Molecular evolution of hepatitis A virus in a human diploid cell line

Abstract

Aim: To investigate the hotspots, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells.

Methods: Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the full-length genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells.

Results: Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-1, other than its progenitor H2w.

Conclusion: Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.

Figure 1

Summary of hotspots and time course of mutations in the process of consecutive passage of HAV in human cell line KMB17. The numbers on the curve indicate total mutations occurring at every 5 passages. Text in the boxes describes hot mutations occurring at the stage of cell culture passage.

Figure 2

Phylogenetic analysis of the complete VP1 region using MEGA software package version 3.1. The numbers at nodes indicate bootstrap percentages after 1000 replications of bootstrap sampling. The bar indicates genetic distance.