Demonstration of Aleutian disease virus-specific lymphocyte response in mink with progressive Aleutian disease: comparison of sapphire and pastel mink infected with different virus strains

Lymphocyte blastogenesis was used to study the antiviral lymphocyte response of sapphire (Aleutian) and pastel (nonAleutian) mink inoculated with Pullman or Utah 1 Aleutian disease virus (ADV). Both mink genotypes developed a virus-specific response when inoculated with Utah 1 ADV. In contrast, after inoculation of Pullman ADV, sapphire mink had a positive virus-specific response, whereas pastel mink did not. Response occurred late after infection (8 wk) and correlated with the development of progressive Aleutian disease (AD). The response to keyhole limpet hemocyanin (KLH) and concanavalin A (Con A) was also determined. Most mink of either genotype, inoculated with either virus strain, maintained an anti-KLH response during disease. Most mink also responded to Con A, although some exhibited suppressed Con A response late in the disease course. These results indicated that mink develop an anti-ADV lymphocyte response during progressive AD and are not immunosuppressed with regard to other antigens or mitogens.     

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.     

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.     

Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink.

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.     

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.     

Reduced severity of lesions in mink infected transplacentally with Aleutian disease virus.

Inoculation of mink late in the second trimester of pregnancy with Aleutian disease virus (ADV) produces a persistent infection in the offspring. When these mink were analyzed at 83 days of age and compared with adolescent mink infected for a similar length of time, the transplacentally infected mink show: 1) a marked reduction in plasmacytosis, immunoglobulin level and specific ADV antibody; 2) increased amounts of infectious ADV and numbers of cells containing viral antigen; 3) a marked reduction in immune complex glomerulonephritis and absence of immune complex arteritis; 4) free ADV antigen in the glomeruli; and 5) a striking accumulation of eosinophils in the tissues. The findings suggest that the degree of ADV expression is partially immunologically controlled.     

Pathogenesis of Aleutian mink disease parvovirus infection: effects of suppression of antibody response on viral mRNA levels and on development of acute disease.

We suppressed the B-cell development and antibody response in mink by using treatment with polyclonal anti-immunoglobulin M (anti-IgM) to study the effects of antiviral antibodies on development of Aleutian mink disease parvovirus (ADV)-induced disease in more detail. Newborn mink kits were injected intraperitoneally with 1 mg of either anti-IgM or a control preparation three times a week for 30 to 34 days. At 21 days after birth, groups of mink kits were infected with the highly virulent United isolate of ADV. At selected time points, i.e., postinfection days 9, 13, 29, and 200, randomly chosen mink kits were sacrificed, and blood and tissues were collected for analyses. The efficacy of immunosuppressive treatment was monitored by electrophoretic techniques and flow cytometry. Effects of treatment on viral replication, on viral mRNA levels, and on development of acute or chronic disease were determined by histopathological, immunoelectrophoretic, and molecular hybridization techniques. Several interesting findings emerged from these studies. First, antiviral antibodies decreased ADV mRNA levels more than DNA replication. Second, suppression of B-cell development and antibody response in mink kits infected at 21 days of age resulted in production of viral inclusion bodies in alveolar type II cells. Some of these kits showed mild clinical signs of respiratory disease, and one kit died of respiratory distress; however, clinical signs were seen only after release of immunosuppression, suggesting that the production of antiviral antibodies, in combination with the massive amounts of free viral antigen present, somehow is involved in the induction of respiratory distress. It is suggested that the antiviral antibody response observed in mink older than approximately 14 days primarily, by a yet unknown mechanism, decreases ADV mRNA levels which, if severe enough, results in restricted levels of DNA replication and virion production. Furthermore, such a restricted ADV infection at low levels paves the way for a persistent infection leading to immunologically mediated disease. The potential mechanisms of antibody-mediated restriction of viral mRNA levels and mechanisms of disease induction are discussed.     

Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink.

The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV-seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed.     

Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines.

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.     

Immunoenzyme Western blotting analysis of antibody specificity in Aleutian disease of mink, a parvovirus infection.

Aleutian disease virus (ADV), an autonomous parvovirus, persistently infects mink and induces very high levels of virus-specific antibody. All strains of ADV infect all mink, but only highly virulent strains cause progressive disease in non-Aleutian mink. The development of antibody to individual ADV proteins was evaluated by Western blotting by using the sera of 22 uninfected mink and 163 naturally or experimentally infected mink. ADV has virion proteins of 86,000 and 78,000 daltons that are closely related. A new, possibly nonvirion protein of 143,000 daltons was observed, as well as a known nonvirion protein of 71,000 daltons. Sera from mink experimentally or naturally infected with ADV of high or low virulence generally reacted about equally with all four proteins. The only exceptions noted were that 8 of 15 sera of mink infected transplacentally preferentially reacted with the two virion proteins and sera from mink with the monoclonal gammopathy of Aleutian disease reacted preferentially with either virion (10 of 12) or nonvirion (2 of 12) proteins.     

Aleutian mink disease parvovirus in wild riparian carnivores in Spain

Serious declines in populations of native European mink (Mustela lutreola) have occurred in Europe. One responsible factor may be infectious diseases introduced by exotic American mink (Mustela vison). In order to investigate a possible role for Aleutian mink disease parvovirus (ADV), we surveyed native riparian carnivores and feral American mink. When serum samples from 12 free-ranging European and 16 feral American mink were tested, antibodies to ADV were detected from three of nine European mink. ADV DNA was detected by polymerase chain reaction in whole cell DNA from four of seven carcasses; two American mink, one European mink and a Eurasian otter (Lutra lutra). Lesions typical of Aleutian disease were present in one of the American mink. A portion of the ADV VP2 capsid gene was sequenced and the results suggested that two sequence types of ADV were circulating in Spain, and that the Spanish ADVs differed from other described isolates from North America and Europe. Future conservation and restoration efforts should include measures to avoid introduction or spread of ADV infection to native animals.     

Aleutian mink disease parvovirus infection of mink macrophages and human macrophage cell line U937: demonstration of antibody-dependent enhancement of infection.

Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I strain, but not the cell culture-adapted ADV-G strain, infects mink peritoneal macrophage cultures and the human macrophage cell line U937 in vitro. However, preincubation of ADV-G with ADV-infected mink serum enhanced its infectivity for U937 cells. the enhancing activity was present in the protein A-binding immunoglobulin G fraction in the serum, but F(ab')2 fragments failed to enhance the infection. On the other hand, the same sera inhibited ADV-G infection of Crandell feline kidney (CRFK) cells. Although U937 cells were not fully permissive for antibody-enhanced ADV-G infection, ADV mRNA expression, genome amplification, and protein expression were identical to those found previously for ADV-Utah I infection of U937 cells. Preincubation of ADV-Utah I with soluble protein A partly inhibited the infection of U937 cells but did not affect infection of CRFK cells. In mink peritoneal macrophages, preincubation with the infected mink serum did not make ADV-G infectious. However, the infectivity for mink macrophages of antibody-free ADV-Utah I prepared from the lungs of infected newborn mink kits was enhanced by ADV-infected mink serum. Moreover, protein A partly blocked ADV-Utah I infection of mink macrophage cultures. These results suggested that ADV-Utah I enters mink macrophages and U937 cells via an Fc receptor-mediated mechanism. This mechanism, antibody-dependent enhancement, may also contribute to ADV infection in vivo. Furthermore, since ADV infection in mink is characterized by overproduction of anti-ADV immunoglobulins, antibody-dependent enhancement may play a critical role in the establishment of persistent infection with ADV in vivo.     

The relationship between capsid protein (VP2) sequence and pathogenicity of Aleutian mink disease parvovirus (ADV): a possible role for raccoons in the transmission of ADV infections.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.     

Cytokine profiles in adult mink infected with Aleutian mink disease parvovirus

The aim of this study was to examine the levels of gamma interferon (IFN-gamma)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8(+)) cells in the blood during the infection. We quantified the percentages of IFN-gamma- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-gamma- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN-gamma and IL-4 in ADV-infected mink was produced by CD8(+) cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.     

Molecular comparisons of in vivo- and in vitro-derived strains of Aleutian disease of mink parvovirus.

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.     

Nucleotide sequence of the 5''-terminal palindrome of Aleutian mink disease parvovirus and construction of an infectious molecular clone.

The 5'-terminal palindrome of the ADV-G strain of Aleutian mink disease parvovirus (ADV) was molecularly cloned and sequenced. A full-length molecular clone of ADV-G, denoted pXVB, was then constructed. When this clone was transfected into cell cultures, infectious ADV could be rescued. Virus derived from pXVB was nonpathogenic for adult mink, as is the parent ADV-G strain.     

Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid

Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.