Molecular characterization of HBV strains circulating among the treatment-naive HIV/HBV co-infected patients of eastern India

Abstract

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.

Figure 1. Phylogenetic analysis of the HBV isolates circulating among the HIV/HBV co-infected population of eastern India.

The phylogenetic tree was constructed based on the complete small S region (nucleotide 155–835 from EcoRI site) of the HBV genome using the neighbor-joining method and bootstrap value of 1000 replicates. The 39 isolates (denoted by EICIS) were analyzed with respect to 42 reference sequences retrieved from the GenBank which are designated by their respective accession numbers along with their HBV genotypes/subgenotypes and country of origin. • represents isolates belonging to HBV/A, ▪ represents HBV/C whereas ▴ represents HBV/D.

Figure 2. Inter-patient genetic distances for different HBV genes and its divergence among different HBV genotypes.

(A) The median genetic distance was significantly higher for the HBV PreS1/S2/Surface region compared to the precore/core regions. (B) The median genetic distance was significantly lower in case of HBV/A compared to HBV/D and HBV/C for both the HBV open reading frames (ORF), thus indicating the low genetic diversity of HBV/A.

Figure 3. Mutations in the Basal Core Promoter/PreCore and the Core regions of the HBV Genome.

(A) The frequency of the major mutations found in the BCP/precore region of the HBV strains circulating among the treatment naive HIV infected cohort of eastern India are presented here. The frequency of the G1896A precore mutation was higher compared to the BCP double mutations in our settings with majority of the strains harboring the C1858T mutation. (B) The frequencies of the amino acid mutations found in the immune-active regions of the core gene are shown. The major mutations in the MHC class I restricted (amino acids 18–27, 88–96, 130–140, 141–151) and MHC class II-restricted (amino acids 1–20, 50–69, 81–105, 117–131, 141–165) T-cell epitopes of core antigen includes the V27I and T12S respectively.

Figure 4. Schematic representation of the PreS deletions and the mutations in the small surface gene of the HBV strains isolated from the HIV/HBV co-infected patients of eastern India.

(A) The PreS deletions are indicated by dashed lines (–). The numbers ahead of each deletion indicates the starting point of the respective amino acid deletions whereas the numbers besides the triangle (▴) indicates the length of each deletion observed in the co-infected individuals. (B) The major mutations occurring frequently within the small surface gene of HBV are shown here. The substitutions marked with a star (*) are mainly contributed due to the presence of the HBV/D2 sequences.