Impact of immune escape mutations and N-linked glycosylation on the secretion of hepatitis B virus virions and subviral particles: Role of the small envelope protein.

Hepatitis B virus (HBV) expresses three co-terminal envelope proteins: large (L), middle (M), and small (S), with the S protein driving the secretion of both virions and subviral particles. Virion secretion requires N-linked glycosylation at N146 in the S domain but can be impaired by immune escape mutations. An M133T mutation creating a novel glycosylation site at N131could rescue virion secretion of N146Q mutant (loss of original glycosylation site) and immune escape mutants such as G145R. Here we demonstrate that other novel N-linked glycosylation sites could rescue virion secretion of the G145R and N146Q mutants to variable extents. Both G145R and N146Q mutations impaired virion secretion through the S protein. The M133T mutation restored virion secretion through the S protein, and could work in trans. Impaired virion secretion was not necessarily associated with a similar block in the secretion of subviral particles.

Detection and circulation of hepatitis B virus immune escape mutants among asymptomatic community dwellers in Ibadan, southwestern Nigeria.

BACKGROUND: In 2012, the first Nigerian Hepatitis B Virus (HBV) immune escape mutant (IEM) case was detected in a pregnant woman in southwestern Nigeria. Consequently, this study was designed to investigate the presence and possible circulation of IEMs amongst asymptomatic community dwellers in southwestern Nigeria. METHODS: Blood specimens collected from 438 asymptomatic community dwellers were screened for HBsAg using ELISA technique. Subsequently, the S-gene was amplified in HBsAg positive samples by a nested PCR protocol, and amplicons sequenced. Isolates were then subtyped by amino acid residues at positions 122, 127, 134 and 160, and genotyped by phylogenetic analysis. RESULTS: Of the 31 (7.08%) samples positive for HBsAg, the ∼ 408 bp Sgene fragment was successfully amplified and sequenced in 27. Samples obtained from 4 patients could not be amplified due to low titres. Sequence data from only 15 of the isolates could be analysed further as eight of the remaining 12 had multiple peaks while the rest three showed no similarity to any HBV gene when subjected to BLAST analysis. Thirteen of the 15 isolates were identified as genotype E. Eleven of which were subtyped as ayw4 while the remaining two could not be subtyped due to sR122Q/P substitutions. The last two isolates that could not be genotyped and subtyped had other mutations in the "a" determinant associated with IEMs. CONCLUSIONS: This study confirmed presence and circulation of HBV IEM in Nigeria, the country's inclusion in the genotype E crescent, and the value of phylogenetic analysis in HBV identification.

Detection of hepatitis B virus isolates with mutations associated with immune escape mutants among pregnant women in Ibadan, southwestern Nigeria.

Perinatal transmission of hepatitis B virus (HBV) and its associated immune escape mutants (IEMs), is the major vehicle through which a population of chronically infected people who serve as infectious HBV reservoirs is maintained in communities. Therefore, to assess the risk of perinatal transmission, 272 pregnant women attending ante-natal clinics in Ibadan metropolis, southwestern, Nigeria, were screened for HBsAg using ELISA technique. Samples positive for HBsAg were subjected to HBV DNA detection by PCR amplification of the S-gene and amplicon sequencing. Isolates were genotyped and subtyped using a combination of molecular techniques. Fifteen (5.5%) of the pregnant women were positive for HBsAg of which HBV DNA was detected in seven. Five of the isolates were typed as genotype E subtype ayw4 using amino acid residues at positions 122, 127, 134 and 160. Another could only be typed as genotype E subtype ayw4 by further phylogenetic analysis. The remaining one isolate did not belong to any of genotypes A - H. Three of the HBV isolates including the untypable, had mutations in the 'a' determinant associated with IEMs. This study confirms the endemicity of HBV, the risk of perinatal transmission and the circulation of genotype E subtype ayw4 in Nigeria. It further demonstrates the presence of IEMs in Nigeria.

The impact of hepatitis B virus precore/core gene carboxyl terminal mutations on viral biosynthesis and the host immune response.

BACKGROUND: We aimed to elucidate the impact of hepatitis B virus (HBV) precore/core gene mutations on spontaneous hepatitis B e antigen (HBeAg) seroconversion, HBV biosynthesis, and the human immune responses in chronic HBV-infected patients. METHODS: We analyzed the HBV precore/core gene sequences by cloning method in 33 chronic HBV-infected patients during the inflammatory phase before spontaneous HBeAg seroconversion. The impact of the most prevalent mutant on HBeAg biosynthesis was assessed by Western blotting and native agarose gel analysis in Huh7 cells, and the human immune responses were assessed by in vitro stimulation of CD3+ CD8+ T lymphocytes of chronic HBV-infected subjects. RESULTS: The P135Q and G1896A were the most prevalent mutants before HBeAg seroconversion, acting as markers of HBeAg seroconversion (hazard ratios = 2.75 and 4.50; P = .01 and <.001, respectively). The P135Q mutants displayed decreased HBeAg secretion and HBV capsid molecular weight, while showing increased 22 kD HBeAg proprotein accumulation in Huh7 cells. The P135Q mutant peptide induced less interferon-γ expression in CD3+ CD8+ T lymphocytes in HBeAg-negative subjects compared to the wild-type peptide (P = .03). CONCLUSIONS: The HBV P135Q mutant emergence during the inflammatory phase was associated with HBeAg seroconversion. It was associated with altered HBV capsid assembly, HBeAg biosynthesis, and reduced human immune responses following HBeAg seroconversion.