Molecular characterization of hepatitis B virus X gene in chronic hepatitis B patients

BACKGROUND: HBV-X protein is associated with the pathogenesis of HBV related diseases, specially in hepatocellular carcinomas of chronic patients. Genetic variability of the X gene includes genotypic specific variations and mutations emerging during chronic infection. Its coding sequence overlaps important regions for virus replication, including the basal core promoter. Differences in the X gene may have implications in biological functions of the protein and thus, affect the evolution of the disease. There are controversial results about the consequences of mutations in this region and their relationship with pathogenesis. The purpose of this work was to describe the diversity of HBV-X gene in chronic hepatitis patients infected with different genotypes, according to liver disease. METHODS: HBV-X gene was sequenced from chronic hepatitis B patient samples, analyzed by phylogeny and genotyped. Nucleotide and aminoacid diversity was determined calculating intragenetic distances. Mutations at 127, 130 and 131 aminoacids were considered in relation to liver disease. RESULTS: The most prevalent genotype detected in this cohort was F (F1 and F4), followed by D and A. Most of the samples corresponding to genotypes A and F1 were HBeAg(+) and for genotypes D and F4, HBeAg(−) samples were represented in a higher percentage. Intragenetic distance values were higher in HBeAg(−) than in positive samples for all genotypes, and lower in overlapped regions, compared to single codification ones. Nucleotide and aminoacid diversities were higher in HBeAg(−), than in HBeAg(+) samples. CONCLUSIONS: Independently of the infecting genotypes, mutations at any of 127, 130 and/or 131 aminoacid positions and HBeAg(−) status were associated with mild liver disease in this cohort

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro

A population-based study to investigate host genetic factors associated with hepatitis B infection and pathogenesis in the Chinese population

Background Hepatitis B virus (HBV) infection is a significant public health problem that may lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC). Approximately 30% of the world\u27s population has been infected with HBV and approximately 350 million (5–6%) are persistent carriers. More than 120 million Chinese are infected with HBV. The role of host genetic factors and their interactions with environmental factors leading to chronic HBV infection and its complications are not well understood. We believe that a better understanding of these factors and interactions will lead to more effective diagnostic and therapeutic options. Methods/Design This is a population-based, case-control study protocol to enroll 2200 Han Chinese from medical centers in northern and western China. Adult subjects in the following groups are being enrolled: healthy donors (n = 200), HBV infected persons achieving virus clearance (n = 400), asymptomatic HBV persistent carriers (n = 400), chronic hepatitis B cases (n = 400), decompensated liver cirrhosis with HBV infection cases (n = 400), and hepatocellular carcinoma with HBV infection cases (n = 400). In addition, for haplotype inference and quality control of sample handling and genotyping results, children of 1000 cases will be asked to provide a buccal sample for DNA extraction. With the exception of adult patients presenting with liver cirrhosis or HCC, all other cases and controls will be 40 years or older at enrollment. A questionnaire is being administered to capture dietary and environmental risk factors. Both candidate-gene and genome-wide association approaches will be used to assess the role of single genetic factors and higher order interactions with other genetic or environmental factors in HBV diseases. Conclusion This study is designed and powered to detect single gene effects as well as gene-gene and environmental-gene interactions. The identification of allelic polymorphisms in genes involved in the pathway leading to chronic viral infection, liver cirrhosis and, ultimately, hepatocellular carcinoma would provide insights to those factors leading to HBV replication, liver inflammation, fibrosis, and the carcinogenic process. An understanding of the contribution of host genetic factors and their interactions may inform public health policy, improve diagnostics and clinical management, and provide targets for drug development

Inhibition of Bovine Viral Diarrhea Virus RNA Synthesis by Thiosemicarbazone Derived from 5,6-Dimethoxy-1-Indanone▿

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV)