PATHOGENESIS OF ALEUTIAN DISEASE VIRUS INFECTION

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Persistent infection of mink with Aleutian mink disease parvovirus (ADV) causes disturbances of immune regulation, including polyclonal hypergammaglobulinemia, plasmacytosis, immune complex disease, interstitial and glomerulonephritis. The spectrum of findings suggests a cytokine disorder. One focus of this project is to evaluate the role of cytokines in AD. Independent clones of several mink cytokines and two housekeeping genes have been developed. A non-radioactive RNAse protection assay (RPA) for these genes has also been developed. Transcription of ADV and another mink parvovirus, mink enteritis virus (MEV) was compared in synchronized CRFK cells. Pronounced differences in total transcript levels and relative ratios of the various mRNA species were noted. The overall rate of ADV transcription was approximately 1% that of MEV. The R3 transcript, coding for VPs, comprised 80% of MEV mRNA, but only 8% of ADV mRNA. R1, coding for NS1 was between 5 and 10% of the viral RNA for both MEV and ADV. R2 and R2' mRNA, coding for NS2, was about 10% for MEV, but 80% for ADV. Sequence analysis indicates that the ADV R2 could also code for VP as well as NS2, although it would have to initiate by leaky scanning using the P3 rather than the P36 promoter. ADV transcription in vivo was also examined by RPA. Transcripts were readily detected ten days after infection. The overall level of transcription is <1% of the in vitro situation, but the relative ratios of the various ADV mRNAs appeared, in preliminary experiments, to be the same in vivo as in vitro. In order to compare the subcellular localization of ADV proteins and nucleic acids in permissive and restricted infection, specific antisera for nonstructural (NS) and capsid proteins (VP) have been prepared and conjugated to fluorochromes. PCR has been used to make probes for fluorescence in situ hybridization (FISH). These reagents have been used in multi-channel confocal microscopy to characterize permissive ADV infection in CRFK cells. ADV DNA, NS and VP are found in specific intracellular locations that differ during the time course of infection. Furthermore, spatial segregation of replicative intermediates is evident. Preliminary microscopic analysis of mink lymph nodes suggests that the ratio of germinal center light zones to dark zones decreases following ADV infection. This is followed by involution of follicles, thinning of the perifollicular region and thickening of the medullary cords. Functional disruption of lymphoid structure may provide clues to the mechanism of ADV induced immune dysregulation, as it has in HIV infections.