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.     

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.     

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.     

Mink with Aleutian disease have autoantibodies to some autoantigens

Thirty mink infected with Aleutian disease virus (ADV) were found to have elevated levels of antibody to double-stranded DNA (dsDNA) in their sera when compared to 30 healthy mink. The anti-dsDNA antibody levels in the diseased mink were, however, not found to correlate with the total amount of immunoglobulin. This was a common observation for all autoantibodies tested. The concentration of rheumatoid factors of IgG class, but not those of IgM class, was found to be significantly higher in the diseased mink at the chosen level of significance (P less than 0.01). IgG antibodies to thyroglobulin were likewise significantly higher in the ADV-infected mink. Unexpectedly, we found IgG antibodies with specificities for cardiolipin and mitochondrial antigens to be significantly higher in healthy mink than in ADV-infected mink. This difference is especially remarkable since the serum immunoglobulin concentration of the ADV-infected mink was three times higher than the serum immunoglobulin concentration of the normal mink.     

水貂阿留申病毒地方强毒株ADV-LN的分离鉴定

本研究采用对流免疫电泳方法(CIEP)检测辽宁水貂养殖场疑似水貂阿留申病毒(aleutian mink disease virus,ADV)水貂血清抗体,采集抗体阳性水貂的肝脏、脾脏、肾脏和肠系膜淋巴结组织,电镜观察存在细小病毒样颗粒。组织液研磨无菌处理后,接种CRFK细胞,盲传6代,取病毒细胞分离液用PCR方法检测,呈ADV阳性。将病毒分离液纯化后接种健康水貂,隔离观察,接种后3 d即出现食欲减退,贫血,被毛无光泽,后期出现拒食、狂饮、死亡,个别水貂出现神经症状,表现抽搐、痉挛、步态蹒跚、共济失调,证明分离获得的病毒为ADV强毒株,命名为ADV-LN株。     

Isolation and Identification of Local Aleutian Mink Virus Virulent Strain ADV-LN

Mink suspected infection aleutian mink disease virus (ADV) from mink breeding areas in Liaoning province were tested with CIEP method.The mink with antibody to ADV were selected and culled.Liver,spleen,kidney and mesenteric lymph node samples were taken for pathological examination and the viruses were observed under electron microscope.The grinded tissue fluid filter was added penicillin and treptomycin and inoculated into CRFK cells and passaged by 6 times for virus isolation.And cells cultures were identified as ADV by PCR.Then they were inoculated into healthy mink.Three days later,the mink showed clinical signs,which including the loss of appetite,anemia,hair dull,antifeedant and binge drinking.Some minks showed neurological symptoms,manifested symptoms of convulsions,cramps,staggering gait,ataxia,or hind limb paralysis and died.The virus strains isolated and identified were named as the ADV-LN.     

Detection of antibody in Aleutian disease of mink: comparison of enzyme-linked immunosorbent assay and counterimmunoelectrophoresis

Experiments were undertaken to investigate the potential of the enzyme-linked immunosorbent assay (ELISA) as a screening test for the diagnosis of the 2 known naturally occurring forms of Aleutian disease of mink. Anti-Aleutian disease virus (ADV) antibody activity was not detectable in the sera of mink with nonprogressive Aleutian disease despite the demonstration of antibody by counterimmunoelectrophoresis (CIEP) in the same sera. Anti-ADV antibody was detectable in 93% of sera from mink at various stages of experimentally induced progressive Aleutian disease. False-negative reactions occurred in sera which demonstrated high anti-ADV antibody titers by CIEP. As a consequence of the high prevalence of false-negative reactions, the ELISA was not considered to be an effective screening test. However, using CIEP as an indicator of ADV infection, the ELISA may be useful in differentiating mink with nonprogressive Aleutian disease from mink with progressive Aleutian disease.     

Effect of a valine residue at codon 352 of the VP2 capsid protein on in vivo replication and pathogenesis of Aleutian disease parvovirus in mink

OBJECTIVE: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10. ANIMALS: 32 eight-month-old female sapphire mink (Mustela vison). PROCEDURE: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy. RESULTS: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V. CONCLUSIONS AND CLINICAL RELEVANCE: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.     

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.     

水貂阿留申病毒结构蛋白与非结构蛋白的研究进展

水貂阿留申病毒(Aleutian mink disease virus,ADV)是一种主要侵染水貂的自主复制型细小病毒,是一种在水貂中广泛存在的重要病原体。病毒粒子的蛋白分为结构蛋白(VP1、VP2)和非结构蛋白(NS1、NS2)两类。VP1蛋白对病毒粒子产生感染性有重要作用;VP2蛋白是主要免疫功能区,能刺激机体产生中和抗体;NS1和NS2主要参与病毒的复制和基因的表达调节。文中对近年来国内外学者关于水貂阿留申病毒结构蛋白和非结构蛋白的研究情况进行归纳和总结。     

Mink infected with aleutian disease virus have an elevated level of CD8-positive T-lymphocytes

Lymphocytes, monocytes, granulocytes, B-lymphocytes and CD8-positive T-lymphocytes of non-infected mink and mink infected with Aleutian disease virus (ADV) were measured by flow cytometry. The gammaglobulin levels of the sera were also measured. Besides development of hypergammaglobulinaemia in the infected mink, the most pronounced finding was that the number of CD8-positive lymphocytes doubled on average during development of Aleutian disease, while the number of B-lymphocytes did not change dramatically. The enhanced CD8 frequency was still apparent 6 months after initial ADV infection of the mink. The present experiments contribute to a better understanding of the immune deficiency stage seen in mink infected with ADV.     

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.     

Persistent viral shedding during asymptomatic Aleutian mink disease parvoviral infection in a ferret.

A 2-year-old domestic ferret that appeared clinically healthy was repeatedly seropositive for Aleutian mink disease parvovirus (ADV) over a 2-year observation period. Antibody titers, determined by counter-immunoelectrophoresis, ranged from 1024 to 4096. Viral DNA also was identified in serum, urine, feces, and blood cell fractions by polymerase chain reaction analysis. Ultimately, DNA in situ hybridization revealed ADV DNA in histologic sections of various tissues and organs. These data indicate that this asymptomatic ferret was persistently infected with ADV.     

猪瘟兔化弱毒E2基因重组杆状病毒的构建及抗体制备

为制备猪瘟特异性抗体,将猪瘟兔化弱毒E2基因序列修饰后克隆到pFastBac1质粒载体,经转座、转染后构建重组杆状病毒,并用纯化的重组猪瘟E2蛋白免疫大耳白兔制备特异性抗体。实验结果表明成功获得能分泌重组猪瘟E2蛋白的杆状病毒,使用纯化的重组猪瘟E2蛋白制备的猪瘟抗体具有良好的特异性和敏感性,为猪瘟病毒荧光抗体染色液的制备奠定了基础。     

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.     

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.     

水貂阿留申病病毒CIEP抗原结合蛋白初探

以水貂阿留申病病毒对流免疫电泳(CIEP)细胞抗原为材料,经酶印迹(Westemblotting)测定,水貂阿留申病病毒CIEI细胞抗原与多克隆阳性血清反应,分子量为60000,50000和25000,而与CIEP阴性的抗水貂阿留申病病毒的单克隆抗体(Y—2—9)反应,分子量为60000,50000.因此初步确定水貂阿留申病病毒CIEP细胞抗原决定族位于分子25000蛋白上.     

Isolation of virus and antibody containing immune complexes from mink with Aleutian disease by affinity chromatography of equine complement clq

Affinity chromatography on immobilized equine complement Clq was used for the isolation of complement-binding immune complexes in sera of mink infected with Aleutian disease virus. Immune complexes were isolated and quantitated from 4 of 5 infected mink, as early as 2 weeks after infection and before hypergammaglobulinemia had appeared. The quantity of immunoglobulin G in these immune complexes ranged from 180 to 370 micrograms/ml serum. There were no Clq-binding immune complexes found in mink which were negative for Aleutian disease antibody. Using 125I-labeled BSA-anti-BSA complexes, we demonstrated that the affinity columns bound selectively immune complexes which had formed in antibody excess, whereas immune complexes in antigen excess were not bound. By neutralization of sensitized virus with anti-mink IgG serum, non Clq-binding immune complexes were also detected, which indicates that circulating immune complexes in persistently infected mink are heterogeneous as far as their reactivity with equine Clq is concerned.