HBV infection is an epidemic in China. In Northwest China, the prevalence of HBV infection is much higher compared to other areas. In previous HBV studies in Northwest China, phylogenetic analysis of the HBV S region was employed to reveal the HBV genotypes . However, the complete characteristics of epidemic HBV strains have not been well studied, especially in those strains in asymptomatic carriers. In this study, 242 complete HBV genome sequences were isolated from asymptomatic carriers who underwent physical examinations in the local centers for disease prevention and control. Based on these sequences, consensus sequences of genotypes B, C and D were determined and compared with each other, and moreover, all the sequences were screened for clinically relevant mutations. This study, which presents the HBV genomic background of early stage infection, will contribute to the establishment of a reliable virus evolution history and provide vital genomic baseline references for further clinical studies.
In China, HBV genotypes B and C are suggested to be the major genotypes in the population . Since the time Genotype D was first reported by Fan J et al. in the Qinghai-Tibet Plateau in 1997, it has never been considered a major genotype in China [5, 14]. However, in this study, 54 of 242 (22.31%) HBV sequences were identified as genotype D in Northwest China, which was higher than expected. It can be inferred that genotype D may play a more important role in HBV prevalence in western China than previously expected.
The HBV genotype consensus sequence is important in establishing an HBV genotype sequence motif and inferring the genotype-dependent function of various HBV domains. Mutation Master is a reliable server that rapidly provides a visual display of consensus sequences and genetic variability, using multiple sequence alignments . In this study, Mutation Master Server was used to analyze HBV sequences of different genotypes for consensus sequences and genetic variability. By comparing different genotype consensus sequences, we found that most of the genotype-dependent variability was concentrated in the spacer region, which has not previously been reported.
The P ORF and S ORF are the most important for the prevention and therapy of HBV infection. In the P ORF, 28 genotype-dependent positions were detected in the reverse transcriptase region, where there is a functional domain in which reverse transcription and synthesis of the second DNA strand occurs. Positions within this region may be influenced and selected during antiviral treatment by using nucleoside/nucleotide analogs, such as lamivudine . The presence of HBV genotype-dependent variability in this therapeutic target region suggests the role of clinical treatment selection in the evolution of different HBV genotypes. In clinically relevant mutation screening analysis, no HBV antiviral drug resistance mutations were observed, which was consistent with a similar HBV genetic diversity study conducted in American blood donors . Drug-resistant HBV variants may be inefficient in transmission and/or establishment of a chronic infection or may be underrepresented in the pool of HBV that is actively transmitted by sexual or parenteral routes .
In the S region, 23 genotype-dependent positions were detected, and 9 of these positions were located in the major hydrophilic region. The small S protein is encoded by the S region and is a major component of the viral envelope that plays an important role in the host immune response. The major hydrophilic region encompasses aa101-aa160 of the S protein and is exposed on the surface to both virions and subviral particles. This region is highly immunogenic and is potentially under the selective pressure of the immune system . The presence of HBV genotype variability in this immune-dominant region suggests the role of immune selection in the evolution of different HBV genotypes. Deletion mutations in preS regions were much less prevalent (6.61%) compared to previously reported results in Chinese chronic HBV infections and HCC (20%) [18, 19]. This observation is consistent with these mutations leading to impaired virus particle secretion and thus being negatively selected during the transmission of HBV. According to a similar study of HBV genetic diversity in American blood donors, the prevalence of the well-known neutralization escape mutation G145R in the HBV envelope protein was as high as 22% . However, in contrast to those results, in our study, the G145R mutation was heavily underrepresented (2.07%), from which it can be inferred that the genotype B, C and D strains in Northwest China may have less likelihood of vaccine escape . Furthermore, compared to the G145R mutation, several mutations within the major hydrophilic region were detected at high prevalence, such as M134Y and S143T/L. It can be inferred that these mutations may have been strongly selected in long-term infected carriers, in whom a strong antibody response develops .
This study has several limitations. First, the analysis was restricted to HBsAg-positive infections as detected by current blood HBV-screening assays. Consequently, infections by highly divergent variants that would not be detected by these assays would not be identified. Second, cases with low HBV qualification were not selected for complete HBV genome amplification by PCR due to amplification and sequencing accuracy, and moreover, a moderate proportion of cases were not able to be characterized due to failure of complete genome amplification by PCR. Third, bulk PCR product sequencing was performed in this study and therefore may not have detected cases of dual infection, minor populations of drug resistance or immune escape variants represented in viral quasi-species.