村田 貴之

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.

BACKGROUND: A substantial portion of gastric cancer (GC) is linked to Epstein-Barr virus (EBV) infection. The characteristics of this viral genome, such as specific viral strains and large structural variations, influence the progression of diseases like nasopharyngeal carcinoma and hematological malignancy. However, the EBV genomes from GC have not been thoroughly characterized. METHODS: Our study involved 849 consecutive GC patients diagnosed at Nagoya City University Hospital, Japan (NCU cohort). We detected EBV from formalin-fixed, paraffin-embedded sections using a novel direct PCR-based rapid detection method. Additionally, we analyzed 142 EBV whole genomes (125 newly sequenced) from GC, comparing them with 205 genomes from other EBV-associated diseases. RESULTS: We identified 32 (3.8%) patients associated with EBVaGC in the NCU cohort. Moreover, the direct PCR identified several GC specimens containing EBV-infected lymphocytes or their follicles. The dominant viral strain in GC was type 1 EBV, prevalent in most parts of the world, and no GC-specific strain was identified. We found no significant associations between single-nucleotide variants in the viral genome and GC. Structural variations of the EBV genome were infrequent in GC (4 cases, 2.1%), contrasting with EBV-associated hematological malignancy, which frequently carries large deletions. CONCLUSIONS: This study is the first to uncover the genomic variations of EBV in GC. While EBV is definitively linked to GC, the characteristics of its genomes do not strongly correlate with disease development or progression. Our findings on viral genomes supplement the current understanding of human genomes in EBVaGC.

Major histocompatibility complex class II (MHC-II) is the most significant genetic risk factor for systemic lupus erythematosus (SLE), but the nature of the self-antigens that trigger autoimmunity remains unclear. Unusual self-antigens, termed neoself-antigens, are presented on MHC-II in the absence of the invariant chain essential for peptide presentation. Here, we demonstrate that neoself-antigens are the primary target for autoreactive T cells clonally expanded in SLE. When neoself-antigen presentation was induced by deleting the invariant chain in adult mice, neoself-reactive T cells were clonally expanded, leading to the development of lupus-like disease. Furthermore, we found that neoself-reactive CD4+ T cells were significantly expanded in SLE patients. A high frequency of Epstein-Barr virus reactivation is a risk factor for SLE. Neoself-reactive lupus T cells were activated by Epstein-Barr-virus-reactivated cells through downregulation of the invariant chain. Together, our findings imply that neoself-antigen presentation by MHC-II plays a crucial role in the pathogenesis of SLE.