Takayuki Murata

Epstein-Barr virus (EBV) infects over 90% of humans and is associated with both hematological and epithelial malignancies. Here, we analyzed 990 EBV genomes (319 newly sequenced and 671 from public databases) from patients with various diseases to comprehensively characterize genomic variations, including single nucleotide variations (SNVs) and structural variations (SVs). While most SNVs were a result of conservative evolution and reflected the geographical origins of the viral genomes, we identified several convergent SNV hotspots within the central homology domain of EBNA3B, the transactivation domain of EBNA2, and the second transmembrane domain of LMP1. These convergent SNVs appear to fine-tune viral protein functionality and immunogenicity. SVs, particularly large deletions, were frequently observed in chronic active EBV disease (28%), EBV-positive diffuse large B-cell lymphoma (48%), extranodal NK/T-cell lymphoma (41%), and Burkitt lymphoma (25%), but were less common in infectious mononucleosis (11%), post-transplant lymphoproliferative disorder (7%), and epithelial malignancies (5%). In hematological malignancies, deletions often targeted viral microRNA clusters, potentially promoting viral reactivation and lymphomagenesis. Non-deletion SVs, such as inversions, were also prevalent, with several inversions disrupting the C promoter to suppress latent gene expression, thereby maintaining viral dormancy. Furthermore, recurrent EBNA3B deletions suggested that this viral transcription factor functions as a tumor suppressor. EBNA3B knockout experiments in vitro revealed downregulation of human tumor suppressors, including PTEN and RB1, which could explain the enhanced lymphomagenesis observed in EBNA3B-deficient lymphoblastoid cell line xenografts. Our findings highlight both disease-specific and general contributions of EBV genomic alterations to human cancers, particularly in hematological malignancies.

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.

Patients with hematologic diseases have experienced coronavirus disease 2019 (COVID-19) with a prolonged, progressive course. Here, we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels persisted for >4 weeks, and two of them succumbed to COVID-19. The autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping cytomegalovirus, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and an animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D, and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent the acquisition of drug resistance and improve outcomes, such as the selection of appropriate treatment strategies for lymphoma considering patients' immune status and the institution of early intensive antiviral therapy.

BACKGROUND AND AIMS: HBV leads to severe liver diseases, such as cirrhosis and HCC. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. APPROACH AND RESULTS: We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed HBeAg and HBsAg production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. CONCLUSIONS: Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.