Viral Quasi-Species Evolution During Hepatitis Be Antigen Seroconversion

Background & Aims: Although viral quasi-species evolution may be related to pathogenesis of disease, little is known about this in hepatitis B virus (HBV); consequently, we aimed to evaluate the evolution of HBV quasi-species in patients with well-characterized clinical phenotypes of chronic hepatitis B. Methods: Four cohorts of well-defined clinical phenotypes of chronic hepatitis B, hepatitis Be antigen (HBeAg) seroconverters (spontaneous seroconverters and interferon-induced seroconverters) and nonseroconverters (controls and interferon nonresponders) were followed during 60 months on average. Serum from 4 to 5 time points was used for nested polymerase chain reaction, cloning, and sequencing of the precore/core gene (20 clones/sample). Only patients with genotype B were used. Sequences were aligned using Clustal X, then serial-sample unweighted pair grouping method with arithmetic means phylogenetic trees were constructed using Pebble 1.0 after which maximum likelihood estimates of pairwise distances under a GTR + I + G model was assessed. Viral diversity and substitution rates were then estimated. Results: Analysis of 3386 sequences showed that HBeAg seroconverters had 2.4-fold higher preseroconversion viral sequence diversity (P = .0183), and 10-fold higher substitution rate (P < .0001) than did nonseroconverters, who had persistently low viral diversity (3.6 × 10^-3 substitutions/site) and substitution rate (2.2 × 10^-5 substitutions · site^-1 · month^-1). After seroconversion, there was a striking increase in viral diversity. Most seroconverters had viral variants that showed evidence of positive selection, which was seen mainly after seroconversion. 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 to increased immune reactivity that drives escape mutations.