Early on treatment detection of nucelos(t)ide analogue resistant minor variants of Hepatitis B virus (HBV) with ultra deep-pyrosequencing (UDPS) in chronic hepatitis B(CHB) patients

Introduction: UDPS allows the detection of minor viral variants during the natural course and antiviral treatment in HBV-infected patients. It could be shown that double infections with two HBV genotypes and minor strains carrying resistance mutations not detected by clonal sequencing could be analyzed. Here we tested a method for analyzing multiple HBV genotypes in a single sample as well as its potential to identify resistance mutations in patients months before viral breakthrough.

Patients and methods: A custom HBV sample was constructed by equimolar pooling of PCR amplicons derived from viral nucleic acid extracts from the serum of three different CHB patients. They had been identified as HBV genotypes B and D. Two samples were described as genotype D (one as wild-type, one as carrier of the resistance mutation rtM204V), and one sample as genotype B (wild-type). The genotype D wild-type developed lamivudine (LMV) and adefovir (ADV) resistance later on treatment. UDPS was performed on a GS Flx 454 sequencer (Roche). Sequencing data was analyzed using GenomiX Workbench (CLC Bio).

Results: Sequence analysis showed 37,07% of reads mapping to HBV genotype B (subtype adw), 61.99% to genotype D (subtypes w3 and ayw3) while the remaining 0,96% where scattered among other genotypes. Single nucleotide polymorphism (SNP) analysis of the sequences mapped to distinct genotypes clearly discriminated between wild-type and mutated HBV strains. For one patient, previously described as infected with wild-type HBV genotype D, a minor variant (coverage 35, frequency 100%) in the viral quasi species showed mutations at position rtM204 and rtI233, mediating viral resistance to LMV and ADV. Follow-up of this patient revealed a viral breakthrough with a major species resistant to LMV and ADV (rtM204V, rtI233V) 14 months later.

Conclusions: UDPS is a feasible method for the identification of HBV viral sequences including genotype and resistance mutations. The detection of minor variants carrying resistance-bearing mutations more than a year prior to viral breakthrough could constitute a new tool for individualizing antiviral treatment, especially with low genetic barrier nucleos(t)ide analogues. Whether the occurrence of minor quasi-species with resistance mutations could also be reflected by serological parameters like serum HBsAg is currently investigated.