Characterization and Clinical Significance of Natural Variability in Hepatitis B Virus Reverse Transcriptase in Treatment-Naive Chinese Patients by Sanger Sequencing and Next-Generation Sequencing

Abstract

Mutations in hepatitis B virus (HBV) reverse transcriptase (RT) are associated with nucleos(t)ide analogue (NA) resistance during long-term antiviral treatment. However, the characterization of mutations in HBV RT in untreated patients has not yet been well illustrated. The objective of this study was to investigate the characterization and clinical significance of natural variability in HBV RT in treatment-naive patients. HBV RT sequences were analyzed in 427 patients by Sanger sequencing and in 66 patients by next-generation sequencing. Primary or secondary NA resistance (NAr) mutations were not found, except A181T in RT (rtA181T) by Sanger sequencing, but they were detected by next-generation sequencing. Mutations were found in 56 RT amino acid (aa) sites by Sanger sequencing, 36 of which had mutations that could lead to changes in B or T cell epitopes in the RT or S protein. The distribution of mutations was diverse in different sections within the RT region. Multiple mutations showed significant association with HBV DNA, HBsAg, HBeAg, age, and severity of liver fibrosis. Mutations at rt251, rt266, rt274, rt280, rt283, rt284, and rt286 were found most in the advanced liver disease (ALD) group by next-generation sequencing. The present study demonstrates that next-generation sequencing (NGS) was more suitable than Sanger sequencing to monitor NAr mutations at a low rate in the treatment-naive patients, and that mutations in the RT region might be involved in the progression to ALD.

Mutational characterization of HBV reverse transcriptase gene and the genotype-phenotype correlation of antiviral resistance among Chinese chronic hepatitis B patients

“…Through Sanger sequencing, our study assessed the influence on the types or frequency of mutations by HBV genotype, and discovered 14 genotype-dependent nucleotide polymorphic sites and 5 genotype-dependent AA polymorphic sites (Figure 1, Table S1, Table S2). In these findings, rt221, rt224, rt238 were identified to be genotype-dependent sites, as coincided with the reported literature [14,20]. rt222, rt223 were newly identified in Fujian, China and rarely reported in any other areas of China, strongly suggesting that geographical distribution of HBV genotypes would also be a contributing factor in HBV genome sequence discrepancies.…”

“…This discrepancy exists between studies correlated with the geographical or ethnic background of patients as well as viral genotype, implying a high genetic variability of HBV between various epidemic viral strains in different regions. Moreover, it was still noted that the direct-sequencing as population-based sequencing approach, might be restricted to detect major quasispecies more than 20% of the entire HBV populations, thus possibly leading to the undetection of the strains that were minor but functionally critical [19,20]. To confirm these minor strains, a high throughput sequencing detection may need to be performed.…”

<p>Prevalence of Potential Resistance Related Variants Among Chinese Chronic Hepatitis B Patients Not Receiving Nucleos(T)ide Analogues</p>

“…Some of these positions had been reported previously, but others (rt82, rt214, and rt242) were not found in the corresponding genotype from previous studies. 5,15 The rt242 variant was only found in genotype B in a previous study 5 but was found only in genotype C in our study. Therefore, we speculated that different genotypes possessed different RT variants due to differences in evolutionary characteristics.…”

“…Additionally, another study reported that AA polymorphic positions showed discrepancies in different areas in China. 15 These data indicated that the definition of genotype variants needs to take into account the reference sequences of different genotypes in different areas and the consensus sequence derived from local HBV isolates. Moreover, researchers have also suggested that some AA polymorphic positions could contribute to decreased viral replication and affect drug treatment outcomes.…”

“…[6][7][8][9][10] Several previous studies have demonstrated that the classic DR variants also exist in chronic hepatitis B (CHB) patients not receiving NAs. [11][12][13] However, the results were quite discrepant among different areas and countries, 5,14,15 and the prevalence of putative resistant variants and pretreatment variants among NAs-treated and untreated CHB patients were not well defined.…”