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Cumulative viral evolutionary changes in chronic hepatitis B virus infection precedes hepatitis B e antigen seroconversion
  1. Yan Cheng1,
  2. Stephane Guindon2,
  3. Allen Rodrigo3,
  4. Lin Ying Wee2,
  5. Masafumi Inoue4,
  6. Alex J V Thompson5,
  7. Stephen Locarnini5,
  8. Seng Gee Lim1,6
  1. 1Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
  2. 2Department of Statistics, University of Auckland, Auckland, New Zealand
  3. 3Department of Biology, National Evolutionary Synthesis Center, Duke University, Durham, North Carolina, USA
  4. 4Experimental Therapeutic Center, Agency for Science, Technology and Research, Singapore
  5. 5Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, Monash University, Melbourne, Australia
  6. 6Department of Gastroenterology and Hepatology, National University Health System, Singapore, Singapore
  1. Correspondence to Professor Seng Gee Lim, Department of Gastroenterology and Hepatology, National University Health System, Tower Block level 10, 1E Kent Ridge Road, Singapore 119228, Singapore; mdclimsg{at}


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 non-seroconverters (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.

  • Hepatitis B

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