Although hepatitis A virus (HAV) is a common cause of acute hepatitis in the United States, no vaccine is available for prevention of this infection. While virus adapted to growth in cell culture has reduced virulence, such virus is incompletely attenuated for man. However, because cell culture-adapted HAV is at least partially attenuated, characterization of mutations related to cell culture adaptation would form a basis for understanding HAV attenuation at the molecular level. Inasmuch as cDNA from wild-type, HM-175 strain HAV has been cloned and extensively sequenced, cDNA made from a cell-culture adapted HM-175 variant would be cloned into the plasmid vector pBR322. A cDNA sub-clone library would be constructed in the single-stranded M13mp8/9 vector and the nucleotide sequence of the cDNA determined using a shotgun approach. This sequence would be compared with that established for wild-type HM-175 virus to determine the nature of mutations accompanying adaptation to cell culture. To establish the biologic significance of mutations identified in this virus variant, a second, independently isolated, cell culture-adapted variant of the same HAV strain would be examined for consonant changes. This would be accomplished by primer-extension of virion RNA or by cloning and sequencing of cDNA. Simultaneously, construction of plasmids containing complete copies of wild-type or cell culture-adapted HAV cDNA would be attempted and their infectivity determined by DNA transfection in a modified radioimmunofocus assay. Genomic regions involved in cell culture adaptation would then be precisely mapped by transfection with recombinant plasmids containing wild-type and cell culture-adapted cDNA sequences, using rapid growth in BS-C-1 cells as a positive selection factor. These experiments should result in recombinant virus variants with defined mutations that could be further characterized for attenuation in non-human primate models of hepatitis A (animal experiments not proposed as part of this study), and should permit the eventual development of new virus variants with more complete or more stable attenuation characteristics.
