Molecular characterization of hepatitis B virus in Bangladesh reveals a highly recombinant population

Abstract

The natural history and treatment outcome of hepatitis B viruses (HBV) infection is largely dependent on genotype, subgenotype, and the presence or absence of virulence associated mutations. We have studied the prevalence of genotype and subgenotype as well as virulence and drug resistance associated mutations and prevalence of recombinant among HBV from Bangladesh. A prospective cross-sectional study was conducted among treatment naïve chronic HBV patients attending at Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh for HBV viral load assessment between June and August 2015. Systematical selected 50% of HBV DNA positive patients (every second patient) were enrolled. Biochemical and serological markers for HBV infection and whole genome sequencing (WGS) was performed on virus positive sample. Genotype, subgenotype, virulence, nucleos(t)ide analogue (NA) resistance (NAr) mutations, and the prevalence of recombinant isolates were determined. Among 114 HBV DNA positive patients, 57 were enrolled in the study and 53 HBV WGS were generated for downstream analysis. Overall, 38% (22/57) and 62% (35/57) of patients had acute and chronic HBV infections, respectively. The prevalence of genotypes A, C, and D was 18.9% (10/53), 45.3% (24/53), and 35.8% (19/53), respectively. Among genotype A, C and D isolates subgenotype A1 (90%; 9/10), C1 (87.5%; 21/24) and D2 (78.9%; 15/19) predominates. The acute infection, virulence associated mutations, and viral load was higher in the genotype D isolates. Evidence of recombination was identified in 22.6% (12/53) of the HBV isolates including 20.0% (2/10), and 16.7% (4/24) and 31.6% (6/19) of genotype A, C and D isolates, respectively. The prevalence of recombination was higher in chronic HVB patients (32.2%; 10/31 versus 9.1%; 2/22); p<0.05. NAr mutations were identified in 47.2% (25/53) of the isolates including 33.9% novel mutations (18/53). HBV genotype C and D predominated in this population in Bangladesh; a comparatively high prevalence of recombinant HBV are circulating in this setting.

Fig 1. A midpoint rooted tree showing the relationship between the Bangladeshi HBV genome sequences with 103 reference sequences.

The tree was constructed using RAxML v7.2.8 available in Geneious software using GTR+G+I nucleotide substitution model with 1000 bootstrapping replicates. Bootstrap values greater than 70% are shown at the branch nodes. The Bangladeshi HBV strains are presented as BD HBV followed by isolate number, genotype and subgenotype and reference genomes are presented as genotype, subgenotype followed by Gene Bank accession number and country of origin. The scale bar indicates the number of nucleotide substitution. (A) presents the full-length genome excluding the recombination susceptible region (1–1272 and 2028–3215 bp). Isolates with recombination are marked with a asterics and are clustered with major parent genotype. (B) present the recombination susceptible region (1273 to 2027 bp). Isolates with recombination are marked with a asterics and are clustered with minor parent genotype.

Fig 2. Schematic representation of the mosaic structure of the HBV recombinant sequences of the present study.

The recombinant sequences are divided into four groups (G1 to G4); based on the major and minor parent of recombination fragments. The recombinant between C/B, D/C, A/C, D/B genotypes were presented as Group1, 2, 3, and 4 respectively. Genotype A sequence is presented in blue, genotype B in red, genotype C in gray and genotype D in green. A linear physical map of the HBV genome including the position of different gene(s) is depicted. Numbering starts from the hypothetical EcoRI restriction site.