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

Abstract

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.

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine

“…The virus encoded by this cDNA has 21 nucleotide changes compared with the wild-type HM175 virus (8) (Fig. 2) and in cell culture (18) and in animals (5) has characteristics similar to those of the passage 32 virus (Fig. 1).…”

“…In addition, new MRC-5-specific mutations in the 2B-2C region and a cluster of mutations acquired in the 5Ј NC region during passage in AGMK cells (bases 124, 131 to 134, 152, and 203) ( Fig. 2) were found to increase the efficiency of growth in MRC-5 cells, but only when combined with the MRC-5-specific cluster of 5Ј NC mutations (18). Studies of HAV growth in fetal rhesus kidney 4 (FRhK-4) cells previously demonstrated that a few mutations which had a major impact on growth could be functionally replaced by a large number of mutations which individually had only a small impact (14).…”

“…1). Neither the plasmidderived virus nor the passage 32 virus grew efficiently in MRC-5 cells (17,18). Therefore, the AGMK-adapted virus was serially passaged in MRC-5 cells to adapt the virus to growth in these cells.…”

“…In the presence of the AGMK-specific mutations, a cluster of four mutations in the 5Ј noncoding (NC) region (bases 591, 646, 669, and 687) ( Fig. 2) was shown to increase greatly the efficiency of growth in the MRC-5 cells (18). In addition, new MRC-5-specific mutations in the 2B-2C region and a cluster of mutations acquired in the 5Ј NC region during passage in AGMK cells (bases 124, 131 to 134, 152, and 203) ( Fig.…”

Translation initiation of viral mRNAs

“…In common to PV, HAV RNA was shown to have acquired mutations in its IRES element that enhance replication by facilitating cap-independent translation in a celltype-specific fashion. Interestingly, passage of HAV in different cell types engendered different sets of mutations; however, all adaptive events were clustered within the IRES [139][140][141].…”