Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

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.     

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.     

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.     

Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins.

We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.     

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.     

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.     

Some molecular-biological and immunological aspects of Aleutian disease of mink

Information on the development of the infectious process in the Aleutian disease of minks (ADM) on the molecular level, are updated. In particular, the decisive role played by the ABM virus infection of the cells of the immune system (B lymphocytes, macrophages, follicular dendritic cells) and the possible involvement of the mechanism of the antibody-dependent aggravation of this infection are pointed out. In addition, the role of the weak ADM virus promoter p36, the immune status of minks and their age in the determination of the acute or "slow" character of the course of the disease are considered.     

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture.

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.     

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.     

High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.     

Mink farms predict Aleutian disease exposure in wild American mink

Background

Infectious diseases can often be of conservation importance for wildlife. Spillover, when infectious disease is transmitted from a reservoir population to sympatric wildlife, is a particular threat. American mink (Neovison vison) populations across Canada appear to be declining, but factors thus far explored have not fully explained this population trend. Recent research has shown, however, that domestic mink are escaping from mink farms and hybridizing with wild mink. Domestic mink may also be spreading Aleutian disease (AD), a highly pathogenic parvovirus prevalent in mink farms, to wild mink populations. AD could reduce fitness in wild mink by reducing both the productivity of adult females and survivorship of juveniles and adults.

Methods

To assess the seroprevalence and geographic distribution of AD infection in free-ranging mink in relation to the presence of mink farms, we conducted both a large-scale serological survey, across the province of Ontario, and a smaller-scale survey, at the interface between a mink farm and wild mink.

Conclusions/Significance

Antibodies to AD were detected in 29% of mink (60 of 208 mink sampled); however, seroprevalence was significantly higher in areas closer to mink farms than in areas farther from farms, at both large and small spatial scales. Our results indicate that mink farms act as sources of AD transmission to the wild. As such, it is likely that wild mink across North America may be experiencing increased exposure to AD, via disease transmission from mink farms, which may be affecting wild mink demographics across their range. In light of declining mink populations, high AD seroprevalence within some mink farms, and the large number of mink farms situated across North America, improved biosecurity measures on farms are warranted to prevent continued disease transmission at the interface between mink farms and wild mink populations.     

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.     

Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus.

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.