Hepatitis B virus covalently closed circular DNA formation in murine hepatic cells uncovers a late entry block

Eliminating cccDNA to cure hepatitis B virus infection

Identification of NTCP as an HBV Receptor: The Beginning of the End or the End of the Beginning?

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Hepatitis B virus (HBV) infection is one of the most serious health problems. HBV infection leads to serious morbidity and mortality due to the complications of chronic HBV infection. For example, HBV chronic infection is responsible for most of the cases of hepatocellular carcinomas. HBV is a small DNA virus and considered the prototype of family Hepadnaviridae. HBV genome is composed of partially double-stranded, relaxed-circular DNA (rcDNA), of 3.2 kb, in which the plus strand is incomplete, while the minus strand is complete, and the viral polymerase is attached to the 5' end of the minus strand. In the infected cells, HBV rcDNA genome is converted to a plasmid-like, covalently closed circular DNA (cccDNA) in the nucleus. CccDNA is considered the template for all HBV RNAs. Current chronic HBV infection treatments include pegylated interferon-[alpha], which has severe side effects, and nucleos(t)ide reverse transcriptase inhibitors which have no effect on cccDNA, which is highly stable, and consequently, fail to completely cure most patients. Thus, targeting cccDNA is the key element for curing chronic HBV infection. Studying host factors that have a role in HBV infection and replication may help develop new strategies to cure chronic HBV infection. Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on HBV infection is not known and its role on replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines providing strong evidence for the involvement of MOV10 in restricting HBV replication cycle. We demonstrated that MOV10 does not interact with HBV core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. Further studies are still needed to investigate the exact mechanism of which MOV10 restricts HBV. As mentioned earlier, HBV rcDNA is converted in the nucleus of infected cell to cccDNA. To be converted to cccDNA, rcDNA undergoes some modifications including the removal of HBV polymerase from the 5' end of the minus-strand DNA, the plus-strand DNA should be completed, and both strands should be covalently ligated to form the closed, plasmid-like form. The exact kinetics of cccDNA formation are not clear yet. We studied the kinetics of early events following HBV infection. Our results demonstrated that HBV DNA can be detected in the nuclei of infected cells within 2 hours post-infection by qPCR and microscopy. Also, we were able to detect HBV cccDNA within 6 hours post-infection using qPCR. Early detection of nuclear HBV DNA and cccDNA formation after infection by qPCR and microscopy will help study the early HBV infection events, and screen new cccDNA formation inhibitors with an easy and fast way.

Most cases of hepatocellular carcinoma (HCC) are associated with cirrhosis related to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Changes in the time trends of HCC and most variations in its age-, sex-, and race-specific rates among different regions are likely to be related to differences in hepatitis viruses that are most prevalent in a population, the timing of their spread, and the ages of the individuals the viruses infect. Environmental, host genetic, and viral factors can affect the risk of HCC in individuals with HBV or HCV infection. This review summarizes the risk factors for HCC among HBV- or HCV-infected individuals, based on findings from epidemiologic studies and meta-analyses, as well as determinants of patient outcome and the HCC disease burden, globally and in the United States.

Occult HBV infection—both hidden and mysterious

Natural history of chronic hepatitis B in Euro-Mediterranean and African Countries

Epidemiology of hepatitis B in Europe and worldwide.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is particularly prevalent in China. China is also a hyperendemic area for hepatitis B virus (HBV) infection. Although a strong association between HBV infection and HCC has been established previously, the role of hepatitis C virus (HCV) infection and the interaction between HBV and HCV in the development of HCC has not been adequately explored. The major objective of this study is to determine the relationship between HBV or HCV infection and HCC by use of case-control study in Henan, China. In all, 152 HCC patients and 115 control patients were collected from four hospitals in Henan, China between January 1994 and October 1995. The demographic characteristics of the two groups were comparable. In further analysis, a 1:1 pair-matched case-control study was performed. Of 152 HCC patients, 113 were randomly selected to be pair-matched by sex and age (+/-5 years) to controls with non-hepatic disease. All the cases and controls were interviewed during hospitalization by two specially trained interviewers using a standard questionnaire. All sera were tested for HBV and HCV markers. Odds ratios (OR) and 95% CI for HCC risk factors were calculated by logistic regression model controlling for possible confounding factors such as sex and age. The multivariate analysis was done on the basis of the univariate analysis. The results of this study indicated that the prevalence of hepatitis B surface antigen (HBsAg) and antibody to HCV (anti-HCV) were much higher in HCC patients (63.2% and 11.2% respectively) than in the control patients (5.2%, 3.5%). The difference between two groups was significant (P < 0.05). Risk factor analysis revealed that both HBV and HCV infection were important factors for HCC in Henan, China and HBV appeared to have a key role in the development of HCC. Odds ratios of HBsAg and HBV infection were 28.82 (95% CI: 11.18-78.78) and 31.22 (95% CI: 13.86-72.15), respectively. Moreover, the risk of developing HCC increased significantly and showed an additive effect when both viral markers of HBV and HCV infection were considered (OR = 42.85). Results from the 1:1 pair-matched case-control study also showed that HBV infection was an important risk factor for HCC, which was consistent with the results from the group-matched case-control study. This is the first reported case-control study of HCC in Henan, China. This study provides further evidence that chronic HBV infection is strongly associated with the development of HCC among this population. Our results have demonstrated that HCV and HBV infection are independent and probably additive risk factors for HCC.

Chronic hepatitis B virus (HBV) infection remains a significant public health burden with no cure currently available. The research to cure HBV has long been hampered by the lack of immunocompetent small animal models capable of supporting HBV infection. Here, we set out to explore the feasibility of the golden Syrian hamster as an immunocompetent small rodent model for HBV infection. We first started with in vitro assessments of the HBV replication cycle in primary hamster hepatocytes (PHaHs) by adenoviral HBV (Ad-HBV) transduction. Our results demonstrated that PHaHs support HBV reverse transcription and subsequent cccDNA formation via the intracellular recycling pathway. Next, with luciferase reporter assays, we confirmed that PHaHs support the activities of all HBV major promoters. Then, we transduced PHaHs with an adenoviral vector expressing HBV receptor human Na+/taurocholate cotransporting polypeptide NTCP (Ad-huNTCP), followed by HBV inoculation. While the untransduced PHaHs did not support HBV infection, Ad-huNTCP-transduced PHaHs supported de novo cccDNA formation, viral mRNA transcription, and expression of viral antigens. We then humanized the amino acid (aa) residues of hamster NTCP (haNTCP) critical for HBV entry, aa84-87 and aa157-165, and transfected HepG2 cells with constructs expressing wild-type haNTCP and humanized-haNTCP, H84R/P87N and H84R/P87N/G157K/M160V/M165L, respectively, followed by HBV inoculation. The results showed that the humanization of H84R/P87N alone was sufficient to support HBV infection at a level comparable to that supported by huNTCP. Taken together, the above in vitro evidence supports the future direction of humanizing haNTCP for HBV infection in vivo.IMPORTANCEOne of the biggest challenges in developing an HBV cure is the lack of immunocompetent animal models susceptible to HBV infection. Developing such models in mice has been unsuccessful due to the absence of a functional HBV receptor, human NTCP (huNTCP), and the defect in supporting viral cccDNA formation. In search of alternative models, we report herein multiple lines of in vitro evidence for developing a golden Syrian hamster model for HBV infection. We demonstrate that the primary hamster hepatocytes (PHaHs) support HBV replication, transcription, and cccDNA formation, and PHaHs are susceptible to de novo HBV infection in the presence of huNTCP. Furthermore, expressing hamster NTCP with two humanized residues critical for HBV entry renders HepG2 cells permissive to HBV infection. Thus, our work lays a solid foundation for establishing a gene-edited hamster model that expresses humanized NTCP for HBV infection in vivo.

Transfer of HBV Genomes Using Low Doses of Adenovirus Vectors Leads to Persistent Infection in Immune Competent Mice

Time-dependent events in natural history of occult hepatitis B virus infection: the importance of population-based long-term follow-up study with repeated measurements

Expanded screening for chronic hepatitis B virus infection in China

Mortality among hemodialysis patients in Europe, Japan, and the United States: Case-mix effects

Be cautious for exceptional results in evaluating the effect of adolescent booster of hepatitis B vaccine