Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain.

The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures. Currently, the mechanism underlying its attenuation phenotype remain largely unknown. In this study, we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques. The recovered H2 strain (H2ic) from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production, similar to the parental H2 strain. Additionally, H2ic did not cause disease in Ifnar1^-/- C57 mice, consistent with the H2 strain. To explore the cell-adaptive mutations of the H2 strain, chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone. The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic. Other chimeric viruses containing the 2B, 2C, or 3A proteins from H2w failed to be recovered. Furthermore, there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses. These results demonstrate that adaptive mutations in the 2B, 2C, and 3A proteins are essential for efficient replication of the H2 strain in cell cultures. Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice. Together, this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.