cis-acting sequences in the Aleutian mink disease parvovirus late promoter important for transcription: comparison to the canine parvovirus and minute virus of mice.

We are currently investigating the regulation of transcription of the Aleutian mink disease parvovirus (ADV). ADV causes a chronic immune complex-mediated condition known as classical Aleutian disease, characterized by slow viral replication. This slow replication is an intrinsic property of ADV and distinguishes it from the more prototypic parvoviruses such as minute virus of mice (MVM) and canine parvovirus (CPV). We have previously suggested a role for the weak ADV promoters in the slow replication and thereby the absence of acute cytopathology and instead establishment of persistent ADV infection with progressive immune complex-mediated chronic lesions. In this study, we have mapped the cis-acting sequences around the ADV P36 promoter responsible for both constitutive transcription and transactivation mediated by the nonstructural protein 1. The mapping was performed by using endpoint deletions of the ADV P36 promoter and by making chimeras between the ADV P36 and MVM P38 promoters. We found the weak constitutive activity of the ADV P36 promoter to be caused by suboptimal promoter proximal sequences, while the low level of transactivation was caused mainly by an upstream region including sequences with homology to the transactivation responsive element (tar) of the H-1 parvovirus (M.-L. Gu, F.-X. Chen, and S. L. Rhode, Virology 187:10-17, 1992). We also found the corresponding regions in the MVM and CPV P38 promoters to be important for transactivation of these promoters by making 5' deletions of the promoter region. In addition, it was found that MVM tar-like and upstream sequences could transfer high nonstructural protein 1 responsiveness to the ADV promoter even though the distance between the tar-like element and the TATA box was significantly changed. On the basis of comparative data for ADV, MVM, CPV, and H-1, a new clustered motif (TTGGTT) is proposed to be the responsive cis-acting element for transactivation. Homology comparison of the specific transcriptional elements of the ADV P36, MVM P38, and CPV P38 promoters suggests that few, but crucial, changes in the ADV P36 promoter and upstream region are responsible for the weak constitutive activity and low level of transactivation of the ADV P36 promoter.

Identification of Aleutian mink disease parvovirus transcripts in macrophages of infected adult mink.

We examined Aleutian mink disease parvovirus (ADV) mRNA expression in lymph nodes of adult mink infected with ADV by Northern (RNA) blot and in situ hybridization. In Northern blot analysis, ADV transcripts were detected in the poly(A) RNA fraction extracted from mesenteric lymph nodes of two of five mink 10 days after intraperitoneal inoculation with the virulent Utah I strain of ADV. In strand-specific in situ hybridization, ADV DNA and mRNA were detected in some macrophagelike cells located in the medullary sinus in mesenteric lymph node sections from two of six infected mink by using biotinylated probes. In suspensions of lymph node cells, about 30% of the cells phagocytic for latex particles contained ADV DNA and about 1% of these cells contained ADV mRNA. In peritoneal exudate cells, about 20% of the macrophagelike cells contained ADV DNA and about 2% of these cells contained ADV mRNA. These results indicated that some macrophages in ADV-infected mink contained ADV mRNA and were target cells in ADV infection.

Nucleotide sequence analysis of Aleutian mink disease parvovirus shows that multiple virus types are present in infected mink.

Different isolates of Aleutian mink disease parvovirus (ADV) were cloned and nucleotide sequenced. Analysis of individual clones from two in vivo-derived isolates of high virulence indicated that more than one type of ADV DNA were present in each of these isolates. Analysis of several clones from two preparations of a cell culture-adapted isolate of low virulence showed the presence of only one type of ADV DNA. We also describe the nucleotide sequence from map units 44 to 88 of a new type of ADV DNA. The new type of ADV DNA is compared with the previously published ADV sequences, to which it shows 95% homology. These findings indicate that ADV, a single-stranded DNA virus, has a considerable degree of variability and that several virus types can be present simultaneously in an infected animal.

Virus-specific B-lymphocytes are probably the primary targets for Aleutian disease virus.

368 1- to 5-year-old mink of wild-type or black genetic background were infected with Aleutian disease virus (ADV) naturally or using virus-containing immune complexes or purified virus. Thirty of the mink were immunized with dinitrophenol-conjugated ovalbumin (DNP-OA) before and during infection. Blood samples were taken at monthly intervals. We found that weak (and transient) monoclonal or oligoclonal immunoglobulin components were present in the plasma or serum approximately 1 month after infection, as judged by zone electrophoresis. In a few cases, we found quite stable myeloma-like hypergammaglobulinemia, which usually occurs much later in the infection. All sera with monoclonal immunoglobulin components and most of the sera with immunoglobulins of restricted heterogeneity were analysed by crossed serum line immunoelectrophoresis. In all cases, the distinct immunoglobulins were found to have antibody activity to ADV proteins. In the few sera from DNP-OA-immunized mink showing restricted immunoglobulin heterogeneity, this was also the case. The findings from the study imply that ADV-specific B lymphocytes are probably the primary targets for ADV. The resulting ADV replication introduces a "pseudo-transformation" stage, so that the infected B lymphocytes proliferate and differentiate to an extreme degree. The mechanism behind this B-cell pseudotransformation ability of ADV is a puzzle. It may, however, be important, that the p75/85 structural polypeptides of ADV contain an amino acid sequence almost identical to the GTP-binding pocket of the Ras oncogene.

Violet mink develop an acute disease after experimental infection with Aleutian disease virus (ADV) isolate ADV SL3.

Six-Aleutian (aa)-genotype violet mink were infected intraperitoneally with the Aleutian Disease Virus (ADV) bone marrow derived isolate ADV SL3. All animals developed virus-specific antibodies and hypergammaglobulinaemia. Mortality during the fourteen week duration of the infection was 50%. The virus induced (histo)pathological lesions typical for Aleutian Disease. By immunohistochemical examination using a virus capsid-specific monoclonal antibody viral antigen was detected in lymph nodes, spleen, kidneys and once in hepatic Kupffer cells. By Southern blot and in situ hybridization studies with strand-specific RNA probes able to distinguish viral replicative forms from merely sequestered genomic DNA, ADV replication was detected in mesenteric lymph nodes and spleen. In one mink DNA replicative forms were also found in bone marrow cells or mononuclear cells of the peripheral blood, respectively. Only single-stranded viral DNA was detected in liver, kidney, gut and lung of infected animals. From Southern blot hybridization results a different, possibly organ-specific permissiveness of ADV in vivo is suggested.

Mechanisms contributing to the virus persistence in Aleutian disease.

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.

Detection of Aleutian disease virus DNA in tissues of naturally infected mink.

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.

Analysis of Aleutian disease of mink parvovirus infection using strand-specific hybridization probes.

A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.

Studies on the sequential development of acute interstitial pneumonia caused by Aleutian disease virus in mink kits.

We studied different parameters during the development of acute interstitial pneumonia in mink kits caused by neonatal infection with Aleutian disease virus (ADV). When histological lesions, presence of intranuclear inclusion bodies, and intranuclearly localized ADV antigen were correlated with levels of single-stranded virion and duplex replicative forms of ADV DNA in the different tissues, it was concluded that the lung, probably alveolar type II cells, is the major primary target for viral replication and cytopathology. The presence of the duplex dimeric replicative-form DNA, a strong marker of parvovirus replication, was also observed in low amount in the mesenteric lymph node, suggesting replication of ADV in this organ, although no viral cytopathology could be demonstrated. Moreover, a few intranuclear inclusion bodies were demonstrated in kidney and liver from affected kits, but intranuclearly localized ADV antigen could not be demonstrated in liver sections, and neither could duplex dimer replicative-form DNA, suggesting that these organs are nevertheless not a major site of ADV replication. When the data were compared with results previously reported for ADV-infected adult mink and ADV-infected permissive cell cultures, the data suggested that the pattern of ADV replication in alveolar type II cells is similar to that seen in infected cell cultures but that the replication in the other kit organs resembles the restricted pattern seen in adult mink.

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.

Demonstration of non-degraded Aleutian disease virus (ADV) proteins in lung tissue from experimentally infected mink kits. Brief report.

Isolates of ADV replicate to rather high quantities in lungs from neonatally infected mink kits. The virus was analysed for polypeptide composition, and for the first time high molecular weight polypeptides have been observed in in vivo produced ADVs. These polypeptides are analogous to those of in vitro produced ADVs. The molecular weights of the structural polypeptides of the low virulence Pullman ADV and the highly virulent DK and Utah I isolates of ADV were found to be 88kD and 78kD and in vivo produced ADV-G polypeptides were found to be 85kD and 75kD, the same molecular weights as those described for in vitro produced ADV-G. Presence of the ADV coded, non-structural polypeptide with the molecular weight of 71kD (p71) was also demonstrated in the lung tissue from mink kits.

Experimental transmission of Aleutian disease virus (ADV) to different animal species.

Two animals from each of 8 different species (mink, Finn raccoon, cat, dog, ferret, blue fox, mouse and rabbit) were inoculated with the highly virulent Utah I strain of ADV. Only the mink developed hypergammaglobulinemia. All animals produced antibodies to ADV antigens, but with different titres. Mink sera had much higher antibody titres than the other animal sera. Antibodies to the ADV-coded, non-structural polypeptide (p71) were found in mink, Finn raccoons and dogs only. Presence of this kind of antibodies was taken as evidence of ADV replication, because p71 was not present in the ADV inoculum. The animals were killed 4 weeks after virus inoculation. Homogenates of different organs from mink, Finn raccoons, ferrets, dogs, mice and the cat were shown to infect ADV-negative mink, which developed antibodies to ADV following inoculation. We conclude from the present studies that mink and Finn raccoons are potential threats as ADV transmitters. Cats, ferrets, dogs and mice may be considered potential ADV reservoirs, because they possibly harbour ADV for 4 weeks or longer. Blue foxes and rabbits did not seem to be a risk factor for ADV transmission.

Aleutian disease in mink: virology, immunology and pathogenesis.

Aleutian disease is an immunological disease of mink caused by a persistent virus infection. Recently Aleutian disease viral antigen has been extracted from tissue of mink early in the course of infection and the Aleutian disease virus has been isolated. The virus particles were similar in morphology and size to certain parvovirus having a 23 nm diameter, spherical shape and icosahedral capsid. Recent developments in serological techniques, i.e. immunofluorescence, complement fixation, countercurrent electrophoresis and immunodiffusion, have enabled rapid progress toward understanding the nature of the disease. It is suggested that the hypergammaglobulinemia is due to overproduction of IgG antibody specific for the Aleutian disease virus. The glomerulonephritis, vasculitis, positive antiglobulin test, systemic proliferation of lymphocytes and generalized plasmacytosis in Aleutian disease are believed to be the results of a continuing host antiviral immune response, the persistence of the virus in the presence of high level of specific antibody and the formation and deposition of the immune complexes.

Acute interstitial pneumonitis caused by Aleutian disease virus in mink kits.

In four Danish mink ranches acute interstitial pneumonitis caused excessive mortality among kits within the first 2 1/2 months after parturition. The disease was found to be due to an Aleutian disease virus (ADV) and could be reproduced experimentally in neonatal kits by inoculation with material from spontaneous cases, as well as with other strains of ADV. Experimental reproduction was only possible in kits from dams free of Aleutian disease (AD) whereas kits from dams experimentally or naturally infected with ADV developed no lung changes. Presently available evidence indicates that the initial lung lesions result from primary viral injury to type II alveolar cells, and that immune mechanisms, essential for the development of traditional AD, are not involved in the pathogenesis.

Aleutian disease virus, a parvovirus, is proteolytically degraded during in vivo infection in mink.

The polypeptides of the highly virulent mink-passaged Utah I and the nonvirulent cell culture-adapted ADV-G strain of Aleutian disease virus (ADV) were compared. When CRFK cells infected with either Utah I or ADV-G were analyzed by immunoprecipitation, both viruses induced proteins with molecular weights characteristic of the ADV-G 85,000 ( 85k )- and 75k-dalton structural proteins (p85 and p75) as well as the 71k -dalton nonvirion protein p71 . However, when Utah I, Pullman ADV, and DK ADV (a Danish isolate of ADV) were purified from infected mink, only polypeptides with molecular weights between 27k and 30k could be identified. In addition, trypsin treatment of ADV-G degraded p85 and p75 to smaller antigenic proteins with molecular weights of 24k and 27k, similar to those found for the virulent in vivo viruses. The effect of proteolytic treatment of ADV was then studied in detail. Purification of Utah I ADV from mink organs in the presence of protease inhibitor did not prevent the appearance of the low-molecular-weight proteins and ADV-G proteins were not degraded upon purification from a homogenate of normal mink organs, suggesting that artifactual proteolysis was not occurring. When a serum pool from terminally diseased mink was analyzed by radioimmunoassay for antibody reactivity against trypsinized and nontrypsinized ADV-G, five times higher reactivity was found for the trypsinized ADV-G than for the nontrypsinized ADV-G, an effect which could not be elicited by chymotrypsin or V8 protease treatment, implying that in vivo-produced ADV was being modulated in vivo by trypsin or a trypsin-like enzyme. Trypsinization was shown not to cause a change in ADV virion density, but to decrease the in vitro infectivity of ADV-G for CRFK cells. These studies suggested that during infection of mink ADV proteins are degraded to highly antigenic smaller polypeptides.

Royal pastel mink respond variously to inoculation with Aleutian disease virus of low virulence.

Information was sought on the varied responses of royal pastel mink (a non-Aleutian genotype) to Aleutian disease virus of low virulence. Thus, of 20 yearling female pastel mink inoculated subcutaneously with a large amount of the Pullman strain of Aleutian disease virus, only 3 succumbed to the disease. Of the other 17 mink, 3 had neither viremia nor a rise in level of serum gamma globulin during the 24 weeks after inoculation. The other 14 mink were viremic for variable periods during the first 12 weeks. In only five mink was the viremia accompanied by elevated levels of serum gamma globulin, usually from week 8 on. Of the 16 subclinically infected mink that did not succumb to intercurrent disease and otherwise remained healthy, 9 were examined at 19 to 31 months for persisting virus. In only one mink, small amounts were detected in the mesenteric lymph node and spleen nearly 28 months after inoculation. The other seven mink that survived the infection were not protected when challenged 31 months later with a small amount of the highly virulent Utah-1 strain. Even though still poorly understood, these varied responses of the royal pastel mink to infection with Aleutian disease virus of low virulence have important pathogenetic and epidemiological implications.

Immune complexes in Aleutian disease: demonstration of antibody on isolated virus.

The specific binding of Staphylococcal protein A for mammalian immunoglobulin G was used to demonstrate IgG associated with Aleutian disease virus (ADV) when isolated from infected mink tissues. Protein A specifically bound to mink serum Ig with no reaction with other serum or tissue proteins. Protein A labeled with 131Iodine reacted with crude virus preparations but not with virus that had been purified by freon extraction to the point where it became reactive with antibody by counterimmunoelectrophoresis. Binding to purified ADV was restored when the purified virus was first reacted with antibody. Results of urea treatment indicated this as an alternative method for isolation of ADV free from antibody.

Aleutian disease virus in B and T lymphocytes from blood and spleen and in bone marrow cells from naturally infected mink.

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.

Comparative pathogenicity of four strains of Aleutian disease virus for pastel and sapphire mink.

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.