STRUCTURE AND FUNCTION OF THE ADV GENOME

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The purpose of this project is to study various aspects of genome structure and function of the Aleutian mink disease parvovirus (ADV). A major effort is to determine which segments of the ADV genome govern pathogenicity and the ability of various virus isolates to replicate permissively in cell culture. Using a series of segmental exchanges between a full-length infectious molecular clone of the nonpathogenic ADV-G strain and a partial (15-88 map unit) clone of pathogenic ADVs, we have found that clones containing the 53-65 mu segment of pathogenic ADVs express viral antigen in cell culture but do not produce infectious virus in cell culture. This segment is part of the coding region for the capsid proteins and only 4 amino acid differences occur between ADV-G and the pathogenic ADVs; this segment does not contain the hypervariable region. Using a mutagenesis strategy based on the polymerase chain reaction we have mutated on e of these 4 amino acids and found that virus rescued from this clone retains the characteristics of the parent ADV-G. Several of the chimeric clones yielded ADV that could be rescued in cell culture and these viruses were assayed for the ability to induce disease in adult or newborn mink. No virus that replicated in cell culture caused any significant disease in either adult or newborn mink. We have continued to study the expression of ADV proteins in eukaryotic expression systems. We have found that synthesis of empty ADV particles was directed by a recombinant vaccinia virus that contained the coding sequences for both ADV capsid proteins (VV-IL1). These particles could be purified and banded at a density of ca. 1.33 g/ml in cesium chloride, identical to empty ADV particles produced in standard ADV infections. VV- IL1 was injected into mice; 8/8 mice produced detectable antibody as assayed by indirect immunofluorescence, but only 2/8 produced neutralizing antibody. Vaccination of adult mink with either VV-IL1 or WR vaccinia virus led to an accelerated from of ADV when the mink were challenged with a low dose of ADV.