No abstract

No abstract

A DNA sequence of 4,592 nucleotides (nt) was derived for the nonpathogenic ADV-G strain of Aleutian mink disease parvovirus (ADV). The 3'(left) end of the virion strand contained a 117-nt palindrome that could assume a Y-shaped configuration similar to, but less stable than, that of other parvoviruses. The sequence obtained for the 5' end was incomplete and did not contain the 5' (right) hairpin structure but ended just after a 25-nt A + T-rich direct repeat. Features of ADV genomic organization are (i) major left (622 amino acids) and right (702 amino acids) open reading frames (ORFs) in different translational frames of the plus-sense strand, (ii) two short mid-ORFs, (iii) eight potential promoter motifs (TATA boxes), including ones at 3 and 36 map units, and (iv) six potential polyadenylation sites, including three clustered near the termination of the right ORF. Although the overall homology to other parvoviruses is less than 50%, there are short conserved amino acid regions in both major ORFs. However, two regions in the right ORF allegedly conserved among the parvoviruses were not present in ADV. At the DNA level, ADV-G is 97.5% related to the pathogenic ADV-Utah 1. A total of 22 amino acid changes were found in the right ORF; changes were found in both hydrophilic and hydrophobic regions and generally did not affect the theoretical hydropathy. However, there is a short heterogeneous region at 64 to 65 map units in which 8 out of 11 residues have diverged; this hypervariable segment may be analogous to short amino acid regions in other parvoviruses that determine host range and pathogenicity. These findings suggested that this region may harbor some of the determinants responsible for the differences in pathogenicity of ADV-G and ADV-Utah 1.

Strand-specific hybridization probes were used in in situ molecular hybridization specifically to localize cells containing replicative intermediates of Aleutian disease of mink parvovirus (ADV). When adult mink of Aleutian genotype were infected with ADV Utah I, the largest number of cells positive for viral replication (i.e., containing replicative-form DNA and RNA) were found in the mesenteric lymph nodes and spleens at 10 days after infection. The localization of positive cells in the middle of germinal centers suggested that they were B lymphoblasts. Circulating leukocytes and bone marrow cells also contained viral RNA, but the levels of replicative-form DNA were below detectability. The levels of viral DNA and RNA in adult mink cells replicating ADV were decreased compared with those in permissively infected cell cultures or neonatal mink, suggesting that the replication of ADV in adult mink might be semipermissive or restricted at some early stage of viral gene expression. The low level of viral replication and transcription in lymphoid cells might provide a mechanism for the development of immune disorders and for the maintenance of persistent infection. Single-stranded virion DNA was found in other organs, but the strand-specific probes made it possible to show that this DNA represented virus sequestration. In addition, glomerular immune complexes containing virion DNA were detected, suggesting that ADV virions, or perhaps free DNA, may have a role in the development of ADV-induced glomerulonephritis.

We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8°C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 x 106. In addition, two major virusassociated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [3S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus. ADV antibodies as described previously (4). Serum gamma globulin levels were determined as described previously (4). Virus strains. An isolate of Utah-I ADV adapted to growth at 31.8°C in Crandall feline kidney (CRFK) cells by John Gorham, Washington State University, Pullman, was obtained from him at passage levels 7 and 5 in CRFK cells. This cell culture isolate of Utah-I ADV is designated ADV-Gorham (ADV-G) here. The previously described CRFK cell-adapted isolate of Utah-I ADV (30) was obtained from David Porter,