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The present study addressed the causal role of Aleutian mink disease parvovirus (ADV) in acute interstitial pneumonia in mink kits. All the examined isolates of ADV caused interstitial pneumonia in newborn kits, although the severity of disease and the mortality varied. These findings indicate that ADV is the direct causal agent of this disease in mink kits and that cofactors, which could have been present in the original ADV-K isolate, do not play a role. Acute interstitial pneumonia characterized by hypertrophy and hyperplasia of alveolar type II cells, intranuclear viral inclusions, interstitial edema, and hyaline membrane formation was experimentally reproduced in mink kits infected as newborns with five different isolates of ADV. Four hundred forty-nine newborn mink kits were included in the study, of which 247 were necropsied. The lesions caused by the different isolates were indistinguishable by histopathologic examination, but the incidence (50-100%) and severity (mortality of 30-100%, n = 218) of disease among the mink kits varied. Also, the content of ADV antigens in the lungs of infected kits varied among the groups. According to these features, the examined isolates could be placed in groups of high and low virulence. ADV-K, ADV-Utah I, and ADV-DK were in a highly virulent group producing a mortality of 90-100% (n = 110) in mink inoculated as newborns. ADV-GL and ADV-Pullman belonged to a group of low virulence, with an incidence of clinical disease of 50-70% and a mortality of approximately 30-50% (n = 118) in kits inoculated as newborns. The mortality in the control group receiving a mock inoculum was around 12% (n = 34). The period from infection to development of fatal disease varied from approximately 12 days for the highly virulent isolates up to around 20 days for the isolates of low virulence. The 107 mink kits that survived inoculation with ADV as newborns developed lesions typical of classical Aleutian disease irrespective of the ADV isolate used. The lesions consisted of chronic immune complex-mediated glomerulonephritis and infiltrations with mononuclear cells, including plasma cells in lung, liver, spleen, kidney, mesenteric lymph node, and intestine. Surviving kits also had hypertrophy of the bronchus-associated lymphoid tissue and focal subpleural, intraalveolar accumulations of large cells with foamy cytoplasm, so-called lipid pneumonia.

Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages.

A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.

By using strand-specific in situ hybridization and immunohistochemistry, evidence for replication of the Aleutian mink disease parvovirus was observed in cells resembling macrophages and cells resembling follicular dendritic cells at 10 days after infection but only in macrophages at 60 days. Sequestration of the Aleutian mink disease parvovirus in larger numbers of macrophages and follicular dendritic cells was noted at both 10 and 60 days.

DNA from one cell culture-adapted and two pathogenic strains of Aleutian disease of mink parvovirus (ADV) was molecularly cloned into the vectors pUC18 and pUC19. The DNA from the two pathogenic strains (ADV-Utah I and ADV-Pullman) was obtained from virus purified directly from the organs of infected mink, whereas the DNA from the nonpathogenic ADV-G was derived from cell culture material. The cloned segment from all three viruses represented a 3.55-kilobase-pair BamHI (15 map units) to HindIII (88 map units) fragment. Detailed physical mapping studies indicated that all three viruses shared 29 of 46 restriction endonuclease recognition sites but that 6 sites unique to the pathogenic strains and 5 sites unique to ADV-G were clustered in the portion of the genome expected to code for structural proteins. Clones from all three viruses directed the synthesis of two ADV-specific polypeptides with molecular weights of approximately 57 and 34 kilodaltons. Both species reacted with sera from infected mink as well as with a monoclonal antibody specific for ADV structural proteins. Because production of these ADV antigens was detected in both pUC18 and pUC19 and was not influenced by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, their expression was not regulated by the lac promoter of the pUC vector, but presumably by promoterlike sequences found within the ADV DNA. The proteins specified by the clones of ADV-G were 2 to 3 kilodaltons smaller than those of the two pathogenic strains, although the DNA segments were identical in size. This difference in protein molecular weights may correlate with pathogenicity, because capsid proteins of pathogenic and nonpathogenic strains of ADV exhibit a similar difference.

Organs of naturally infected mink were examined for the presence of Aleutian disease virus (ADV) DNA by in situ hybridization. Spleen, lymph nodes, thymus, bone marrow, kidney, liver, lung and small intestine were found to be positive for ADV to differing extents. Infected lymphoid organs showed a focal distribution of positive cells. Southern blot analysis of DNA extracted from infected organs revealed replicative forms of viral DNA in spleen and bone marrow samples only. These findings are consistent with a lymphotropism of ADV in vivo. Compared to the situation after experimental infection of mink these results indicate additional sites of virus replication and/or persistence of the naturally occurring disease.

In the nuclei of 4% of peripheral blood or spleen mononuclear cells (MNC), Aleutian disease virus(ADV)-specific antigens were found by a direct immunofluorescence test. The MNC were further fractionated by nylon wool, affinity chromatography using Staphylococcus aureus protein, or Percoll gradient techniques. ADV and specific antigens were detected in MNC fractions enriched in either the B or T lymphocytes. In the bone marrow, up to 40% antigen-positive cells were demonstrated over a period of 15 months. These findings were confirmed by the detection of infectious virus in the MNC of blood and spleen and in bone marrow cells. Adherent cells from mink and control cells from ADV-negative ferrets were negative in both tests. These findings indicate that ADV exhibits a lymphotropism and can persist in the B- and T-cell fractions from ADV-infected mink over a long period of time. Furthermore, co-cultivation of mink MNC and bone marrow cells with the CCC clone 81 cells was shown to be reproducible method for the detection of ADV in persistently infected mink.

A rocket line immunoelectrophoretic assay (RLIE) was developed for the simultaneous quantification of viral antigens and antiviral antibodies of the important mink parvovirus, Aleutian disease virus (ADV). The sensitivity of the RLIE assay was found to be 5 log2 higher than that of the counter current immunoelectrophoresis which is the assay routinely used for diagnostic purposes.

Virus was isolated from infected mink organs by a combination of tissue homogenization, fluorocarbon extraction and ultracentrifugation. The final preparation was analysed by crossed immunoelectrophoresis and electronmicroscopy. Virions had a capsid diameter of 22 nm. Preparative agarose electrophoresis separated virions from contaminating ferritin. Crossed immunoelectrophoresis of virus gave a single precipitate with sera from infected mink. Crossed immunoelectrophoretic analysis with intermediate gels showed that a part of the virus preparation was complexed with antibody. Serum from a certain mink was found to contain precipitating antibody to (poly)nucleotid. Virus and virus-antibody complexes were found to focus at pH 4.0-4.4 in isoelectric focusing. In SDS-polyacrylamide gel-electrophoresis the main virus protein was found to have a molecular weight of 69000. This study gives further support to the classification of aleutian disease virus as a parvovirus.

Aleutian disease virus, the causative agent of a persistent infection in mink, was isolated in a continuous line of feline renal cells when the cultures were maintained at reduced temperature (31.8 degrees). After serial in vitro passage of the virus at this temperature it had an optimum replication temperature of 37 degrees. An immunofluorescence focus assay was found to be suitable for virus quantitation. The cultured virus reproduced Aleutian disease in mink, and the virus could be reisolated from the mink 10--180 days after inoculation. The properties of the virus suggest that it is a member of the parvovirus group.

Information was sought on the comparative pathogenicity of four North American strains (isolates) of Aleutian disease virus for royal pastel (a non-Aleutian genotype) and sapphire (an Aleutian genotype) mink. The four strains (Utah-1, Ontario [Canada], Montana, and Pullman [Washington]), all of mink origin, were inoculated intraperitoneally and intranasally in serial 10-fold dilutions. As indicated by the appearance of specific antibody (counterimmunoelectrophoresis test), all strains readily infected both color phases of mink, and all strains were equally pathogenic for sapphire mink. Not all strains, however, regularly caused Aleutian disease in pastel mink. Infection of pastel mink with the Utah-1 strain invariably led to fatal disease. Infection with the Ontario strain caused fatal disease nearly as often. The Pullman strain, by contrast, almost never caused disease in infected pastel mink. The pathogenicity of the Montana strain for this color phase was between these extremes. These findings emphasize the need to distinguish between infection and disease when mink are exposed to Aleutian disease virus. The distinction has important implications for understanding the natural history of Aleutian disease virus infection in ranch mink.

Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.