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 (A GMK) cells, was adapted to growth in MRC-5 cells. The nucleotide seque nce of the MRC-5 cell-adapted virus was determined and compared with t he known sequence of the AGMK cell-adapted virus. Thirteen unique muta tions, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5' nonco ding region (NC), and three of the remaining nine mutations encoded am ino acid changes. Infectious chimeric cDNAs were constructed from infe ctious cDNA clones of the AGMK cell-adapted and wild-type HM-175 virus es and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. Th e viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotyp es in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could he divided into three s ets. 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 regio n that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups i n the 5' NC region and one in the 2C gene were required to increase gr owth dramatically in MRC-5 cells.