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Objective To examine viral evolutionary changes and their relationship to hepatitis B e antigen (HBeAg) seroconversion. Design A matched case-control study of HBeAg seroconverters (n=8) and non-seroconverters (n=7) with adequate stored sera before seroconversion was performed. Nested PCR, cloning and sequencing of hepatitis B virus (HBV) precore/core gene was performed. Sequences were aligned using Clustal X2.0, followed by construction of phylogenetic trees using Pebble 1.0. Viral diversity, evolutionary rates and positive selection were then analysed. Results Baseline HBV quasispecies viral diversity was identical in seroconverters and non-seroconverters 10 years before seroconversion but started to increase approximately 3 years later. Concurrently, precore stop codon (PSC) mutations appeared. Some 2 years later, HBV-DNA declined, together with a dramatic reduction in HBeAg titres. Just before HBeAg seroconversion, seroconverters had HBV-DNA levels 2 log lower (p=0.008), HBeAg titres 310-fold smaller ( p=0.02), PSC mutations > 25% ( p<0.001), viral evolution 8.1-fold higher ( p=0.01) and viral diversity 2.9-fold higher (p<0.001), compared to non-seroconverters, with a 9.3- fold higher viral diversity than baseline ( p=0.011). Phylogenetic trees in seroconverters showed clustering of separate time points and longer branch lengths than non-seroconverters ( p=0.01). Positive selection was detected in five of eight seroconverters but none in nonseroconverters ( p=0.026). There was significant negative correlation between viral diversity (rs=-0.60, p<0.001) and HBV-DNA or HBeAg (rs=-0.58, p=0.006) levels; and positive correlation with PSC mutations (rs=0.38, p=0.009). Over time, the significant positive correlation was viral diversity (rs=0.65, p<0.001), while negative correlation was HBV-DNA (rs=-0.627, p<0.001) and HBeAg levels (rs=-0.512, p=0.015). Conclusions Cumulative viral evolutionary changes that precede HBeAg seroconversion provide insights into this event that may have implications for therapy. [ABSTRACT FROM AUTHOR]