C.-T. Bock

Hepatitis B virus‐induced hepatocellular carcinoma: functional roles of MICA variants

Hepatitis B virus infection is a high‐risk factor for hepatocellular carcinoma. The human major histocompatibility complex class I chain‐related gene A (MICA) is a ligand of the NKG2D receptor that modulates the NK and T‐cell‐mediated immune responses and is associated with several diseases. This study determined the effects of MICA polymorphisms during HBV infection and HBV‐induced HCC. We conducted a case–controlled study in a Vietnamese cohort and genotyped ten functional MICA polymorphisms including the microsatellite motif in 552 clinically classified hepatitis B virus patients and 418 healthy controls. The serum soluble MICA levels (sMICA) were correlated with MICA variants and liver enzyme levels. We demonstrated a significant contribution of MICA rs2596542G/A promoter variant and nonsynonymous substitutions MICA‐129Met/Val, MICA‐251Gln/Arg, MICA‐175Gly/Ser, triplet repeat polymorphism and respective haplotypes with HBV‐induced HCC and HBV persistence. The circulating sMICA levels in HBV patient groups were elevated significantly compared with healthy controls. A significant contribution of studied MICA variants to sMICA levels was also observed. The liver enzymes alanine amino transferase (ALT), aspartate transaminase (AST), total bilirubin and direct bilirubin were positively correlated with sMICA levels suggesting sMICA as a biomarker for liver injury. We conclude that MICA polymorphisms play a crucial role in modulating innate immune responses, tumour surveillance and regulate disease susceptibility during HBV infection.

Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg‐positive patients

Hepatitis D virus (HDV) infection is acquired as a co‐ /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10–20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty‐one HDV isolates in Vietnamese HBsAg‐positive patients. HDV subgenomic and full‐length genome sequences were obtained using newly established HDV‐specific RT‐PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full‐length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg‐positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro‐epidemiology of HDV and subsequent pathophysiology in chronic HBV‐ /HDV‐related liver diseases.

Molecular phenotypes of human parvovirus B19 in patients with myocarditis

AIM To investigate molecular phenotypes of myocardial B19V-infection to determine the role of B19V in myocarditis and dilated cardiomyopathy (DCM). METHODS Endomyocardial biopsies (EMBs) from 498 B19V-positive patients with myocarditis and DCM were analyzed using molecular methods and functional experiments. EMBs were obtained from the University Hospitals of Greifswald and Tuebingen and additionally from 36 German cardiology centers. Control tissues were obtained at autopsy from 34 victims of accidents, crime or suicide. Identification of mononuclear cell infiltrates in EMBs was performed using immunohistological staining. Anti-B19V-IgM and anti-B19V-IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). B19V viral loads were determined using in-house quantitative real-time polymerase chain reaction (PCR). For B19V-genotyping a new B19V-genotype-specific restriction fragment length polymorphism (RFLP)-PCR was established. B19V-genotyping was verified by direct DNA-sequencing and sequences were aligned using BLAST and BioEdit software. B19V P6-promoter and HHV6-U94-transactivator constructs were generated for cell culture experiments. Transfection experiments were conducted using human endothelial cells 1. Luciferase reporter assays were performed to determine B19V-replication activity. Statistical analysis and graphical representation were calculated using SPSS and Prism5 software. RESULTS The prevalence of B19V was significantly more likely to be associated with inflammatory cardiomyopathy (iCMP) compared to uninflamed DCM (59.6% vs 35.3%) (P < 0.0001). The detection of B19V-mRNA replication intermediates proved that replication of B19V was present. RFLP-PCR assays showed that B19V-genotype 1 (57.4%) and B19V-genotype 2 (36.7%) were the most prevalent viral genotypes. B19V-genotype 2 was observed more frequently in EMBs with iCMP (65.0%) compared to DCM (35%) (P = 0.049). Although there was no significant difference in gender-specific B19V-loads, women were more frequently infected with B19V-genotype 2 (44.6%) than men (36.0%) (P = 0.0448). Coinfection with B19V and other cardiotropic viruses was found in 19.2% of tissue samples and was associated with higher B19V viral load compared to B19V-monoinfected tissue (P = 0.0012). The most frequent coinfecting virus was human herpes virus 6 (HHV6, 16.5%). B19V-coinfection with HHV6 showed higher B19V-loads compared to B19V-monoinfected EMBs (P = 0.0033), suggesting that HHV6 had transactivated B19V. In vitro experiments confirmed a 2.4-fold increased B19V P6-promoter activity by the HHV6 U94-transactivator. CONCLUSION The finding of significantly increased B19V loads in patients with histologically proven cardiac inflammation suggests a crucial role of B19V-genotypes and reactivation of B19V-infection by HHV6-coinfection in B19V-associated iCMP. Our findings suggest that B19V-infection of the human heart can be a causative event for the development of an endothelial cell-mediated inflammatory disease and that this is related to both viral load and genotype.

Hepatitis E Virus Superinfection and Clinical Progression in Hepatitis B Patients

Hepatitis E virus (HEV) infection may cause acute hepatitis and lead to hepatic failure in developing and developed countries. We studied HEV seroprevalences in patients with hepatitis B virus (HBV) infection to understand the consequences of HEV superinfection in a Vietnamese population. This cross-sectional study was conducted from 2012 to 2013 and included 1318 Vietnamese patients with HBV-related liver diseases and 340 healthy controls. The case group included patients with acute (n=26) and chronic hepatitis B (n=744), liver cirrhosis (n=160), hepatocellular carcinoma (n=166) and patients with both liver cirrhosis and hepatocellular carcinoma (n=222). Anti-HEV IgG and IgM antibodies were assessed in patients and controls by ELISA. HEV-RNA was identified by PCR assays and sequencing. Seroprevalences of anti-HEV IgG among hepatitis B patients and controls were 45% and 31%, respectively (adjusted P=0.034). Anti-HEV IgM seroprevalences were 11.6% and 4.7% in patients and controls, respectively (adjusted P=0.005). Seroprevalences were higher among the elder individuals. When stratifying for patient groups, those with liver cirrhosis had the highest anti-HEV IgG (52%) and anti-HEV IgM (19%) seroprevalences. Hepatitis B patients with current HEV infection had abnormal liver function tests compared to patients with past or without HEV infection. One HEV isolate was retrieved from a patient with both liver cirrhosis and hepatocellular carcinoma and identified as HEV genotype 3. This study indicates high prevalences of HEV infection in Vietnamese HBV patients and among healthy individuals and shows that HEV superinfection may influence the outcome and progression of HBV-related liver disease.Hepatitis E virus (HEV) infection may cause acute hepatitis and lead to hepatic failure in developing and developed countries. We studied HEV seroprevalences in patients with hepatitis B virus (HBV) infection to understand the consequences of HEV superinfection in a Vietnamese population. This cross-sectional study was conducted from 2012 to 2013 and included 1318 Vietnamese patients with HBV-related liver diseases and 340 healthy controls. The case group included patients with acute (n = 26) and chronic hepatitis B (n = 744), liver cirrhosis (n = 160), hepatocellular carcinoma (n = 166) and patients with both liver cirrhosis and hepatocellular carcinoma (n = 222). Anti-HEV IgG and IgM antibodies were assessed in patients and controls by ELISA. HEV-RNA was identified by PCR assays and sequencing. Seroprevalences of anti-HEV IgG among hepatitis B patients and controls were 45% and 31%, respectively (adjusted P = 0.034). Anti-HEV IgM seroprevalences were 11.6% and 4.7% in patients and controls, respectively (adjusted P = 0.005). Seroprevalences were higher among the elder individuals. When stratifying for patient groups, those with liver cirrhosis had the highest anti-HEV IgG (52%) and anti-HEV IgM (19%) seroprevalences. Hepatitis B patients with current HEV infection had abnormal liver function tests compared to patients with past or without HEV infection. One HEV isolate was retrieved from a patient with both liver cirrhosis and hepatocellular carcinoma and identified as HEV genotype 3. This study indicates high prevalences of HEV infection in Vietnamese HBV patients and among healthy individuals and shows that HEV superinfection may influence the outcome and progression of HBV-related liver disease.

Treatment of severe, nonfulminant acute hepatitis B with lamivudine vs placebo: a prospective randomized double‐blinded multicentre trial

Acute hepatitis B virus (aHBV) infection can lead to fulminant liver failure, which likely is prevented by early lamivudine therapy. Even nonfulminant but severe acute hepatitis B can lead to significant morbidity and impaired quality of life. Therefore, lamivudine was evaluated in patients with severe aHBV in a placebo‐controlled trial. Patients with severe aHBV infection (ALT >10× ULN, bilirubin >85 μm, prothrombin time >50%) were prospectively treated with lamivudine 100 mg/day or with placebo within 8 days after the diagnosis. The primary end point was time to bilirubin <34.2 μm. Secondary end points were time to clear HBsAg and HBV‐DNA, development of anti‐HBs and normalization of ALT. Eighteen cases were randomized to lamivudine, 17 to placebo. 94% of patients were hospitalized. No individual progressed to hepatic failure; all but one patient achieved the primary end point. Due to smaller than expected patient numbers, all study end points did not become statistically significant between treatment arms. Median time end points [in days] were bilirubin <34.2 μm (26.5 vs 32), ALT normalization (35 vs 48) and HBsAg clearance (48 vs 67) referring to earlier recovery under lamivudine, in contrast to loss of HBV‐DNA (62 vs 54) and development of anti‐HBs (119 vs 109). In all but two patients (one in every group), HBsAg clearance was reached in the study. Adverse events occurred more frequently during lamivudine therapy, but did not reach statistical significance. Lamivudine may ameliorate severe aHBV infection, but limited patient numbers prevented definite conclusions.

Optimisation of quantitative miRNA panels to consolidate the diagnostic surveillance of HBV-related hepatocellular carcinoma

Background Circulating microRNAs (miRNA) are biomarkers for several neoplastic diseases, including hepatocellular carcinoma (HCC). We performed a literature search, followed by experimental screening and validation in order to establish a miRNA panel in combination with the assessment of alpha-fetoprotein (AFP) levels and to evaluate its performance in HCC diagnostics. Methods Expression of miRNAs was quantified by quantitative PCR (qPCR) in 406 serum samples from 118 Vietnamese patients with hepatitis B (HBV)-related HCC, 69 patients with HBV-related liver cirrhosis (LC), 100 chronic hepatitis B (CHB) patients and 119 healthy controls (HC). Results Three miRNAs (mir-21, mir-122, mir-192) were expressed differentially among the studied subgroups and positively correlated with AFP levels. The individual miRNAs mir-21, mir-122, mir192 or the triplex miRNA panel showed high diagnostic accuracy for HCC (HCC vs. CHB, AUC = 0.906; HCC vs. CHB+LC, AUC = 0.81; HCC vs. CHB+LC+HC, AUC = 0.854). When AFP levels were ≤20ng/ml, the triplex miRNA panel still was accurate in distinguishing HCC from the other conditions (CHB, AUC = 0.922; CHB+LC, AUC = 0.836; CHB+LC+HC, AUC = 0.862). When AFP levels were used in combination with the triplex miRNA panel, the diagnostic performance was significantly improved in discriminating HCC from the other groups (LC, AUC = 0.887; CHB, AUC = 0.948; CHB+LC, AUC = 0.887). Conclusions The three miRNAs mir-21, mir-122, mir-192, together with AFP, are biomarkers that may be applied to improve diagnostics of HCC in HBV patients, especially in HBV-related LC patients with normal AFP levels or HCC patients with small tumor sizes.

P641 HIGH PREVALENCE OF HEPATITIS D VIRUS (HDV) IN VIETNAMESE HBsAg-POSITIVE PATIENTS AND A NATURAL INTER-GENOTYPIC RECOMBINANT OF HDV-GENOTYPES 1 AND 2

P640 GENE MUTATION DETECTION ANALYSIS OF SWINE HEPATITIS E VIRUS PROPAGATED IN THE PRIMARY-CULTURED HUMAN HEPATOCYTES Y. Oshiro, H. Yasue, S. Ideno, S. Hattori, K. Sakai, S. Osari, K. Takeuchi, K. Nagata, N. Ohkohchi. Department of Surgery, Division of Gastroenterological and Hepatobiliary Surgery, and Organ Transplantation, University of Tsukuba, Animal Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan Blood Products Organization, Kobe, Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan E-mail: oshiro@md.tsukuba.ac.jp

Viral evolution in chronic hepatitis B: a branched way to HBeAg seroconversion and disease progression?

Evolution is a natural event that enables the animal and plant kingdoms to adapt and survive to changing environmental effects. The same evolutionary pressures also help to shape micro-organisms, including viruses. The evolutionary significance of virus infections has been a subject of discussion for decades1; however, modern genomic analysis became an important focus of research when it became apparent that genetic variants of viruses could be linked to pathogenesis and disease progression. The first questions relating virus diversity to disease progression were provided by Pierre Lepine in 1938 who reported on the evolution of different strains of rabies viruses and a link between different characteristics of infectivity and virulence.2 Recent reports have also revealed the importance of viral evolution and genetic diversity in the pathogenesis of viral diseases, in particular, of RNA (influenza virus, HCV)3 ,4 and retrovirus infections (HIV).5 Hepatitis B virus (HBV) utilises a reverse transcription strategy that, together with an error-prone viral polymerase, has the potential to generate a large number of genetic variants. Chronic HBV (CHB) infection follows four complex dynamic phases, namely immune tolerance, immune clearance, immune control/inactive state and immune escape/reactivation that evolve over several decades. This scenario is usually accompanied by HBeAg seroconversion. The frequency and severity of hepatitis flares can predict disease progression, while early HBeAg seroconversion confers a favourable outcome, and late or absent HBeAg seroconversion results in progression to severe liver disease, such as cirrhosis. …