A17 Molecular characteristics of hepatitis B virus (HBV) isolated from chronic hepatitis B patients in South Vietnam

Chronic infection with hepatitis B virus (HBV) is a major health problem due to its worldwide distribution and its long-term effects. Vietnam is country with a high HBV burden and the prevalence of chronic HBV infection in general population is 8.8–12.3%. In East Asia, the most common HBV genotypes are B and C. Despite high prevalence of HBV, data on HBV (genotype and subgenotype, virulence markers, drug resistance mutations and prevalence of recombinant strains) is limited in Vietnam. There are only few reports on HBV genotypes in Vietnam, mostly based on pre-S/S gene sequences. We have analyzed whole genome sequence of 98 HBV isolates from chronic HBV patients attending at Hospital for Tropical Disease, Ho Chi Minh City, Vietnam, who were under treatment for 1–6 years. HBV genome was amplified in 4 overlapping fragments (777–1,136 bp) and the amplicons were subjected to deep sequencing by using Illumina MiSeq system. Sequence assembly, genome analysis and phylogenetic analysis were performed within Geneious package. A sequence was assigned to a certain genotype and subgenotype if it was contained within a well-supported phylogenetic cluster (bootstrap value >75%) and the intra-genotypic nucleotide divergence was <7.5 and >4.5%, respectively. Mutations in Basal core promoter (BCP), pre-CORE, and CORE gene regions were determined by comparing with reference sequences. Finally, screening of minor (sub-consensus) variants was performed using the SNP detection tool available in Geneious. 1% frequency and 500-fold coverage were chosen as cut-off values. Among the isolates, 71.43% were genotype B, 27.55% were genotype C and one isolate was a recombinant (between B and C). Among genotype B isolates, 65 were subgenotype B4 (92.86%) and 5 were B2 (7.14%). 92.6% of subgenotype C belong to C1, 3.7% is subgenotype C2 and the remaining 3.7% to C3. Mutations G1752A, T1753C, G1757A, A1762G/T, G1764A and C1766G on BCP and CORE were found in 76 of 98 (77.55%) strains; and in 96.3% of subgenotype C (26/27) isolates. Stop codon mutation on Pre-Core, G1896A, was found in 23 of 98 patients (23.47%); among them, 20 of 23 (86.96%) are genotype B. Mutations related to antiviral drug resistance with frequency more than 1% in the reverse transcriptase (RT) domain of Polymerase gene were identified. 83.67% sequences have more than one mutation. 50% sequences have mutation at rtI169V/M, and all belong to sub-genotype B. Mutations at rtM204I/V and rtM250I were present in 23.47% and 11.22% of isolates (23 and 11 of 98, respectively), and was higher in sub-genotype C (P-values = 0.06 and <0.01, respectively). Our study provides high precision whole genome sequences of the Vietnamese HBV population and shows potential information related to treatment prediction for HBV infection as well as viral evolution. A further study with larger sample size and intensive analysis combined with clinical information promises more useful data for treatment and invention.