The Phylogenetic investigation and determination of hepatitis B virus genotypes and subtypes in Tabriz during 2021

Abstract

The goal of this study was to gain a broader insight into the evolution of HBV in East Azerbaijan and phylogenetic analysis based on the sequence of the surface protein of the virus (HBsAg) to determine its genotype and subgenotypes. Strains with mutations in HBsAg antigenic epitopes have the ability to escape from routine diagnosis, so it is important to identify them with new methods and how to treat and prevent them. The possible findings of this research will be important in the public health system to improve the process of prevention and treatment. Materials and Methods: In this cross-sectional descriptive study, after obtaining ethical consent, blood samples were prepared from 95 patients with chronic hepatitis B referred to the central laboratory of the province in Tabriz, and the serum separated from the samples was used to check the presence of serological markers of hepatitis B, HBsAg, HBeAg, Anti -HBe and Anti-HBc were used using the ELISA technique in the same laboratory as well as the virology laboratory of the Faculty of Medicine in Tabriz. Also, serum biochemical markers related to the liver such as ALT, AST and ALP were measured with an autoanalyzer. Hepatitis B virus genome was extracted from serum by DNA extraction kit (QIAGEN). The genome extraction product from all the collected samples was checked to determine the presence of HBV-DNA through Nested-PCR with specific primers. The positive samples for the presence of HBV-DNA were identified as HBV patients and were quantitatively examined by real-time PCR to determine the viral load. Then the HBsAg gene was sequenced in HBV patients and the results after sequencing were used to analyze the virus genotype and possible mutations using bioinformatics methods. Results: In all identified patients, hepatitis B virus genotype was type D and ayw2 (94.7%) subtypes were the dominant subtypes, ayw3 (3.1%), ayw4 (2.1%). After matching the sequence of patient samples with the reference sequence, it was determined that there were a total of 169 nucleotide substitutions in HBV strains (silent and aberrant mutations), among which 81 (48%) were aberrant mutations and 88 (52%) were silent mutations. In 31 samples (32.6%), none of the nucleotide or amino acid changes were observed. In eight samples (8.4%) out of 95 samples, at least one mutation was observed in the HBsAg "a" determinant region. 58 mutations (71.6%) of 81 amino acid mutations were seen in immunological epitopes, 16 mutations (27.5%) in B cell epitopes, 17 mutations (29.3%) in T helper epitopes and 25 mutations (43.1%) occurred in CTL epitopes. The prevalence of mutations in the MHR region was 19.7%.