Volume 133, Issue 3, Pages 951-958 (September 2007) Viral Quasi-Species Evolution During Hepatitis Be Antigen Seroconversion  Seng Gee Lim, Yan Cheng, Stephane Guindon, Bee Leng Seet, Lay Yong Lee, Peizhen Hu, Shanthi Wasser, Frank Josef Peter, Theresa Tan, Matthew Goode, Allen Gerard Rodrigo  Gastroenterology  Volume 133, Issue 3, Pages 951-958 (September 2007) DOI: 10.1053/j.gastro.2007.06.011 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 ALT and HBV DNA levels in IFN-induced seroconverters (IRs) and IFN nonresponders (NRs). Serum ALT (in IU/L), a marker of liver cell damage, normalizes after HBeAg seroconversion, and HBV DNA (log10 copies/mL) during 5 time points showing >3 log reduction after HBeAg seroconversion. The group of IFN-induced seroconverters (IRs) is red and IFN nonresponders (NRs) is black. Empty squares and asterisks represent serum ALT. Filled squares and diamonds represent HBV DNA. The approximate time of HBeAg seroconversion (SC) is indicated by a vertical arrow. AP = .002 comparing time point I or III with time points IV and V. BP = .041 comparing time point I with the rest time points. CP = .0012 comparing time point II with time points III, IV, and V. DP < .0001 comparing seroconverters with nonseroconverters. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Viral genetic diversity (DNA) in the 4 phenotypic groups. Viral genetic diversity in seroconverters (SS and IR) is significantly higher than that of nonseroconverters (CC and NR). The higher viral genetic diversity before seroconversion (SC) accelerates even further after SC. Viral genetic diversity remains persistently low in nonseroconverters. Approximate time of HBeAg SC is indicated by a vertical arrow. EP < .0001 comparing time point I or III with time points IV and V in seroconverters. FP < .0001 comparing seroconverters with nonseroconverters. GP = .0032 comparing seroconverters with nonseroconverters. HP= .0451 comparing spontaneous seroconverters with IFN-seroconverters. IP = .0183 comparing seroconverters with nonseroconverters. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Viral genetic diversity (DNA) in precore/core sequence (nt 1839–2306) compared with control sequences. We determined the viral genetic diversity of the nonoverlapping region of the precore/core sequences (nt 1839–2306) compared with control sequences. Control 1 sequence is the sequence coding for the precore signal peptide which permits targeting of the precursor of HBeAg to the endoplasmic reticulum (nt 1814–1870), and control 2 sequence is the arginine-rich domain sequence of core which is required for HBV DNA binding to core (nt 2371–2452), which is also part of the overlapping region of core and polymerase gene. (A) Nonseroconverter controls; (B) spontaneous seroconverters. KP = .006 comparing nonoverlapping region with control region 1. LP = .0001 comparing nonoverlapping region with control region 2. MP = .003 comparing nonoverlapping region with control region 1. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Viral evolution rate (substitution rate) in the 4 phenotypic groups. The viral evolutionary rate is significantly higher in seroconverters (SS and IR) than in nonseroconverters (SS and IR). No significant alteration was observed in viral evolutionary rate over time within each group. JP < .0001 comparing seroconverters with nonseroconverters. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Representative sUPGMA phylogenetic trees of HBV DNA sequences for each clinical group showing complex trees in seroconverters but not in nonseroconverters. HBV precore/core sequences without overlapping region of HBx and polymerase from time points I (red filled circle), II (blue inverted triangle), III (green filled square), IV (purple triangle), and V (blue filled diamond) samples are phylogenetically analyzed, and their positions are displayed on the trees. Scale bar represents 0.005% genetic variation. Nonseroconverter patients (CC, NR) show similar simple branching patterns with shorter branch lengths than did the seroconverters. In contrast, seroconverter patients (IR, SS) were more complex, forming clusters over time with longer branch lengths. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Frequency of patients under positive selection in the 4 phenotypic groups. The start codon of the core amino acid sequence is counted as 1. The overlapping region with HBV X and P open reading frame at the 5′ and 3′ ends were removed for the analysis. There were 22 episodes of positive selection in seroconverters at 9 amino acid (aa) sites, with site aa 13 and 135 appearing to have the higher frequency of selection. Gastroenterology 2007 133, 951-958DOI: (10.1053/j.gastro.2007.06.011) Copyright © 2007 AGA Institute Terms and Conditions