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 x 10(-3) substitutions/site) and substitution rate (2.2 x 10(-5) substitutions x site(-1) x 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.

Are in vitro hepatitis B core promoter mutations important for clinical alterations in viral load?

In vitro studies of HBV core promoter mutations in hepatoma cell lines suggest that some mutations in core promoter transcription factor binding sites result in reduced core promoter activity and viral replication. We sought to validate this hypothesis using clinical samples with viral load differences before and after HBeAg seroconversion. A consensus sequence for transcription factor binding sites/regulatory regions was constructed based on published studies. Serum from two time points in 33 seroconverters and 10 interferon non-responders (controls) were utilized. Genotyping, HBV DNA quantification and direct sequencing of core promoter were performed. There were 216 new mutations following HBeAg seroconversion but few in controls. Mutations or mismatches to consensus transcription factor/regulatory region sequences clustered at nucleotide positions appeared genotype-specific, non-group specific or baseline mismatches and were discounted as having significant impact on viral replication. Only a few mutations in three seroconverters (9.1%) were specific, while 39.4% had no new mutations that could be attributed to reduction in viral load following HBeAg seroconversion. In 51.5% of patients, mutations were of uncertain significance because they occurred in demonstrated non-critical clustered nucleotide positions. Core promoter mutations post-seroconversion did not correlate with in vitro induced mutations that reduced the promoter activity.