HEPATITIS A VIRUS: GROWTH CHARACTERISTICS, PURIFICATION, AND CAPSID GENE ORDER (PEPTIDES, IMMUNOREACTIVITIES, POLYPEPTIDES).
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PublisherThe University of Arizona.
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AbstractA human isolate of hepatitis A virus (HAV) strain HAS-15 was adapted to rapid growth FRhK-4 cells and a one-step growth curve was determined. Detectable virion production was absent for approximately 20 h post-infection (p.i.) and was followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10⁹ radioimmunofocus-forming units (RFU) per milliliter was achieved and remained essentially constant for a period of up to 14 days p.i. An adsorption study with HAV HAS-15 using FRhK-4 cells demonstrated greater than 99.9% of infectious virus adsorbed at 25 C in less than 20 min. Milligram amounts of purified HAV HAS-15 were obtained from persistently-infected RFhK-4 cells. The HAV polypeptides were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to nitocellulose for detection by an enzyme-linked immunotransfer blot (EITB) procedure. HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. EITB reactivities of HAV specific anti-peptide sera have allowed the identification of the gene order for the larger HAV P1 gene products and the determination of the following molecular weights: HAV VP2 or 1B (MW 27,000), HAV VP3 or 1C (MW 29,000), and HAV VP1 or 1D (MW 33,000). The disposition of the HAV capsid polypeptides with respect to the virion external surface was evaluated by EITB reactivity of HAV polypeptides with specific antisera. Hyperimmune rabbit anti-157S HAV and human IgM reacted with VP1, VP2, and VP3, while IgG reacted predominantly with VP1 and VP2. Further evaluation of the HAV virion structure was attempted by examining the relative accessibility of the virion polypeptides to various labeling reagents. Reaction of intact virions with Iodogen resulted in the predominant labeling of VP1 while labeling of VP2 and 3 was barely detectable. Selective labeling of VP1 under controlled conditions, combined with the anti-HAV IgG immunologic reactivity against VP1 and VP2, suggests that these two capsid components are more exposed on the virion surface and may play an important role in the generation of neutralizing antibodies.
Degree ProgramMolecular and Cellular Biology