hNTCP­expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs.

Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co­transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single­copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP­recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP­expressing PPHs with pooled HBV­positive serum and purified particles was determined. The potential use of HBV­infected hNTCP­expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post­entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e­antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP­expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV­positive sera, but were poisoned by raw HBV­positive sera. The use of HBV­infected hNTCP­expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP­expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

Hepatitis B virus infection: Defective surface antigen expression and pathogenesis.

Hepatitis B virus (HBV) infection is a global public health concern. HBV causes chronic infection in patients and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Thus, understanding HBV-related pathogenesis is of particular importance for prevention and clinical intervention. HBV surface antigens are indispensable for HBV virion formation and are useful viral markers for diagnosis and clinical assessment. During chronic HBV infection, HBV genomes may acquire and accumulate mutations and deletions, leading to the expression of defective HBV surface antigens. These defective HBV surface antigens have been found to play important roles in the progression of HBV-associated liver diseases. In this review, we focus our discussion on the nature of defective HBV surface antigen mutations and their contribution to the pathogenesis of fulminant hepatitis B. The relationship between defective surface antigens and occult HBV infection are also discussed.

Genetic variation of hepatitis B virus and its significance for pathogenesis.

Hepatitis B virus (HBV) has a worldwide distribution and is endemic in many populations. Due to its unique life cycle which requires an error-prone reverse transcriptase for replication, it constantly evolves, resulting in tremendous genetic variation in the form of genotypes, sub-genotypes, and mutations. In recent years, there has been considerable research on the relationship between HBV genetic variation and HBV-related pathogenesis, which has profound implications in the natural history of HBV infection, viral detection, immune prevention, drug treatment and prognosis. In this review, we attempted to provide a brief account of the influence of HBV genotype on the pathogenesis of HBV infection and summarize our current knowledge on the effects of HBV mutations in different regions on HBV-associated pathogenesis, with an emphasis on mutations in the preS/S proteins in immune evasion, occult HBV infection and hepatocellular carcinoma (HCC), mutations in polymerase in relation to drug resistance, mutations in HBV core and e antigen in immune evasion, chronicalization of infection and hepatitis B-related acute-on-chronic liver failure, and finally mutations in HBV x proteins in HCC.

The protamine-like DNA-binding protein P6.9 epigenetically up-regulates Autographa californica multiple nucleopolyhedrovirus gene transcription in the late infection phase.

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1]. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication[31]. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection (hpi), whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9's co-localization with DNA is temporally and spatially synchronized with P6.9's impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.

Putative phosphorylation sites on WCA domain of HA2 is essential for Helicoverpa armigera single nucleopolyhedrovirus replication.

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality. The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain, in which phosphorylation status are supposed to be critical in respect to actin polymerization. In the present study, two putative phosphorylation sites ((232)Thr and (250)Ser) and a highly conserved Serine ((245)Ser) on the WCA domain of HA2 were mutated, and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome. Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at (245)Ser can produce infectious virions, both (232)Thr and (250)Ser mutations were lethal to the virus. However, actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus, which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.

Novel evidence suggests Hepatitis B virus surface proteins participate in regulation of HBV genome replication.

Naturally occurring mutations in surface proteins of Hepatitis B virus (HBV) usually result in altered hepatitis B surface antigen (HBsAg) secretion efficiency. In the present study, we reported two conserved residues, M75 and M103 with respect to HBsAg, mutations of which not only attenuated HBsAg secretion (M75 only), but also suppressed HBV genome replication without compromising the overlapping p-gene product. We also found M75 and M103 can initiate truncated surface protein (TSPs) synthesis upon over-expression of full-length surface proteins, which may possibly contribute to HBV genome replication. However, attempts to rescue replication-defective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful, which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.