Hepatitis B Virus Reverse Transcriptase Mutations In The Natural History Of Chronic Hepatitis B

Objective:Mutations in hepatitis B virus (HBV) are frequently observed during chronic HBV infection. Despite extensive investigation of natural HBV mutants, naturally occurring mutations in HBV reverse transcriptase (RT) region have not been well characterized. This study aimed to investigate the frequency of natural RT mutation, and identify the locations and origin of mutations within the RT region along the natural course of chronic HBV infection.Methods:In this cross-sectional study, we analyzed mutations at the RT region in three phases of chronic HBV infection (immune-tolerant phase, IT, n=10; immune-active phase, IA, n=9; and inactive carriers phase, IC, n=9) by polymerase chain reaction (PCR) cloning and sequence analysis. Further, we located mutations found in the RT functional domains and inter-domains, and immune epitopes (HLA Ⅰ and HLAⅡ epitopes). We also identified the mutations in the open reading frame (ORF) of main S gene that is fully overlapped with the RT region.Results:Atotal of 575 full-length RT nucleotide sequences (IT:224, IA: 196, IC:155) were successfully generated from 28 treatment-naive subjects (53.57% males; median age,28 years; B-genotype,75% and C-genotype, 25%). The number of clones containing RT mutations in IT, IA and IC group were 16.07%(range:4.17%-52.00%),44.90%(range:16.67%-69.57%), and 43.20%(range:15.00%-100%), respectively. Quasispecies analysis further demonstrated that the extents of RT variation were significant different between IT phase and IA, IC phases (p<0.05). Increased frequencies of RT mutations over time were confirmed by the decreased RT protein homology from IT to IA/IC (IT:94.5%, IA:89.2%, and IC:89.5%, p<0.05). A total of 63 amino acid substitutions (IT:19, IA:37, and IC:36) were identified in this cohort. Frequencies and locations of these mutations varied significantly depending on the different RT domains and immune epitopes (HLAI and HLA Ⅱ epitopes, and "a" determinant). The nucleot(s)ide analogues(NAs) related mutations (rtI169T, rtA181T, and rtS202G) were detected in 8 (1.39%,8/575) clones from two patients (7.14%,2/28).More frequent mutations were detected in the ORF of S gene from the same clones. The majority of RT mutations were shared with genetic changes in the main S gene.Conclusion:Our results suggested significant differ of HBV RT naturally mutations and a strong conservative tendency in the RT region despite significant impact from frequent mutations in the S gene to maintain the essential RT functions in the HBV lifecycle. However, once this conservative protection was compromised to allow more frequent mutations at RT functional domains, HBV replication competence could be weakened.