The Role of HBV Viral Quasispecies in HBeAg Seroconversion

Seng-Gee Lim*. National University Health System, Singapore

Hepatitis B virus (HBV) viral quasispecies may have important roles in pathogenicity similar to quasispecies in hepatitis C and HIV. We had previously characterized the evolution of HBV quasispecies in HBeAg seroconverters compared to non-seroconverters. We examined cohorts of well defined clinical phenotypes of chronic hepatitis B, HBeAg seroconverters and non-seroconverters with and without interferon therapy who were followed over 60 months. Serum was used for nested PCR, cloning and sequencing of the precore/core gene (20 clones/sample). Only patients with genotype B were used. Sequences were aligned using ClustalX, then sUPGMA phylogenetic trees were constructed using Pebble 1.0 following which maximum likelihood estimates of pairwise distances under a GTR+I+G model was assessed. Viral diversity and substitution rates were then estimated. Results of analysis of 3386 sequences showed that HBeAg seroconverters had 2.4-fold higher pre-seroconversion viral sequence diversity (p=0.0183), and 10-fold higher substitution rate (p<0.0001) than non-seroconverters, who had persistently low viral diversity (3.6×10^-3 substitutions/site) and substitution rate (2.2×10^-5 substitutions/site/month). Following seroconversion, there was a striking increase in viral diversity. Most seroconverters had viral variants that showed evidence of positive selection, which was seen mainly postseroconversion. Conclusions: The high viral diversity before a reduction in HBV DNA and before HBeAg seroconversion, could either be related to occurrence of stochastic mutations that lead to a break in immune tolerance, or increased immune reactivity that drives escape mutations. In a follow-up study, we examined the long term evolution of HBV quasispecies and whether the increase in viral diversity could be confirmed.

Furthermore, we sought to determine if this increase in viral diversity could result in creation of new CpG motifs, which could stimulate Toll Receptor 9. TLR9 is one member of a family of highly conserved molecular pattern recognition receptors-toll like receptors, and is stimulated by unmethylated CpG dinucleotides resulting in activation of the innate immune response and consequently adaptive immune response. In this study, we analyzed if there were any CpG sites in the HBV genome of chronic hepatitis B patients and their relationship with HBeAg seroconversion, using Eight HBeAg seroconverters followed up 6–10 years before seroconversion were included. Serum samples of 5 to 9 time points before HBeAg seroconversion were used in the study. Seven non-seroconverters with matching follow up period and stored serum samples were involved as controls. An identical methodology was used to evaluate 20 clones per serum sample and analysed using the same methodology as previously. HBV CpGs status was analyzed with the sequence alignments.

Results: The HBV quasispecies viral diversity gradually increased from 1.4×10^-3 to 1.2×10^-2 within 9 years before HBeAg seroconversion. Positive selection was detected in 5 of 8 seroconverters in the study period. New CpG motifs also emerged before HBeAg seroconversion. The increase of viral diversity was closely correlated with the reduction of HBV DNA, HBeAg levels and increase in frequency of precore stop codon mutation.