Molecular evolution and phylodynamics of hepatitis B virus infection circulating in Iran.

Previous local and national Iranian publications indicate that all Iranian hepatitis B virus (HBV) strains belong to HBV genotype D. The aim of this study was to analyze the evolutionary history of HBV infection in Iran for the first time, based on an intensive phylodynamic study. The evolutionary parameters, time to most recent common ancestor (tMRCA), and the population dynamics of infections were investigated using the Bayesian Monte Carlo Markov chain (BMCMC). The effective sample size (ESS) and sampling convergence were then monitored. After sampling from the posterior distribution of the nucleotide substitution rate and other evolutionary parameters, the point estimations (median) of these parameters were obtained. All Iranian HBV isolates were of genotype D, sub-type ayw2. The origin of HBV is regarded as having evolved first on the eastern border, before moving westward, where Isfahan province then hosted the virus. Afterwards, the virus moved to the south and west of the country. The tMRCA of HBV in Iran was estimated to be around 1894, with a 95% credible interval between the years 1701 and 1957. The effective number of infections increased exponentially from around 1925 to 1960. Conversely, from around 1992 onwards, the effective number of HBV infections has decreased at a very high rate. Phylodynamic inference clearly demonstrates a unique homogenous pattern of HBV genotype D compatible with a steady configuration of the decreased effective number of infections in the population in recent years, possibly due to the implementation of blood donation screening and vaccination programs. Adequate molecular epidemiology databases for HBV are crucial for infection prevention and treatment programs.

HBsAg mutations related to occult hepatitis B virus infection in HIV-positive patients result in a reduced secretion and conformational changes of HBsAg.

BACKGROUND: Occult hepatitis B infection (OBI) is a frequent finding in human immunodeficiency virus (HIV)-infected patients. While several related mutations in the hepatitis B virus (HBV) genome have been reported, their distinct impact on HBsAg synthesis is largely obscure. METHODS: Thirty-one (18%) out of 172 HIV-infected patients, who were selected from HBsAg-negative patients, were positive for HBV-DNA assigned as being OBI-positive. We generated a series of expression constructs of variant HBsAg with "a" determinant amino acid substitutions including P127L, P127T, S136Y, and P127T + S136Y using site-directed mutagenesis. The expression of variant HBsAg was examined by transient transfection in hepatoma cells, followed by HBsAg immunoassay and immunofluorescence stained with specific anti-HBs antibodies. The potential impact of amino acid substitutions at different positions for conformational changes in the HBsAg was investigated using bioinformatics. RESULTS: All variants comprising either single or combined mutations resulted in significantly reduced HBsAg detection in supernatants and in cell lysates of hepatoma cells transfected with the constructs. Moreover, intracellular immunofluorescence staining of cytoblocks showed perinuclear and cytoplasmic fluorescence of HBsAg constructs with significantly diminished fluorescent intensity in comparison to the wild type. Altered protein conformations by predictive models, indicating an impaired detection by the host's immune response as well as by commercial antibody-based test assays. CONCLUSION: Mutations in the "a" determinant region of HBV as often found in OBI remarkably impair the detection of HBsAg from serum and infected cells, emphasizing the relevance of alternative methods such as HBV-DNA quantification for high-risk groups like HIV-infected individuals. J. Med. Virol. 89:246-256, 2017. © 2016 Wiley Periodicals, Inc.

Identification of occult hepatitis B virus (HBV) infection and viral antigens in healthcare workers who presented low to moderate levels of anti-HBs after HBV vaccination.

BACKGROUND: Worldwide, healthcare workers (HCWs) show different levels of response to hepatitis B virus (HBV) vaccine. One of the factors associated with vaccine unresponsiveness may be the existence of current or past HBV infection. Regardless of the presence of HBsAg (overt infection), occult HBV infection (OBI, defined as presence of HBV DNA in the absence of HBsAg) might also account for some non- or hypo-response cases. METHODS: Sera from 120 HBsAg-negative HCWs with low and moderate levels of anti-HBs, <10 IU/mL (group I) and <100 IU/mL (group II) respectively, were selected and were examined for OBI by sensitive real-time PCR regardless of HBV serological profiles. Direct sequencing on surface genes was carried out in OBI-positive cases. RESULTS: Four (3.3%) were positive for OBI. All were negative for anti-HBc. Two of the positive cases had moderate levels of anti-HBs (>10 to <100 IU/mL). No significant differences were found between the two groups in terms of risk factors or serological data. No mutations were found in surface proteins of OBI cases. CONCLUSION: OBI in these subjects might be due to other factors rather than presence of "a" determinant mutations. Healthcare workers with inadequate to moderate levels of anti-HBs (<100 IU/mL) following vaccination, regardless of their serological profile for HBV, should be tested for the presence of HBV DNA by sensitive molecular tests. Anti-HBc is not a reliable marker for suspicion of OBI, especially in high-risk group individuals.

Evolution of hepatitis B virus surface gene and protein among Iranian chronic carriers from different provinces.

BACKGROUND AND OBJECTIVES: Iranian chronic HBV carrier's population has shown a unique pattern of genotype D distribution all around the country. The aim of this study was to explore more details of evolutionary history of carriers based on structural surface proteins from different provinces. MATERIALS AND METHODS: Sera obtained from 360 isolates from 12 Different regions of country were used for amplification and sequencing of surface proteins. A detailed mutational analysis was undertaken. RESULTS: The total ratio for Missense/Silent nucleotide substitutions was 0.96. Sistan and Kermanshah showed the lowest rate of evolution between provinces (P = 0.055). On the other hand, Khorasan Razavi and Khoozestan contained the highest ratio (P = 0.055). The rest of regions were laid between these two extremes. Azarbayjan and Guilan showed the highest proportion of immune epitope distribution (91.3% and 96%, respectively). Conversely, Sistan and Tehran harbored the least percentage (66.6% and 68.8%, respectively). Kermanshah province contained only 5.2%, whereas Isfahan had 54.5% of B cell epitope distribution. In terms of T helper epitopes, all provinces showed a somehow homogeneity: 22.58% (Fars) to 46.6% (Khuzestan). On the other hand, distribution of substitutions within the CTL epitopes showed a wide range of variation between 6.6% (Khuzestan) and 63% (Kermanshah). CONCLUSION: Further to low selection pressure found in Iranian population, the variations between different regions designate random genetic drift within the surface proteins. These finding would have some applications in terms of specific antiviral regimen, design of more efficient vaccine and public health issues.