus infections are a major cause of severe gastroenteritis in children. Human rotavirus strains with the unconventional G8P[14] genotype have sporadically been detected in diarrheic patients in different parts of the world. However, full genomes of only two human G8P[14] strains from Africa (North Africa) have been sequenced, and the origin and evolutionary patterns of African G8P[14] strains remain to be elucidated. Methods In this study, we sequenced the full genome of an African G8P[14] strain (RVA/Human-wt/KEN/A75/2000/G8P[14]) identified in archival stool samples from a diarrheic child in Kenya. Results Full genome-based analysis of strain A75 revealed a unique genogroup constellation, G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3, with the I2-R2-C2-M2-A11-N2-T6-E2-H3 part being common among rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis showed that all the 11 genomic segments of strain A75 are closely related to segments found in artiodactyl rotavirus strains, and likely strain A75 derived from spillover transmission of an artiodactyl rotavirus strain to humans. Conclusion This is the first report on a full genome-based characterization of a human G8P[14] strain from East Africa. This study demonstrates the diversity of human G8P[14] strains in Africa and contributes to the elucidation of their spreading and evolution, which includes zoonotic transmission from artiodactyls.