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Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

ABSTRACT: Many studies have identified mutations in the hepatitis B surface antigen (HBsAg) as important factors limiting the ability of commercial serological assays to detect this viral antigen. However, an association between mutations in the HBsAg gene and the occurrence of occult HBV infection (OBI) in patients has not been established.To detect hepatitis B virus (HBV) DNA in patients with anti-HBc as a unique serological marker, a previously published, cost-effective TaqMan-based real-time polymerase chain reaction (PCR) test with minor groove binding probes was adapted for use in this study. The current study also aimed to investigate HBsAg mutations and genotypes of HBV in OBI at the Viral Hepatitis Ambulatory Clinic in Rio de Janeiro to determine any possible association.Intra-assay and inter-assay reproducibility were determined, and the mean coefficient of variation values obtained were 2.07 and 3.5, respectively. Probit analysis indicated that the 95% detection level was 25 IU/mL. The prevalence of OBI was investigated in 35 serum samples with an 'anti-HBc alone' profile from individuals who attended our clinic between 2011 and 2013.HBV DNA was detected in only one sample, resulting in an OBI rate of 2.9%. Nucleotide sequencing of the pre-S/S region was performed to genotype and analyse mutations within the HBsAg gene of this HBV DNA. The HBV in the OBI case was classified as sub-genotype A1, and a sequence analysis of the small S gene revealed 12 mutations in the major hydrophilic region compared to the consensus A1 sequence. Most of these mutations occurred in amino acid residues that have been reported as clinically relevant because they have been implicated in vaccine escape and/or inability to detect HBsAg by commercial serological assays.Our study suggests the importance of specific HBsAg mutations, different from those in D, B, and C genotypes, in sub-genotype A1 HBV associated with OBI.

SUBMITTER: Almeida RW

PROVIDER: S-EPMC5452485 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

Almeida Ricardo Wagner de RW Mello Francisco Campello do Amaral FCDA Menegoy Isabelle Vasconcelos IV Santo Márcia Paschoal do Espírito MPDE Ginuíno Cléber Ferreira CF Sousa Paulo Sérgio Fonseca de PSF Villar Livia Melo LM Lampe Elisabeth E Lewis-Ximenez Lia Laura LL

Memorias do Instituto Oswaldo Cruz 20170701 7

<h4>Background</h4>Many studies have identified mutations in the hepatitis B surface antigen (HBsAg) as important factors limiting the ability of commercial serological assays to detect this viral antigen. However, an association between mutations in the HBsAg gene and the occurrence of occult HBV infection (OBI) in patients has not been established.<h4>Objectives</h4>To detect hepatitis B virus (HBV) DNA in patients with anti-HBc as a unique serological marker, a previously published, cost-effe ...[more]

PMID: 28591309

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Project description:The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7-338.3% vs. extracellular) and its fluorescence intensity (1.5-2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.

Dataset Information

Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

Publications

Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

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