Attenuated hepatitis A virus: genetic determinants of adaptation to growth in MRC-5 cells

A live candidate hepatitis A virus vaccine, developed from the HM-175 strain and adapted to growth in primary African green monkey kidney (AGMK) cells, was adapted to growth in MRC-5 cells. The nucleotide sequence of the MRC-5 cell-adapted virus was determined and compared with the known sequence of the AGMK cell-adapted virus. Thirteen unique mutations, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5 ' noncoding region (NC), and three of the remaining nine mutations encoded amino acid changes. Infectious chimeric cDNAs were constructed from infectious cDNA clones of the AGMK cell-adapted and wild-type HM-175 viruses and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. The viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotypes in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could be divided into three sets. Two of these were located in the 5 ' NC region, and the third was located in the 2C nonstructural gene. The mutations in the 5 ' NC region that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups in the 5 ' NC region and one in the 2C gene were required to increase growth dramatically in MRC-5 cells. Hepatitis A virus (HAV) continues to cause significan