Publications by authors named "Masaya Sugiyama"

149 Publications

Case Report: Changes in Cytokine Kinetics During the Course of Disease in a Japanese Patient With Multisystem Inflammatory Syndrome in Children.

Front Pediatr 2021 21;9:702318. Epub 2021 Jul 21.

Department of Pediatrics, Center Hospital of the National Center for Global Health and Medicine, Tokyo, Japan.

Multisystem inflammatory syndrome in children (MIS-C) is a severe disease that is reportedly linked to coronavirus disease 2019. Affected patients present with gastrointestinal symptoms and cardiovascular dysfunction, in addition to Kawasaki disease-like features, suggesting the potential for overlapping disease mechanisms. Kawasaki disease has been reported among individuals of East Asian ethnicities, whereas there is minimal clinical literature regarding the occurrence of MIS-C among individuals of Asian ethnicities. A few reports thus far have described changes in cytokine kinetics during the course of disease in patients with MIS-C. We followed the temporal cytokine kinetics in a 9-year-old Japanese girl who exhibited a classical trajectory of MIS-C. The patient exhibited right cervical swelling and pain, abdominal pain, vomiting, and lip reddening, which developed 31 days after she was diagnosed with severe acute respiratory syndrome coronavirus-2 infection. The patient was diagnosed with Kawasaki disease on her fifth day of illness; because she fulfilled the criteria for MIS-C, she was also diagnosed with this disease on her fifth day of illness. Her fever rapidly resolved upon administration of intravenous immunoglobulin, aspirin, and prednisolone. On the patient's sixth day of illness, she developed acute myocarditis, which was treated with two diuretics and one vasodilator; the myocarditis ameliorated within a few days. Analyses of temporal kinetics for 71 serum cytokines revealed several patterns of cytokine changes that were consistent with the patient's clinical course of disease. Importantly, there was a clear distinction between cytokines that did and did not decrease rapidly following post-treatment fever resolution. These findings may be useful for the assessment of disease status and selection of therapy in patients with similar symptoms; they may also provide insights for basic and clinical research regarding MIS-C.
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http://dx.doi.org/10.3389/fped.2021.702318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8335158PMC
July 2021

Vaccine temperature management in Lao People's Democratic Republic: A nationwide cross-sectional study.

Heliyon 2021 Jun 17;7(6):e07342. Epub 2021 Jun 17.

National Center for Global Health and Medicine, 1-21-1 Toyama Shinjuku, Tokyo, 1628655 Japan.

Objective: The objective of the study was to evaluate the duration and frequency of vaccine exposure to suboptimal temperatures during transit from the central vaccine storage in the capital to health centers in Lao PDR.

Methods: Temperature data loggers traveled from the capital to the health centre storages (146) with the vaccines to monitor the vaccine temperature nationwide. One health centre per district was selected using a simple random sampling method for the first round of temperature monitoring. One health centre was selected from every forty-nine high risk districts monitor the trend of vaccine temperature at the health centre storage and during outreach sessions in several districts. Vaccines and temperature data loggers were transported using the normal vaccination transportation.

Findings: Overall, the vaccines were exposed to temperatures >8 °C for an average of 1648 min, equivalent to 9.0% of the observational period, and to temperatures <0 °C for an average of 184 min, equivalent to 1.35% of the study period. The proportion of exposure to temperatures >8 °C was the highest during the transit from the capital to the province. The proportion of exposure to temperatures <0 °C was the highest during storage at district level. Examined by region, vaccines in the northern provinces had higher risk of exposure to temperatures >8 °C; however, the risk of exposure to temperatures <0 °C was scattered nationwide. Moreover, some health centers showed fluctuations in storage temperature.

Conclusions: Challenges associated with cold chain management, and the resulting deterioration of vaccines, might account for outbreaks of vaccine-preventable diseases. The government should examine and invest in suitable technologies and approaches to ensure consistency in cold chain management.
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http://dx.doi.org/10.1016/j.heliyon.2021.e07342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258653PMC
June 2021

FGFR2 maintains cancer cell differentiation via AKT signaling in esophageal squamous cell carcinoma.

Cancer Biol Ther 2021 Jun 5;22(5-6):372-380. Epub 2021 Jul 5.

Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are important for signaling to maintain cancer stem-like cells (CSCs) in esophageal squamous cell carcinoma (ESCC). However, which FGF receptor, 1, 2, 3, 4, and L1, is essential or whether FGFRs have distinct different roles in ESCC-CSCs is still in question. This study shows that FGFR2, particularly the IIIb isoform, is highly expressed in non-CSCs. Non-CSCs have an epithelial phenotype, and such cells are more differentiated in ESCC. Further, FGFR2 induces keratinocyte differentiation through AKT but not MAPK signaling and diminishes CSC populations. Conversely, knockdown of FGFR2 induces epithelial-mesenchymal transition (EMT) and enriches CSC populations in ESCC. Finally, data analysis using The Cancer Genome Atlas (TCGA) dataset shows that expression of FGFR2 significantly correlated with cancer cell differentiation in clinical ESCC samples. The present study shows that each FGFR has a distinct role and FGFR2-AKT signaling is a key driver of keratinocyte differentiation in ESCC. Activation of FGFR2-AKT signaling could be a future therapeutic option targeting CSC in ESCC.
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http://dx.doi.org/10.1080/15384047.2021.1939638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386746PMC
June 2021

Genome-wide copy number variation analysis of hepatitis B infection in a Japanese population.

Hum Genome Var 2021 Jun 8;8(1):22. Epub 2021 Jun 8.

Department of Genome Informatics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Genome-wide association studies have been performed to identify common genetic variants associated with hepatitis B (HB). However, little is known about copy number variations (CNVs) in HB. In this study, we performed a genome-wide CNV analysis between 1830 healthy controls and 1031 patients with HB infection after quality control. Using signal calling by the Axiom Analysis Suite and CNV detection by PennCNV software, we obtained a total of 4494 CNVs across all individuals. The genes with CNVs that were found only in the HB patients were associated with the immune system, such as antigen processing. A gene-level CNV association test revealed statistically significant CNVs in the contactin 6 (CNTN6) gene. Moreover, we also performed gene-level CNV association tests in disease subgroups, including hepatocellular carcinoma patients, liver cirrhosis patients, and HBV carriers, including asymptomatic carriers and patients with HBV-derived chronic hepatitis. Our findings from germline cells suggested that patient-specific CNVs may be inherent genetic risk factors for HB.
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http://dx.doi.org/10.1038/s41439-021-00154-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187437PMC
June 2021

MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter.

J Virol 2021 07 12;95(15):e0076721. Epub 2021 Jul 12.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (NL) reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. HBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Therefore, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis and developing anti-HBV strategies. MafF was shown to suppress transcription from the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1β and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.
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http://dx.doi.org/10.1128/JVI.00767-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274605PMC
July 2021

HLA-A*11:01:01:01, HLA-C*12:02:02:01-HLA-B*52:01:02:02, Age and Sex Are Associated With Severity of Japanese COVID-19 With Respiratory Failure.

Front Immunol 2021 22;12:658570. Epub 2021 Apr 22.

Genome Medical Science Project, National Center for Global Health and Medicine Hospital, Tokyo, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19) was announced as an outbreak by the World Health Organization (WHO) in January 2020 and as a pandemic in March 2020. The majority of infected individuals have experienced no or only mild symptoms, ranging from fully asymptomatic cases to mild pneumonic disease. However, a minority of infected individuals develop severe respiratory symptoms. The objective of this study was to identify susceptible HLA alleles and clinical markers that can be used in risk prediction model for the early identification of severe COVID-19 among hospitalized COVID-19 patients. A total of 137 patients with mild COVID-19 (mCOVID-19) and 53 patients with severe COVID-19 (sCOVID-19) were recruited from the Center Hospital of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan for the period of February-August 2020. High-resolution sequencing-based typing for eight HLA genes was performed using next-generation sequencing. In the HLA association studies, HLA-A*11:01:01:01 [P = 0.013, OR = 2.26 (1.27-3.91)] and HLA-C*12:02:02:01-HLA-B*52:01:01:02 [P = 0.020, OR = 2.25 (1.24-3.92)] were found to be significantly associated with the severity of COVID-19. After multivariate analysis controlling for other confounding factors and comorbidities, HLA-A*11:01:01:01 [P = 3.34E-03, OR = 3.41 (1.50-7.73)], age at diagnosis [P = 1.29E-02, OR = 1.04 (1.01-1.07)] and sex at birth [P = 8.88E-03, OR = 2.92 (1.31-6.54)] remained significant. The area under the curve of the risk prediction model utilizing HLA-A*11:01:01:01, age at diagnosis, and sex at birth was 0.772, with sensitivity of 0.715 and specificity of 0.717. To the best of our knowledge, this is the first article that describes associations of HLA alleles with COVID-19 at the 4-field (highest) resolution level. Early identification of potential sCOVID-19 could help clinicians prioritize medical utility and significantly decrease mortality from COVID-19.
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http://dx.doi.org/10.3389/fimmu.2021.658570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100314PMC
May 2021

Importance of HBsAg recognition by HLA molecules as revealed by responsiveness to different hepatitis B vaccines.

Sci Rep 2021 Mar 2;11(1):3703. Epub 2021 Mar 2.

Genome Medical Science Project, National Center for Global Health and Medicine, Ichikawa, 272-8516, Japan.

Hepatitis B (HB) vaccines (Heptavax-II and Bimmugen) designed based on HBV genotypes A and C are mainly used for vaccination against HB in Japan. To determine whether there are differences in the genetic background associated with vaccine responsiveness, genome-wide association studies were performed on 555 Heptavax-II and 1193 Bimmugen recipients. Further HLA imputation and detailed analysis of the association with HLA genes showed that two haplotypes, DRB1*13:02-DQB1*06:04 and DRB1*04:05-DQB1*04:01, were significantly associated in comparison with high-responders (HBsAb > 100 mIU/mL) for the two HB vaccines. In particular, HLA-DRB1*13:02-DQB1*06:04 haplotype is of great interest in the sense that it could only be detected by direct analysis of the high-responders in vaccination with Heptavax-II or Bimmugen. Compared with healthy controls, DRB1*13:02-DQB1*06:04 was significantly less frequent in high-responders when vaccinated with Heptavax-II, indicating that high antibody titers were less likely to be obtained with Heptavax-II. As Bimmugen and Heptavax-II tended to have high and low vaccine responses to DRB1*13:02, 15 residues were found in the Heptavax-II-derived antigenic peptide predicted to have the most unstable HLA-peptide binding. Further functional analysis of selected hepatitis B patients with HLA haplotypes identified in this study is expected to lead to an understanding of the mechanisms underlying liver disease.
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http://dx.doi.org/10.1038/s41598-021-82986-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925550PMC
March 2021

Transmission dynamics of SARS-CoV-2 on the Diamond Princess uncovered using viral genome sequence analysis.

Gene 2021 May 13;779:145496. Epub 2021 Feb 13.

Genome Medical Sciences Project, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa, Chiba 272-8516, Japan. Electronic address:

An outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred aboard the Diamond Princess cruise ship between her January 20 departure and late February 2020. Here, we used phylodynamic analyses to investigate the transmission dynamics of SARS-CoV-2 during the outbreak. Using a Bayesian coalescent-based method, the estimated mean nucleotide substitution rate of 240 SARS-CoV-2 whole-genome sequences was approximately 7.13 × 10 substitutions per site per year. Population dynamics and the effective reproductive number (R) of SARS-CoV-2 infections were estimated using a Bayesian framework. The estimated origin of the outbreak was January 21, 2020. The infection spread substantially before quarantine on February 5. The R peaked at 6.06 on February 4 and gradually declined to 1.51, suggesting that transmission continued slowly even after quarantine. These findings highlight the high transmissibility of SARS-CoV-2 and the need for effective measures to control outbreaks in confined settings.
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http://dx.doi.org/10.1016/j.gene.2021.145496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880849PMC
May 2021

Hepatic Stellate Cells in Hepatocellular Carcinoma Promote Tumor Growth Via Growth Differentiation Factor 15 Production.

Gastroenterology 2021 04 17;160(5):1741-1754.e16. Epub 2020 Dec 17.

Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan. Electronic address:

Background & Aims: Although the tumor microenvironment plays an important role in tumor growth, it is not fully understood what role hepatic stellate cells (HSCs) play in the hepatocellular carcinoma (HCC) microenvironment.

Methods: A high-fat diet after streptozotocin was administered to HSC-specific Atg7-deficient (GFAP-Atg7 knockout [KO]) or growth differentiation factor 15 (GDF15)-deficient (GFAP-GDF15KO) mice. LX-2 cells, a human HSC cell line, were cultured with human hepatoma cells.

Results: In the steatohepatitis-based tumorigenesis model, GFAP-Atg7KO mice formed fewer and smaller liver tumors than their wild-type littermates. Mixed culture of LX-2 cells and hepatoma cells promoted LX-2 cell autophagy and hepatoma cell proliferation, which were attenuated by Atg7 KO in LX-2 cells. Hepatoma cell xenograft tumors grew rapidly in the presence of LX-2 cells, but Atg7 KO in LX-2 cells abolished this growth. RNA-sequencing revealed that LX-2 cells cultured with HepG2 cells highly expressed GDF15, which was abolished by Atg7 KO in LX-2 cells. GDF15 KO LX-2 cells did not show a growth-promoting effect on hepatoma cells either in vitro or in the xenograft model. GDF15 deficiency in HSCs reduced liver tumor size caused by the steatohepatitis-based tumorigenesis model. GDF15 was highly expressed and GDF15-positive nonparenchymal cells were more abundant in human HCC compared with noncancerous parts. Single-cell RNA sequencing showed that GDF15-positive rates in HSCs were higher in HCC than in background liver. Serum GDF15 levels were high in HCC patients and increased with tumor progression.

Conclusions: In the HCC microenvironment, an increase of HSCs that produces GDF15 in an autophagy-dependent manner may be involved in tumor progression.
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http://dx.doi.org/10.1053/j.gastro.2020.12.015DOI Listing
April 2021

SARS-CoV-2 infection among returnees on charter flights to Japan from Hubei, China: a report from National Center for Global Health and Medicine.

Glob Health Med 2020 Apr;2(2):107-111

Department of Infectious Diseases, Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan.

Due to the significant spread of a new type of coronavirus (SARS-CoV-2) infection (COVID-19) in China, the Chinese government blockaded several cities in Hubei Province. Japanese citizens lost a means of transportation to return back to Japan. The National Center for Global Health and Medicine (NCGM) helped the operation of charter flights for evacuation of Japanese residents from Hubei Province, and this article outlines our experiences. A total of five charter flights were dispatched, and the majority of returnees (793/829 [95.7%]) were handled at NCGM. A large number of personnel from various departments participated in this operation; 107 physicians, 115 nurses, 110 clerical staff, and 45 laboratory technicians in total. Several medical translators were also involved. In this operation, we conducted airborne precautions in addition to contact precautions. Eye shields were also used. The doctors collecting the pharyngeal swab used a coverall to minimize the risk of body surface contamination from secretions and droplets. Enhanced hand hygiene using alcohol hand sanitizer was performed. Forty-eight persons were ultimately hospitalized after the triage at NCGM operation, which was more than the number of persons triaged at the airport ( = 34). Of those hospitalized after NCGM triage, 8.3% (4/48 patients) ultimately tested positive for SARS-CoV-2, significantly higher than the positive rate among subjects not triaged (4/48 [8.3%] 9/745 [1.2%]: = 0.0057). NCGM participated in a large-scale operation to evacuate Japanese nationals from the COVID-19 epidemic area. We were able to establish a scheme through this experience that can be used in the future.
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http://dx.doi.org/10.35772/ghm.2020.01036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731067PMC
April 2020

Prevalence and incidence of HIV-1 infection in a community-based men who have sex with men (MSM) cohort in Ulaanbaatar, Mongolia.

Glob Health Med 2020 Feb;2(1):33-38

AIDS Clinical Center, National Center for Global Health and Medicine, Tokyo, Japan.

The number of HIV-1-infected men who have sex with men (MSM) Mongolian patients started to increase steeply just before 2011. We started collaborative work with community-based organizations that promote safer sex and HIV testing for MSM since mid-2010. Since early 2013, the Mongolian Government has implemented the treat-all strategy for MSM. To determine the efficacy of these countermeasures, we established an MSM cohort in the capital of Mongolia, Ulaanbaatar, in December 2013. HIV antibody was examined at every visit by rapid test. Syphilis was also examined to monitor their sexual behavior. Clients positive for either rapid test were referred to the National Center for Communicable Diseases, Ulaanbaatar, to confirm the results and treatment. Since safer sex promotion is one of the purposes of this cohort, HIV-positive clients were also eligible to participate. A total of 849 MSM were registered and 2,409 HIV/syphilis tests were conducted until December 2017. During this period, 499 (58.8%) clients visited the testing sites repeatedly. Among the 849 clients, HIV-1 infection was confirmed in 83 at registration (prevalence of HIV- 1: 9.8%). One HIV-1 seroconverter was identified (from negative to positive), resulting in incidence of HIV-1 of 0.10/100 person-years (PY). Syphilis was positive in 144 cases at registration (syphilis prevalence: 17.0%), and 53 new syphilis infection cases were diagnosed during the same period, with an incidence of 5.66/100 PY. Despite the high prevalence of HIV-1, the incidence was very low. The results suggest that countermeasures for HIV-1 prevention seem effective in this cohort, however, we still need further strategies for syphilis control.
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http://dx.doi.org/10.35772/ghm.2019.01036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731272PMC
February 2020

Direct hemoperfusion using a polymyxin B-immobilized polystyrene column for COVID-19.

J Clin Apher 2021 Jun 15;36(3):313-321. Epub 2020 Dec 15.

Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan.

Objective: To evaluate the efficacy and safety of direct hemoperfusion using a polymyxin B-immobilized polystyrene column (PMX-DHP) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive pneumonia patients.

Methods: This study was a case series conducted at a designated infectious diseases hospital. Twelve SARS-CoV-2-positive patients with partial pressure of arterial oxygen/percentage of inspired oxygen (P/F) ratio < 300 were treated with PMX-DHP on two consecutive days each during hospitalization. We defined day 1 as the first day when PMX-DHP was performed. PMX-DHP efficacy was assessed on days 7 and 14 after the first treatment based on eight categories. Subsequently, improvement in P/F ratio and urinary biomarkers on days 4 and 8, malfunctions, and ventilator and extracorporeal membrane oxygenation avoidance rates were also evaluated.

Results: On day 14 after the first treatment, disease severity decreased in 58.3% of the patients. P/F ratio increased while urine β2-microglobulin decreased on days 4 and 8. Cytokine measurement pre- and post-PMX-DHP revealed decreased levels of interleukin-6 and the factors involved in vascular endothelial injury, including vascular endothelial growth factor. Twenty-two PMX-DHPs were performed, of which seven and five PMX-DHPs led to increased inlet pressure and membrane coagulation, respectively. When the membranes coagulated, the circuitry needed to be reconfigured. Circuit problems were usually observed when D-dimer and fibrin degradation product levels were high before PMX-DHP.

Conclusions: Future studies are expected to determine the therapeutic effect of PMX-DHP on COVID-19. Because of the relatively high risk of circuit coagulation, coagulation capacity should be assessed beforehand.
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http://dx.doi.org/10.1002/jca.21861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246724PMC
June 2021

Impact of Immune Reconstitution-Induced Hepatic Flare on Hepatitis B Surface Antigen Loss in Hepatitis B Virus/Human Immunodeficiency Virus-1 Coinfected Patients.

J Infect Dis 2021 Jun;223(12):2080-2089

AIDS Clinical Center, National Center for Global Health and Medicine, Tokyo, Japan.

Background: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss.

Methods: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection.

Results: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF.

Conclusions: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.
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http://dx.doi.org/10.1093/infdis/jiaa662DOI Listing
June 2021

Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA.

Virus Res 2020 12 10;290:198191. Epub 2020 Oct 10.

Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan. Electronic address:

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.
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http://dx.doi.org/10.1016/j.virusres.2020.198191DOI Listing
December 2020

Serum CCL17 level becomes a predictive marker to distinguish between mild/moderate and severe/critical disease in patients with COVID-19.

Gene 2021 Jan 14;766:145145. Epub 2020 Sep 14.

Genome Medical Sciences Project, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa, Chiba 272-8516, Japan.

COVID-19, a novel coronavirus-related illness, has spread worldwide. Patients with apparently mild/moderate symptoms can suddenly develop severe pneumonia. Therefore, almost all COVID-19 patients require hospitalization, which can reduce limited medical resources in addition to overwhelming medical facilities. To identify predictive markers for the development of severe pneumonia, a comprehensive analysis of serum chemokines and cytokines was conducted using serial serum samples from COVID-19 patients. The expression profiles were analyzed along the time axis. Serum samples of common diseases were enrolled from a BioBank to confirm the usefulness of predictive markers. Five factors, IFN-λ3, IL-6, IP-10, CXCL9, and CCL17, were identified as predicting the onset of severe/critical symptoms. The factors were classified into two categories. Category A included IFN-λ3, IL-6, IP-10, and CXCL9, and their values surged and decreased rapidly before the onset of severe pneumonia. Category B included CCL17, which provided complete separation between the mild/moderate and the severe/critical groups at an early phase of SARS-CoV-2 infection. The five markers provided a high predictive value (area under the receiver operating characteristic curve (AUROC): 0.9-1.0, p < 0.001). Low expression of CCL17 was specifically observed in pre-severe COVID-19 patients compared with other common diseases, and the predictive ability of CCL17 was confirmed in validation samples of COVID-19. The factors identified could be promising prognostic markers to distinguish between mild/moderate and severe/critical patients, enabling triage at an early phase of infection, thus avoiding overwhelming medical facilities.
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http://dx.doi.org/10.1016/j.gene.2020.145145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489253PMC
January 2021

Modulation of hepatitis B virus infection by epidermal growth factor secreted from liver sinusoidal endothelial cells.

Sci Rep 2020 09 1;10(1):14349. Epub 2020 Sep 1.

Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.
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http://dx.doi.org/10.1038/s41598-020-71453-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462976PMC
September 2020

Orchestration of Intracellular Circuits by G Protein-Coupled Receptor 39 for Hepatitis B Virus Proliferation.

Int J Mol Sci 2020 Aug 7;21(16). Epub 2020 Aug 7.

Genome Medical Science Project, Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan.

Hepatitis B virus (HBV), a highly persistent pathogen causing hepatocellular carcinoma (HCC), takes full advantage of host machinery, presenting therapeutic targets. Here we aimed to identify novel druggable host cellular factors using the reporter HBV we have recently generated. In an RNAi screen of G protein-coupled receptors (GPCRs), GPCR39 (GPR39) appeared as the top hit to facilitate HBV proliferation. Lentiviral overexpression of active GPR39 proteins and an agonist enhanced HBV replication and transcriptional activities of viral promoters, inducing the expression of CCAAT/enhancer binding protein (CEBP)-β (CEBPB). Meanwhile, GPR39 was uncovered to activate the heat shock response, upregulating the expression of proviral heat shock proteins (HSPs). In addition, glioma-associated oncogene homologue signaling, a recently reported target of GPR39, was suggested to inhibit HBV replication and eventually suppress expression of CEBPB and HSPs. Thus, GPR39 provirally governed intracellular circuits simultaneously affecting the carcinopathogenetic gene functions. GPR39 and the regulated signaling networks would serve as antiviral targets, and strategies with selective inhibitors of GPR39 functions can develop host-targeted antiviral therapies preventing HCC.
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http://dx.doi.org/10.3390/ijms21165661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460832PMC
August 2020

High Prevalence of Chronic Viral Hepatitis and Liver Fibrosis Among Mongols in Southern California.

Dig Dis Sci 2021 08 8;66(8):2833-2839. Epub 2020 Aug 8.

Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, 1510 San Pablo Street, 2nd Floor, Los Angeles, CA, 90033, USA.

Background: Mongolia is a highly endemic region for chronic hepatitis B (HBV), hepatitis delta (HDV), and hepatitis C (HCV) infections. Aim of this study was to comprehensively characterize chronic viral hepatitis among Mongols living in Southern California.

Methods: Three screening events were conducted between August and November 2018, with 528 adult Mongols tested for HBV and HCV. HBsAg (+) individuals (CHB) underwent additional testing for HDV RNA and anti-HDV. Liver tests, platelet count, and FibroScan™ were performed on CHB and chronic HCV (CHC) individuals.

Results: Fifty-one out of 534 were HBsAg reactive (9.7%), and all were foreign-born. Mean age of CHB individuals was 37.8 (range 18-69) years. Forty-six out of 51 were HBeAg (-). HBV genotypes were exclusively D2 or A1. Twenty-one out of 51 (41.2%) were anti-HDV (+) and 17/51 (33.3%) were HDV RNA (+). HDV RNA (+) individuals had significantly higher ALT, fibrosis-4 score, and liver stiffness compared to HDV RNA (-) individuals. Incidence of advanced fibrosis was higher in HDV RNA (+) individuals (57% vs. 13%, p = 0.013). Forty-eight (9.1%) individuals were anti-HCV (+) and 19 (3.6%) were HCV RNA (+). Mean age of CHC individuals was 40.2 (range 28-71) years. Prevalence of anti-HCV (+) was higher among those born between 1945 and 1965 versus those born after 1965 (18.8% vs. 7.9%, p = 0.025). Genotype 1b was predominant. Incidence of cirrhosis was 7% among all participants.

Conclusions: Mongols living in the USA are at high risk for CHB and CHC infections. One-third of CHB individuals had CHD superinfection with advanced fibrosis. Universal screening for viral hepatitis in Mongols in the USA is mandatory.
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http://dx.doi.org/10.1007/s10620-020-06499-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868472PMC
August 2021

Evaluation of Coronavirus Disease 2019 Severity Using Urine Biomarkers.

Crit Care Explor 2020 Aug 31;2(8):e0170. Epub 2020 Jul 31.

National Center Biobank Network, National Center for Global Health and Medicine, Tokyo, Japan.

Subjects: Early detection of coronavirus disease 2019 in patients likely to develop severe manifestations enables appropriate interventions, including rapid ICU admission. This study was conducted to determine whether noninvasive urine biomarkers can predict the clinical severity of coronavirus disease 2019.

Interventions: Not applicable.

Measurements And Main Results: This is single-center study, national center hospital designated for infectious disease. Fifty-eight patients who tested positive for severe acute respiratory syndrome coronavirus 2 in respiratory specimens through real-time reverse transcription-polymerase chain reaction were retrospectively studied. Urinary β2-microglobulin, liver-type fatty acid-binding protein were serially measured. Serum interferon-γ and monocyte chemotactic protein-1 were also evaluated. The 58 patients were assigned into three groups. Patients requiring intensive care were assigned to the severe group ( = 12). Patients treated with oxygen were assigned to the moderate group ( = 13). Other patients were assigned to the mild group ( = 33). Urine tests revealed that low β2-microglobulin and liver-type fatty acid-binding protein levels were associated with mild disease, whereas high levels were associated with severe disease. In severe cases, liver-type fatty acid-binding protein tended to be persistently high. The resulting cutoff values were β2-microglobulin; severe versus moderate + mild: 2,457 μg/dL (specificity 76.9% and sensitivity 90.0%, area under the receiver operating characteristic curve 85.9%), liver-type fatty acid-binding protein; severe versus moderate + mild: 22.0 μg/gCre (specificity 84.6% and sensitivity 90%, area under the receiver operating characteristic curve 91.8%). Urinary β2-microglobulin and serum interferon-γ/monocyte chemotactic protein-1 showed a similar trend.

Conclusions: Evaluating urinary biomarkers such as β2-microglobulin and liver-type fatty acid-binding protein may allow determination of coronavirus disease 2019 patients with active cytokines and recognition of patients likely to become critically ill and requiring careful observation and early intervention.
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http://dx.doi.org/10.1097/CCE.0000000000000170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402421PMC
August 2020

Screening siRNAs against host glycosylation pathways to develop novel antiviral agents against hepatitis B virus.

Hepatol Res 2020 Oct 24;50(10):1128-1140. Epub 2020 Aug 24.

Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.

Aim: Hepatitis B virus (HBV) relies on glycosylation for crucial functions, such as entry into host cells, proteolytic processing and protein trafficking. The aim of this study was to identify candidate molecules for the development of novel antiviral agents against HBV using an siRNA screening system targeting the host glycosylation pathway.

Methods: HepG2.2.15.7 cells that consistently produce HBV were employed for our in vitro study. We investigated the effects of siRNAs that target 88 different host glycogenes on hepatitis B surface antigen (HBsAg) and HBV DNA secretion using the siRNA screening system.

Results: We identified four glycogenes that reduced HBsAg and/or HBV DNA secretion; however, the observed results for two of them may be due to siRNA off-target effects. Knocking down ST8SIA3, a member of the sialyltransferase family, significantly reduced both HBsAg and HBV DNA secretion. Knocking down GALNT7, which transfers N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus, also significantly reduced both HBsAg and HBV DNA levels.

Conclusions: These results showed that knocking down the ST8SIA3 and GALNT7 glycogenes inhibited HBsAg and HBV DNA secretion in HepG2.2.15.7 cells, indicating that the host glycosylation pathway is important for the HBV life cycle and could be a potential target for the development of novel anti-HBV agents.
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http://dx.doi.org/10.1111/hepr.13552DOI Listing
October 2020

Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

Genes (Basel) 2020 06 18;11(6). Epub 2020 Jun 18.

Division of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC) worldwide. The integration of HBV genomic DNA into the host genome occurs randomly, early after infection, and is associated with hepatocarcinogenesis in HBV-infected patients. Therefore, it is important to analyze HBV genome integration. We analyzed HBV genome integration in human hepatoma PLC/PRF/5 cells by HBV sequence capture-based next-generation sequencing (NGS) methods. We confirmed the results by using Sanger sequencing methods. We observed that HBV genotype A is integrated into the genome of PLC/PRF/5 cells. HBV sequence capture-based NGS is useful for the analysis of HBV genome integrants and their locations in the human genome. Among the HBV genome integrants, we performed functional analysis and demonstrated the automatic expression of some HBV proteins encoded by HBV integrants from chromosomes 3 and 11 in Huh7 cells transfected with these DNA sequences. HBV sequence capture-based NGS may be a useful tool for the assessment of HBV genome integration into the human genome in clinical samples and suggests new strategies for hepatocarcinogenesis in HBV infection.
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http://dx.doi.org/10.3390/genes11060661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348787PMC
June 2020

Lenvatinib suppresses cancer stem-like cells in HCC by inhibiting FGFR1-3 signaling, but not FGFR4 signaling.

Carcinogenesis 2021 02;42(1):58-69

Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

In hepatocellular carcinoma (HCC), a subset of cells defined by high CD44 and CD133 expression has been reported to possess cancer stem-like cell (CSC) characteristics and to be associated with a poor prognosis. Since the approval of the multikinase inhibitor, lenvatinib, for patients with unresectable HCC, two such inhibitors (sorafenib and lenvatinib) have been employed as first-line systemic chemotherapeutics for these patients. Based on differences in the kinase-affinity profiles between these two drugs, evidence has suggested that both exert different effects on HCC, although these differences are not fully characterized. In this study, using in vitro and a preclinical in vivo xenograft mouse model, we showed that lenvatinib alone (not sorafenib or the cytotoxic agent, 5-fluorouracil) diminished CD44High/CD133High CSCs in HCC. Furthermore, western blotting and reverse transcriptase-polymerase chain reaction analysis revealed that the expression of fibroblast growth factor receptor (FGFR)-1-4 differed between CD44High/CD133High CSCs and control cells. Analysis of the effects of selective FGFR inhibitors and FGFR small interfering RNAs on CSCs in HCC revealed that lenvatinib diminished CSCs in HCC by inhibiting FGFR1-3 signaling, however, FGFR4 signaling was not impacted. Finally, we showed that FGF2 and FGF19 were involved in maintaining CD44High/CD133High CSCs in HCC, potentially, via FGFR1-3. The findings provide novel mechanistic insights into the effects of lenvatinib on CSCs in HCC and provide clues for developing effective targeted therapies against CSCs in HCC.
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http://dx.doi.org/10.1093/carcin/bgaa049DOI Listing
February 2021

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

Hepatology 2021 02 31;73(2):520-532. Epub 2020 Oct 31.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

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.
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http://dx.doi.org/10.1002/hep.31308DOI Listing
February 2021

Prevalence of equine hepacivirus infection in Mongolia.

Virus Res 2020 06 4;282:197940. Epub 2020 Apr 4.

Genome Medical Sciences Project, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa, Chiba, 272-8516, Japan.

Equine hepacivirus (EHV) belongs to the hepacivirus A and is related to hepatitis C virus (HCV). This virus shows hepatic tropism and is known to chronically infect horses. EHV has been reported from various countries, but the prevalence in Mongolia, where large horse populations are pastured, remains unknown. This study collected serum samples from horses in six areas across Mongolia, in order to investigate the status of infection. The possibility of human infection was also examined. The results showed an infection rate among horses of about 40 % in all regions. However, no evidence of EHV viremia was found in human serum. A mutation characteristic of Mongolian EHV was found in the 5'-untranslated region of the viral sequence. Molecular phylogenetic trees for core, NS3, and NS5B sequences showed the formation of two clusters depending on the area from which samples were taken. The same results were obtained from molecular phylogenetic analyses using the full genome. From detailed calculations of genetic diversity calculated using the full genome, EHV appears divisible into two subgenotypes. Blood samples were collected again after a 7-month interval to examine infection persistence. Seventeen of 19 horses retested showed positive results for EHV after 7 months, suggesting a high rate of persistent infection. These results indicate a relatively higher frequency of EHV infection in Mongolia than in Europe or North America, with virus strains divided into at least two subgenotypes.
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http://dx.doi.org/10.1016/j.virusres.2020.197940DOI Listing
June 2020

Full-genome analysis of hepatitis C virus in HIV-coinfected hemophiliac Japanese patients.

Hepatol Res 2020 Jun 31;50(6):763-769. Epub 2020 Mar 31.

AIDS Clinical Center, National Center for Global Health and Medicine, Tokyo, Japan.

Aim: More than 1400 Japanese hemophiliacs acquired HIV infection around 1983 through contaminated blood products imported from the USA, most of whom also acquired hepatitis C virus (HCV) infection. To delineate the HCV genetic relations in HIV-coinfected hemophiliacs, we analyzed stocked plasma samples of the patients seen at the largest referral center for HIV care in Japan.

Methods: Hepatitis C virus full-genome sequences were amplified and determined using next-generation sequencing, and genotyping and phylogenetic analyses of these sequences were carried out. The results of these hemophiliacs were compared with those of previously studied HIV-coinfected Japanese non-hemophiliacs who had undergone similar analysis of HCV full-genome sequences.

Results: From 1997 to the end of 2017, 72 HIV-infected Japanese hemophiliacs regularly visited our outpatient clinic. Of these, 51 patients had detectable plasma HCV-RNA. The HCV full genome was successfully amplified and sequenced in 50 patients. Not only HCV genotypes 1b (28%) and 2a (6%), which are common in Japan, but also HCV genotypes 1a (32%) and 3a (22%) were identified at high frequency. A single case of intergenotypic recombinant form (2b/1a) and a single case of mixed infection (1a and 3a) were also identified. Each sequence derived from hemophiliacs was more than 0.05 genetic distance away from the other sequences in phylogenetic analysis.

Conclusions: Various HCV genotypes were identified in Japanese hemophiliacs, a finding that reflects the HCV genotypic distribution in the USA. The genetic distance among them are the results of viral evolution in each patient plus HCV genetic diversity in the USA.
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http://dx.doi.org/10.1111/hepr.13498DOI Listing
June 2020

Prolyl Isomerase Pin1 Regulates the Stability of Hepatitis B Virus Core Protein.

Front Cell Dev Biol 2020 31;8:26. Epub 2020 Jan 31.

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Japan.

The dynamic interplay between virus and host proteins is critical for establishing efficient viral replication and virus-induced pathogenesis. Phosphorylation-dependent prolyl isomerization by Pin1 provides a unique mechanism of molecular switching to control both protein function and stability. We demonstrate here that Pin1 binds and stabilizes hepatitis B virus core protein (HBc) in a phosphorylation-dependent manner, and promotes the efficient viral propagation. Phos-tag gel electrophoresis with various site-directed mutants of HBc revealed that Thr160 and Ser162 residues within the C terminal arginine-rich domain are phosphorylated concomitantly. GST pull-down assay and co-immunoprecipitation analysis demonstrated that Pin1 associated with phosphorylated HBc at the Thr160-Pro and Ser162-Pro motifs. Chemical or genetic inhibition of Pin1 significantly accelerated the rapid degradation of HBc via a lysosome-dependent pathway. Furthermore, we found that the pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) could dephosphorylate HBc at the Pin1-binding sites, thereby suppressing Pin1-mediated HBc stabilization. Our findings reveal an important regulatory mechanism of HBc stability catalyzed by Pin1 and may facilitate the development of new antiviral therapeutics targeting Pin1 function.
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http://dx.doi.org/10.3389/fcell.2020.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005485PMC
January 2020

A prospective trial of vaccine to prevent hepatitis B virus reactivation after hematopoietic stem cell transplantation.

Bone Marrow Transplant 2020 07 18;55(7):1388-1398. Epub 2020 Feb 18.

Genome Medical Science Project, National Center for Global Health and Medicine, Ichikawa, Japan.

Hepatitis B virus (HBV) reactivation reportedly occurs frequently after hematopoietic stem cell transplantation (HSCT) in resolved HBV-infected patients. Here, 50 patients with resolved HBV infections and scheduled to undergo HSCT were enrolled; all subjects were vaccinated with three doses of hepatitis B vaccine 12 months after HSCT and the incidence of HBV reactivation was monitored. The patients' characteristics were: median age, 61 (34-72) years; male/female, 27/19; allogeneic/autologous, 40/6; bone marrow/peripheral blood stem cells/cord blood, 26/16/4. Of the 46 patients who underwent HSCT, 19 were excluded and did not make it to vaccination due to relapse of underlying disease, HBV reactivation within 12 months of HSCT, or transfer of patients. The remaining 27 were vaccinated 12 months after HSCT and monitored for 2 years. Six showed HBV reactivation, with a 2-year cumulative reactivation incidence of 22.2%; the same incidence was 27.3% only in allogeneic HSCT patients. Factors associated with HBV reactivation included the discontinuation of immunosuppressants (P = 0.0379) and baseline titers of antibody against hepatitis B surface antigen (P = 0.004). HBV reactivation with vaccination following HSCT could occur despite maintenance of serum anti-HBs at more than protective levels.
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http://dx.doi.org/10.1038/s41409-020-0833-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329632PMC
July 2020

Establishment of infectious genotype 4 cell culture-derived hepatitis C virus.

J Gen Virol 2020 02 20;101(2):188-197. Epub 2019 Dec 20.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

To establish infectious genotype 4a (GT4a) cell culture-derived hepatitis C virus (HCVcc), we constructed full-length ED43 and 12 mutants possessing single or double mutations that increase ED43 replicon replication, and performed cell culture after RNA transfection. Sequential long-term culture of full-length ED43 RNA-transfected cells showed increased viral production in two ED43 mutants named ED43 QK/SI and TR/SI among the tested clones. These ED43 mutants possessed a common mutation, R1405G, in the NS3 helicase region and another mutation, D2413G or V2414A, in the NS5a-NS5b cleavage site. Furthermore, serial reinfection of naïve Huh7.5.1 cells accelerated peak HCV production at an earlier time point after every infection. After the fourth infection, we found a common mutation, R1405G, and six additional mutations in both ED43 QK/SI and TR/SI mutants. All seven mutations supported continuous viral production for more than 40 days in both ED43 QS-7M (QK/SI with seven mutations) and ED43 TS-7M (TR/SI with seven mutations). In addition, ED43 TS-7M did not require additional mutations for continuous virus culture up to 124 days. Both ED43 QS-7M and TS-7M were sensitive to the neutralizing E2 antibodies HCV1 and AR3A and the direct-acting antivirals, simeprevir, ledipasvir and sofosbuvir. In conclusion, we established an infectious ED43 strain containing adaptive mutations, which is important for the analysis of HCV genotype-specific pathogenesis, development of pan-genotypic agents and analysis of drug resistance.
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http://dx.doi.org/10.1099/jgv.0.001378DOI Listing
February 2020

MGeND: an integrated database for Japanese clinical and genomic information.

Hum Genome Var 2019 6;6:53. Epub 2019 Dec 6.

1Department of Biomedical Data Intelligence, Graduate School of Medicine, Kyoto University, 53 Shogoin-Kawaharacho. Sakyo-ku, Kyoto, 606-8507 Japan.

To promote the implementation of genomic medicine, we developed an integrated database, the Medical Genomics Japan Variant Database (MGeND). In its first release, MGeND provides data regarding genomic variations in Japanese individuals, collected by research groups in five disease fields. These variations consist of curated SNV/INDEL variants and susceptibility variants for diseases established by genome-wide association study analysis. Furthermore, we recorded the frequencies of HLA alleles in infectious disease populations.
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http://dx.doi.org/10.1038/s41439-019-0084-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897987PMC
December 2019

Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture.

Hepatol Res 2020 Mar 5;50(3):283-291. Epub 2020 Jan 5.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Aim: Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system.

Methods: We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model.

Results: By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3.

Conclusions: We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.
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http://dx.doi.org/10.1111/hepr.13449DOI Listing
March 2020
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