Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

Abstract

Background and AimsAlthough the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity.MethodsTo characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases.ResultsMost genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA.ConclusionOur findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

“…Although it was difficult to detect a small population of variants in the host genome using a traditional approach such as PCR, NGS, owing to its depth, enabled us to detect a novel genomic variation (27). NGS has strongly supported the studies of viral genetic diversity, especially in RNA viruses (28,29), whereas the detection by NGS of quasispecies in DNA virus has been reported less frequently (30)(31)(32)(33). Using PCR analysis, the presence of VP1 quasispecies has been reported in polyomavirus BK (BKV) (34).…”

Accumulation of Somatic Mutations in TP53 in Gastric Epithelium With Helicobacter pylori Infection

“…Massively-parallel sequencing was performed as described previously 19,20 . Fragmented DNA (>5 µg) was used to prepare each DNA sequencing library.…”

Resistant mutations and quasispecies complexity of hepatitis B virus during telbivudine treatment

“…The heterogeneity of viral quasispecies was evaluated by a complexity metric based on the number of variants and their prevalence detected in the population. Quasispecies complexity at the aa and nt level was estimated for each site using S n according to the following formula: S n 52S i ( f i lnf i ), where f i represents the frequency of a particular variant in the quasispecies (Domingo et al, 2006;Nishijima et al, 2012). The mean viral complexity in each sample was determined by calculating the total S n at each position and dividing by the total length nt or aa number.…”

“…In the previous studies, the quasispecies were detected by direct PCR sequencing and the variants in complexity were defined based on the whole length of the reads (Chen et al, 2009;Liu et al, 2011). However, in our study, the complexity was measured by the mean Shannon entropy (S n ) at each nt or aa position following the methods reported previously (Nishijima et al, 2012); (2) the time point of sampling. The quasispecies may show different patterns of evolution at different stages of therapy; (3) different drugs used in the therapy.…”