Detection of inapparent Aleutian disease virus infection in mink.

The normal serum gamma-globulin centration of mink from the Ontario Veterinary College field station was 13.2 +/- 2.6% of total serum proteins. Mink serum gamma-globulin concentrations above 21%, which represented 3 standard deviations above the normal mean, were considered to be hypergammaglobulinemic. About 39% of pastel mink infected naturally with Aleutin disease virus (ADV) exhibited an inapparent or nonprogressive infection. These nonprogressivley infected mink had serum gamma-globulin values below 21% andhad antibody titers less than 256 if tested by the couterimmunoelectrophoresis technique. Mink maintained inapparent infection for at least 10 months after infection with ADV. Neither gross nor histopathologic changes were present in the mink with inapparent ADV infection. The virus persisted in blood, mesenteric lymph nodes, kidney, liver, and spleen of mink with non-progressive infection, although the amount of virus present probably was small.     

Characteristics of inapparent Aleutian disease virus infection in mink.

Inapparent of nonprogressive Aleutian disease virus (ADV) infection is a subclinical but persistent virus infection of mink. Mink with the inapparent type of ADV infection when subjected to stress did not develop the progessive form of the disease. However, when challenged with a large dose of the virus, these mink did develop progressive Aleutian disease indicating that they were not highly resistant to the virus. Sera of mink with either the progressive of the inapparent type of ADV infection did not neutralise the virus. The anti-ADV antibody activity in mink with inapparent type of ADV infection was in the IgG fraction of the serum the same as in mink with progressive Aleutian disease. These data indicate that the resistance of the mink with inapparent infection as compared to mink with progressive Aleutian disease was not due to a difference in the class of immunoglobulin response to the virus. However, mink with progressive Aleutian disease showed a greatly increased immunoglobulin response.     

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.     

Molecular epidemiology of Aleutian mink disease virus in Finland

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. To gain a more detailed view of the molecular epidemiology of mink AMDV in Finland, we phylogenetically analysed 14 new Finnish strains from 5 farms and all 40 strains with corresponding sequences available in GenBank. A part of the major non-structural (NS1) protein gene was amplified and analysed phylogenetically. A rooted nucleotide tree was constructed using the maximum parsimony method. The strains described in this study showed 86-100% nucleotide identity and were nearly identical on each farm. The ratio of synonymous to non-synonymous substitutions was approximately 2.7, indicating a mild purifying selection. Phylogenetic analysis confirmed that AMDV strains form three groups (I-III), all of which contained Finnish strains. The tree inferred that the three lineages of AMDV have been introduced to Finland independently. The analysis suggested that AMDV strains do not cluster into genotypes based on geographical origin, year of isolation or pathogenicity. Based on these data, the molecular clock is not applicable to AMDV, and within this gene area no recombination was detected.     

水貂阿留申病病毒分子生物学研究进展

水貂阿留申病病毒是一种在水貂中广泛存在的重要病原体.该病毒属阿留申病毒属,主要编码4种蛋白(结构蛋白VP1、VP2和非结构蛋白NS1、NS2).VP1蛋白在协助病毒产生感染性方面起着重要作用;VP2蛋白是该病毒的主要免疫原性抗原,能体外中和病毒;NS1和NS2对病毒在宿主细胞中的复制起重要的调节作用.该病毒的分子生物学诊断技术主要有核酸杂交技术、PCR和基因芯片检测技术.     

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.     

Pregnancy rate and foetal mortality in Aleutian disease virus infected mink

It is well known observation that farms contaminated with Aleutian Disease (AD) virus on an average have a lower breeding result than not infected farms. In the Danish eradication programme a farm may be registered as an A-farm, if a number of conditions are fullfilled. Among the conditions are yearly testings by countercurrent immuno-electrophoresis of all breeders or certain groups of these. The testing is carried out by the laboratory of the Danish Fur Breeder’s Association. Starting with the testing of 30.000 samples in 1976 the number of tests were 2.9 millions in 1986. The number of A-farms was more than 1,500 out of 4236 in 1986. The A-farms had in 1986 608,116 breeders with an average breeding result of 4.81 compared to the not tested farms that have an average of 4.37 kits per mated female. While the number of breeders has increased from 1.0 million in 1980 to 2.1 million in 1986, the percentage of infertile females has in the same period decreased from 14.7 to 10.9 with an average breeding result increasing from 4.09 to 4.69 (Anon. 1986).     

Protides of the Mustelidae: immunoresponse of mustelids to Aleutian mink disease virus.

Members of North American Mustelidae were tested for their response to inoculation with 10(6) infective doses of Aleutian disease virus. In subfamily Mustelinae, 3 species in the genus Mustela (M vision, M erminea, and M putorius) and 2 species in genus Martes (Ma pennanti and Ma americana) responded immunologically with some features resembling Aleutian disease in mink. In subfamily Mephitinae, only Mephitis mephitis responded, and others of the subfamily did not, nor did members of subfamilies Melinae and Lutrinae. The responses observed ranged from development of detectable antibody levels determined by counterimmunoelectrophoresis to histopathologic changes typical of Aleutian disease.     

Characterization of deoxyribonucleic acid from cells infected with Aleutian disease virus

Viral DNA was extracted from Crandell feline kidney (CRFK) cells infected with Aleutian disease virus (ADV) and labeled with [ 3H ]thymidine. The sedimentation coefficient in alkaline sucrose gradients was 16S corresponding to a molecular weight of 1.5 X 10(6). The buoyant densities of DNA from infected and control cells were determined by isopyknic sedimentation in CsCl and NaI gradients. Two additional peaks of [ 3H ]DNA were found in infected cells, but not in control cell extracts. Fractionation of this DNA on hydroxylapatite indicated that the new peaks represented a single-stranded component, density 1.728 g/cm3, and a double-stranded component, presumed to be a viral replicative intermediate, density 1.718 g/cm3. The target antigen formation in CRFK cells was measured by gamma-irradiation of ADV and assayed for focus formation. The calculated size of ADV based on these measurements was 1.1 X 10(6). The H-1 parvovirus also was shown to have a size of 1.5 X 10(6) daltons for both antigen and plaque formation. The data indicated similarities existed between ADV and other autonomously replicating parvoviruses in most properties, except that less-than-unit length genome of ADV may be transcribed.     

水貂阿留申病毒诱导猫肾细胞(CrFK)凋亡

为研究水貂阿留申病毒(AMDV)在体外诱导猫肾细胞(CrFK)凋亡情况,用AMDV-G株感染CrFK细胞,采用CCK-8法检测CrFK细胞感染后的细胞存活率,用AO/EB染色法检测CrFK细胞核的形态变化,用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率,并通过分光光度计法检测细胞凋亡执行分子Caspase-3活性。结果显示,AMDV-G株感染可导致CrFK细胞增殖率下降,产生明显的形态学凋亡特征;流式细胞术检测结果显示,AMDV-G株感染可引起CrFK细胞凋亡并随着感染时间的延长凋亡率增加,同时Caspase-3活性显著升高。上述结果表明,AMDV-G株在体外能够诱导CrFK细胞凋亡,为进一步研究AMDV致病机理奠定基础。     

貂阿留申病病毒结构蛋白VP1基因分子特征分析

目的研究貂阿留申病病毒(ADV)结构蛋白VP1基因分子空间结构特征,探讨貂阿留申病病毒致病机制。方法根据Genbank公布的全基因序列设计三对引物,PCR扩增并克隆到pMD-18T载体,阳性重组质粒鉴定、测序验证,拼接后进行生物信息学分析。结果获得了全长2064bpVP1基因,编码688个氨基酸;与细小病毒属中PPV-Nanjing200801相似性最大(51.2%);遗传进化树显示该基因编码蛋白与细小病毒属VP1亲缘关系最近。该蛋白是一种保守不含信号肽的外膜蛋白,具有7个潜在的N-糖基化位点和34个磷酸化位点。二级结构分析显示无规则卷曲含量最高,达68.75%,α螺旋、β折叠分别为16.42%和14.83%;同源建模比对,构建了具有较高合理性和可靠性的三维空间结构。结论预测抗原表位主要位于肽链第254~265、96~112、317~348、514~523、629~645位区段,可为今后开展基因工程疫苗研究奠定基础。     

Infection with Aleutian disease virus-like virus in a captive striped skunk

CASE DESCRIPTION: A 5-month-old captive female striped skunk (Mephitis mephitis) was evaluated because of lethargy, signs of depression, azotemia, and erythema of the skin around the eyes. CLINICAL FINDINGS: Antemortem diagnostic tests revealed renal disease but failed to identify an etiologic agent. A diagnosis of severe nonsuppurative interstitial nephritis was made on the basis of results of histologic examination of renal biopsy specimens. TREATMENT AND OUTCOME: The skunk was administered isotonic fluids SC daily and later every other day because of the handling-related stress. Because of the skunk's deteriorating condition, it was euthanized after 24 days of supportive care. Aleutian disease was diagnosed on the basis of positive results of a PCR assay that targeted the DNA from Aleutian disease virus (ADV); positive results for ADV were also obtained by use of plasma counterimmunoelectrophoresis and an ELISA. Genetic sequencing of the 365-base pair PCR product revealed 90% sequence identity with mink ADV. CLINICAL RELEVANCE: In the skunk of this report, infection with a skunk-specific parvovirus resulted in clinical signs and pathologic changes similar to those associated with ADV infection in mink. For skunks with signs of renal failure, differential diagnoses should include parvovirus infection. In confirmed cases of infection with this ADV-like virus, appropriate quarantine and biosecurity measures should be in place to prevent spread to other susceptible animals within a zoological collection.     

Monitoring chronic infection with a field strain of Aleutian mink disease virus

Aleutian mink disease virus (AMDV) readily spread within farmed mink and causes chronic infections with significant impacts for welfare and economy. In the present study a currently circulating Danish AMDV strain was used to induce chronic experimental infection of farmed mink.PCR was used to detect viral DNA in full blood, organs, faeces and oro-nasal swabs weekly for the first 8 weeks and then biweekly for another 16 weeks after AMDV challenge inoculation of wild type mink. The mink (n = 29) was infected and seroconverted 2–3 weeks after AMDV inoculation and AMDV antibodies persisted during the maximum experimental period of 24 weeks. Viraemia and faecal excretion of viral DNA was detected in the mink (n = 29) at various and intermittent time intervals. Excretion of viral DNA in oro-nasal swabs was detected for 1–8 weeks in 21 mink. This highlights the risk of transmitting AMDV between infected farms.PCR was successfully used to detect viral DNA in organs 8, 16 and 24 weeks after AMDV inoculation with only minor differences between these weeks which is of diagnostic interest.This AMDV challenge model was also used to mimic natural infection of susceptible sapphire mink. Four of 6 sapphire mink were infected indirectly via the AMDV inoculated wild type mink whereas the other 2 sapphire mink remained uninfected.     

Histopathologic and immunohistochemical lesions in liver of mink infected with Aleutian disease virus

Parvovirus of Aleutian disease causes mainly damage to kidneys, but immune complexes deposition and damage may occur also in other organs. In mink farms of Latvia the liver dystrophy or hepatic lipidosis of mink is widely distributed. The goal of this study was to examine probability of liver damage and regeneration of mink infected with Aleutian disease virus. Liver injury was assessed histologically. The mink liver demonstrated inflammation of liver parenchyma and foci of fatty liver. In immunohistochemistry, during liver regeneration the matrix metalloproteinases MMP-9, vascular endothelial growth factor and beta-defensin 2 expressions were lower, but MMP-2 and nerve growth factor receptor p75 expression was increased.     

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.     

水貂阿留申病毒串联表位重组抗原的表达及血清学初步评价

为探究水貂阿留申病毒(ADV)VP2主要抗原表位在水貂阿留申病血清学诊断中的作用,本试验将VP2蛋白主要抗原区的表位基因用柔性肽串联连接,将其克隆到表达载体pET-30a(+)中进行原核表达,并对诱导条件进行优化。利用Ni-NTA His-Bind纯化系统对融合蛋白进行纯化,经SDS-PAGE和Western blot鉴定表达产物,并将融合蛋白作为检测抗原进行CIEP试验。结果显示,融合蛋白在IPTG终浓度为1 mmol/L、37℃诱导表达4 h,蛋白表达量最高,其大小约为30 000,以可溶性表达形式为主;融合蛋白能与6×His单克隆抗体及ADV多克隆抗体发生特异性反应,说明该蛋白具有较好的反应原性;且该蛋白作为对流免疫电泳技术(CIEP)检测抗原与ADV阳性血清之间产生了明显的沉淀线,证实了该蛋白作为CIEP检测抗原的可能性。以纯化后的重组蛋白为抗原初步建立的间接ELISA方法具有较高的准确性。结果表明该蛋白具有良好的血清学检测价值,为水貂阿留申病血清学诊断方法的建立奠定了基础。     

Aleutian mink disease virus in furbearing mammals in Nova Scotia,Canada

Background

Aleutian mink disease virus (AMDV) is widespread among ranched and free-ranging American mink in Canada, but there is no information on its prevalence in other wild animal species. This paper describes the prevalence of AMDV of 12 furbearing species in Nova Scotia (NS), Canada.

Methods

Samples were collected from carcasses of 462 wild animals of 12 furbearing species, trapped in 10 NS counties between November 2009 and February 2011. Viral DNA was tested by PCR using two primer pairs, and anti-viral antibodies were tested by counterimmunoelectrophoresis (CIEP) on spleen homogenates.

Results

Positive PCR or CIEP samples were detected in 56 of 60 (93.3%) American mink, 43 of 61 (70.5%) short-tailed weasels, 2 of 8 (25.0%) striped skunks, 2 of 11 (18.2%) North American river otters, 9 of 85 (10.6%) raccoons, and 2 of 20 (10.0%) bobcats. Samples from six fishers, 24 coyotes, 25 red foxes, 58 beavers, 45 red-squirrels and 59 muskrats were negative. Antibodies to AMDV were detected by CIEP in 16 of 56 (28.6%) mink and one of the 8 skunks (12.5%). Thirteen of the mink were positive for PCR and CIEP, but three mink and one skunk were CIEP positive and PCR negative. Positive CIEP or PCR animals were present in all nine counties from which mink or weasel samples were collected.

Conclusions

The presence of AMDV in so many species across the province has important epidemiological ramifications and could pose a serious health problem for the captive mink, as well as for susceptible wildlife. The mechanism of virus transmission between wildlife and captive mink and the effects of AMDV exposure on the viability of the susceptible species deserve further investigation.