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During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.
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Ciervos , Flavivirus , Metagenómica , Garrapatas , Animales , Metagenómica/métodos , Japón/epidemiología , Ciervos/virología , Flavivirus/genética , Flavivirus/aislamiento & purificación , Flavivirus/clasificación , Garrapatas/virología , Filogenia , Viroma/genética , Virión/genética , Sus scrofa/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Seroepidemiológicos , Genoma ViralRESUMEN
Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.
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Transporte Activo de Núcleo Celular , Núcleo Celular , Flavivirus , Humanos , Flavivirus/metabolismo , Flavivirus/fisiología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/virología , Replicación Viral/fisiología , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Carioferinas/metabolismo , Carioferinas/genética , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Chlorocebus aethiops , Células HEK293RESUMEN
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.
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Virus de la Hepatitis A , Loxapina , Animales , Ratones , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/metabolismo , Biosíntesis de Proteínas , Replicación Viral/genética , ARN/metabolismo , Proteínas Virales/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multi-component protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin (CaM). This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and CaM determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.
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Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.
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Quinasa de la Caseína II , ADN , Factor 1 Regulador del Interferón , Virosis , Cromatina , ADN/genética , Factor 1 Regulador del Interferón/genética , Transducción de Señal/genética , Humanos , Quinasa de la Caseína II/genética , Inmunidad Innata , Virosis/genética , Virosis/inmunologíaRESUMEN
Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.
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Anexina A5 , Hepacivirus , Hepatitis C , Ocludina , Humanos , Anexina A5/genética , Anexina A5/metabolismo , Regulación hacia Abajo , Hepacivirus/fisiología , Ocludina/genética , Ocludina/metabolismo , Isoformas de Proteínas/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Internalización del VirusRESUMEN
Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.
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Anticuerpos Neutralizantes , Bombyx , Virus del Dengue , Ratones Endogámicos BALB C , Proteínas del Envoltorio Viral , Animales , Bombyx/genética , Bombyx/virología , Bombyx/metabolismo , Virus del Dengue/genética , Virus del Dengue/inmunología , Ratones , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/biosíntesis , Anticuerpos Neutralizantes/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/biosíntesis , Vacunas contra el Dengue/inmunología , Vacunas contra el Dengue/genética , Anticuerpos Antivirales/inmunología , Dengue/inmunología , Dengue/virología , Serogrupo , Dominios Proteicos , FemeninoRESUMEN
In 2018, there was a hepatitis A outbreak in Japan, and hepatitis A virus (HAV) infection is considered a sexually transmitted disease. In general, patients with hepatitis A should be given attention, and this disease should be prevented more than ever. The Japan Agency for Medical Research and Development (AMED) Hepatitis A and E viruses (HAV and HEV) Study Group has worked on the project to create "Recent Advances in Hepatitis A Virus (HAV) Research and Clinical Practice Guidelines for HAV Infection in Japan". The group consists of expert hepatologists and virologists who gathered at virtual meeting on August 5, 2023. Data about the pathogenesis, infection routes, diagnosis, complications, several factors for the severities, vaccination, and current and future treatments for hepatitis A were discussed and debated for a draft version. The participants assessed the quality of cited studies. The finalized recommendations are presented in this review. The recent advances in HAV research and clinical practice for HAV infection in Japan, have been reviewed by the AMED HAV and HEV Study Group.
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Acute hepatitis E was considered rare until reports emerged affirming the existence of hepatitis E virus (HEV) genotypes 3 and 4 infections in Japan in the early 2000s. Extensive studies by Japanese researchers have highlighted the pivotal role of pigs and wild animals, such as wild boars and deer, as reservoirs for HEV, linking them to zoonotic infections in Japan. Currently, when hepatitis occurs subsequent to the consumption of undercooked or grilled pork, wild boar meat, or offal (including pig liver and intestines), HEV infection should be considered. Following the approval of anti-HEV immunoglobulin A antibody as a diagnostic tool for hepatitis E by Japan's Health Insurance System in 2011, the annual number of diagnosed cases of HEV infection has surged. Notably, the occurrence of post-transfusion hepatitis E promoted nationwide screening of blood products for HEV using nucleic acid amplification tests since 2020. Furthermore, chronic hepatitis E has been observed in immunosuppressed individuals. Considering the significance of hepatitis E, heightened preventive measures are essential. The Japan Agency for Medical Research and Development Hepatitis A and E viruses (HAV and HEV) Study Group, which includes special virologists and hepatologists, held a virtual meeting on February 17, 2024. Discussions encompassed pathogenesis, transmission routes, diagnosis, complications, severity factors, and ongoing and prospective vaccination or treatments for hepatitis E. Rigorous assessment of referenced studies culminated in the formulation of recommendations, which are detailed within this review. This comprehensive review presents recent advancements in HEV research and Japanese clinical practice guidelines for HEV infection.
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Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.
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Virus de la Encefalitis Japonesa (Especie) , Flavivirus , Proteínas Nucleares , Gránulos de Estrés , Proteína que Contiene Valosina , Proteínas no Estructurales Virales , Replicación Viral , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Flavivirus/genética , Flavivirus/metabolismo , Flavivirus/fisiología , Genoma Viral , Humanos , Proteínas Nucleares/metabolismo , ARN Viral/genética , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo , Proteína que Contiene Valosina/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.
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Hepatitis B , Simportadores , Humanos , Ratones , Animales , Virus de la Hepatitis B/fisiología , Internalización del Virus , Hepatitis B/metabolismo , Hepatocitos/metabolismo , Células Hep G2 , Virus de la Hepatitis Delta/metabolismo , Simportadores/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismoRESUMEN
Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.
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Virus de la Hepatitis A , Hepatitis A , Macrófagos , Esparcimiento de Virus , Animales , Ratones , Linfocitos T CD8-positivos , Heces , Virus de la Hepatitis A/fisiología , Inflamación , Macrófagos/virología , Receptor de Interferón alfa y beta/genética , ARN Viral/genética , Ratones NoqueadosRESUMEN
HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.
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Hepatitis A/metabolismo , Hepatitis A/patología , Factor 3 Regulador del Interferón/metabolismo , Hígado/patología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , TranscriptomaRESUMEN
Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis. To clarify the epidemiology of HEV and characterize the genetic diversity of the virus in Japan, nationwide enhanced surveillance and molecular characterization studies of HEV in Japan were undertaken from 2014 to 2021. In total, 2770 hepatitis E cases were reported, of which 88% were domestic cases, while only 4.1% represented cases following infection abroad. In addition, 57% of domestic infections occurred in males aged in their 40s-70s. For domestic cases, infection via pork meat consumption continued to be the most reported route. Analysis of the 324 sequences detected between 2016 and 2021 showed that the majority of domestic HEV strains belong to Genotype 3a (G3a) and G3b. In contrast, six of eight cases of G1 HEV reflected infection abroad. Our results suggest that HEV is circulating widely in Japan, with genotypes G3a and G3b being most prevalent. Continued surveillance is necessary to monitor future trends and changes in the epidemiology of HEV in Japan.
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Virus de la Hepatitis E , Hepatitis E , Masculino , Humanos , Hepatitis E/epidemiología , Japón/epidemiología , Filogenia , Virus de la Hepatitis E/genética , Genotipo , ARN Viral/genéticaRESUMEN
BACKGROUND AND AIMS: The future development of hepatocellular carcinoma (HCC) in patients after sustained virologic response (SVR) is an important issue. The purposes of this study were to investigate pathological alterations in organelle of the liver of SVR patients and to characterize organelle abnormalities that may be related to carcinogenesis after SVR. METHODS: The ultrastructure of liver biopsy specimens from patients with chronic hepatitis C (CHC) and SVR were compared to cell and mouse models and assessed semi-quantitatively using transmission electron microscopy. RESULTS: Hepatocytes in patients with CHC showed abnormalities in the nucleus, mitochondria, endoplasmic reticulum, lipid droplet, and pericellular fibrosis, comparable to those seen in hepatitis C virus (HCV)-infected mice and cells. DAA treatment significantly reduced organelle abnormalities such as the nucleus, mitochondria, and lipid droplet in the hepatocytes of patients and mice after SVR, and cured cells, but it did not change dilated/degranulated endoplasmic reticulum and pericellular fibrosis in patients and mice after SVR. Further, samples from patients with a post-SVR period of >1 year had significantly larger numbers of abnormalities in the mitochondria and endoplasmic reticulum than those of <1 year. A possible cause of organelle abnormalities in patients after SVR could be oxidative stress of the endoplasmic reticulum and mitochondria associated with abnormalities of the vascular system due to fibrosis. Interestingly, abnormal endoplasmic reticulum was associated with patients with HCC for >1 year after SVR. CONCLUSIONS: These results indicate that patients with SVR exhibit a persistent disease state and require long-term follow-up to detect early signs of carcinogenesis.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Animales , Ratones , Carcinoma Hepatocelular/patología , Antivirales/uso terapéutico , Neoplasias Hepáticas/patología , Hepacivirus , Hepatitis C/tratamiento farmacológico , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Respuesta Virológica Sostenida , Cirrosis Hepática/complicaciones , Orgánulos/patología , Carcinogénesis/patologíaRESUMEN
The incidence of hepatitis A virus (HAV) infection has declined significantly worldwide, including in Japan. A nationwide seroepidemiological study on hepatitis A in Japan has taken place almost every 10 years since 1973, and the last study was performed in 2003. In the present study, we describe the latest seroepidemiological pattern of hepatitis A in Japan using 7867 serum specimens obtained from healthy individuals collected between 2013 and 2017, approximately 10 years after the last study. Among them, 223 were anti-HAV positive. About 68% of individuals aged 60 years and older had anti-HAV antibodies, whereas only 1.1% of those aged below 60 years old had immunity; thus, almost all individuals younger than 60 years of age were HAV susceptible. In comparison with previous investigations, the susceptible population has increased and aged. According to data from the National Epidemiological Surveillance of Infectious Diseases (NESID) program, between 1989 and 2016, the proportion of patients with hepatitis A aged 60 years and older continuously increased with each year. The NESID data also suggested that recently, typical large foodborne outbreaks of hepatitis A have become rare, and cases tend to be reported among at-risk groups; overseas travelers contributed to 25% of hepatitis A cases, and in 2018, the first nationwide hepatitis A outbreak that affected mostly men who have sex with men was reported. The purpose of this study was to determine the current status of HAV infection in Japan, based on both seroepidemiology and the national surveillance data from the NESID.
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Virus de la Hepatitis A , Hepatitis A , Minorías Sexuales y de Género , Masculino , Humanos , Persona de Mediana Edad , Anciano , Femenino , Hepatitis A/epidemiología , Anticuerpos de Hepatitis A , Homosexualidad Masculina , Japón/epidemiología , Estudios Seroepidemiológicos , Susceptibilidad a EnfermedadesRESUMEN
BACKGROUND: Optimal hemostasis provides safety and reliability during neurosurgery which improves surgical outcomes. Previously, artificial cerebrospinal fluid (aCSF) and its component sodium bicarbonate were found to facilitate physiological hemostasis by amplifying platelet aggregation. This study aimed to verify whether aCSF amplifies platelet-dependent hemostasis in the presence of antiplatelet agents. METHODS: We prepared platelet-rich plasma (PRP) or washed platelets using aspirin (acetylsalicylic acid, (ASA)) or normal saline (NS). We evaluated samples treated with a commercially available aCSF solution or NS for amplification of aggregation, activation of integrin αIIbß3, phosphatidylserine (PS) exposure, P-selectin (CD62P) expression, and formation of microparticles (MPs). We assessed the effect of aCSF on in vivo hemostasis in the presence of ASA by measuring the tail bleeding time in ASA-or NS-injected C57BL/6 N mice. RESULTS: Compared with NS, aCSF amplified ASA-inhibited platelet aggregation by recovering platelet activation including PS exposure, MP release, CD62P expression, and integrin αIIbß3 activation. When using washed platelets, aCSF almost completely counteracted the inhibition of platelet aggregation by ASA. Prolonged bleeding time from the amputated tail of ASA-injected mice was significantly shortened by the treatment with aCSF compared to NS. Sodium bicarbonate also directly amplified ASA-inhibited platelet aggregation. CONCLUSIONS: aCSF and sodium bicarbonate facilitate physiological hemostasis through the recovery of inhibited platelet aggregation even in the presence of ASA. The utilization of aCSF in the operative field may be advantageous for facilitating hemostasis in patients with impaired platelet function and contribute to improving outcomes of neurosurgery.
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Aspirina , Agregación Plaquetaria , Animales , Ratones , Aspirina/farmacología , Aspirina/uso terapéutico , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/farmacología , Bicarbonato de Sodio/metabolismo , Bicarbonato de Sodio/farmacología , Reproducibilidad de los Resultados , Ratones Endogámicos C57BL , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Hemostasis/fisiología , Plaquetas/metabolismoRESUMEN
OBJECTIVES: Carotid artery stenting is sometimes adapted for some at-risk cases; however, appropriate treatment timing with stroke onset is controversial. This study aims to identify factors that have an impact on complications and outcomes, especially in patients at high risk. MATERIALS AND METHODS: We examined the characteristics of 152 consecutive patients treated by carotid artery stenting between January 2018 and March 2022 and retrospectively analyzed the risk factors for complications and poor outcomes (modified-Rankin-Scale deterioration), such as patient background, carotid artery stenting risks (access route tortuosity, severe calcification, vulnerable plaque, estimated glomerular filtration rate <30 mL/min/1.73 m2, etc.), characteristics of the stenosis, details of treatment, and treatment timing. RESULTS: The average North American Symptomatic Carotid Endarterectomy Trial criteria score was 68.3% and the lesion length was 20.5±9.7mm. Among patients, 107 (70.4%) had a carotid artery stenting risk. In high-risk carotid artery stenting cases, symptomatic complications occurred in 32 (30.0%), and the 90-day modified Rankin scale score deteriorated in 15 cases (14.0%). Multivariate analysis showed that cases with triple antithrombotic therapy (p=0.003), stenting within 7 days (p=0.0032), and after 28+ days (p=0.0035) of stroke onset were independently associated factors for complications. CONCLUSIONS: This study showed that among risk factors, triple antithrombotic therapy in particular was a risk factor for perioperative complications. Carotid artery stenting for patients with stroke after 28 days of onset affects the prognosis. Therefore, although further study is warranted, waiting more than one month for treatment in patients requiring carotid artery stenting is a potential risk.
Asunto(s)
Estenosis Carotídea , Accidente Cerebrovascular , Humanos , Estenosis Carotídea/complicaciones , Estenosis Carotídea/diagnóstico por imagen , Estenosis Carotídea/terapia , Fibrinolíticos , Estudios Retrospectivos , Stents , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/etiología , Arterias CarótidasRESUMEN
Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I-III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semi-pangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection.IMPORTANCE The HBV preS1 2-47 aa region (preS1/2-47) is essential for virus binding with sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2-47 have been reported to neutralize HBV infection; however, which region in preS1/2-47 contains the critical neutralizing epitope for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2-47 and found that MAbs recognizing the N- or C-terminus of preS1/2-47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current HB vaccines comprising the small S protein.
RESUMEN
BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.