Isolation Of Aleutian Mink Disease Virus, Establishment Of Diagnostic Methods For The Virus, And Study Of The Viral Pathogenicity

Aleutian disease(AD), caused by Aleutian Mink Disease virus(AMDV), is a persistent and progressive disease in mink. AD is distributed in all mink breeding countries, causing considerable economic losses to mink raising industry. The common method for AD control is the elimination of seropositive animals. Studies have shown that the seropositive rate in China was as high as 80%, study the properties of Chinese prevalent strains, including the viral genomic evolution, virus pathogenicity, and development of Chinese strain-specific diagnostic methods will be beneficial for the control of AD in China. This study includes the following sections:(1) Isolation of AMDV and viral genomic evolutionary analysisIn order to isolate AMDV, livers and spleens from serological positive minks(tested by counter immune electrophoresis, CIEP) in Heilongjiang province were collected. The organs were homogenized and inoculated on Crandell feline kidney(Cr FK) cells. Successful viral isolation was confirmed by electron microscope observation and virus-specific genome detection. The genome DNA of the viral strain(namely AMDV-HLJ) was extracted, each fragment of virus genome was amplified through polymerase chain reaction(PCR), partial NS1 and complete VP2 gene were obtained. Phylogenetic analysis showed that AMDV-HLJ was likely had the same evolutionary ancestors with LN1/LN2 strains which were also isolated in northeast China. It was further found that five amino acid sites within the two neutralizing epitopes in VP2 were mutated. These mutations, however, did not influence the antigenicity and pathogenicity of AMDV-HLJ.(2) Study on the pathogenicity of AMDV-HLJ.In order to study the pathogenicity of AMDV-HLJ in vivo, health minks were challenged with the virus. The clinical symptoms, gross lesions of necropsied animals, and histopathological damages were observed and recorded. Results indicated that AMDV-HLJ could cause severe or even fatal disease. Multi-organs damages were found after virus challenge. Histopathologically, organs of the nervous, respiratory, immune, digestive, urinary and reproductive systems showed classical virus-inducing pathological changes. Results also suggested that infection of AMDV could cause secondary bacterial infection. Results of in situ hybridization demonstrated that the viral genome DNA existed in not only mononuclear macrophages but also testicular spermatogenic, alveolar epithelial(type II) and Purkinje cells. These results suggested that AMDV could infect and replicate in different cells, thus may employ different mechanisms to induce damages in the host.(3) Establishment of CIEP to detect AMDV-specific antibodyDue to the advantages of simplicity, accuracy, high efficiency and cost effectiveness, CIEP is considered as the gold standard to screen the virus-infected animals. Nevertheless, the antigen for CIEP used in China is made from foreign viral strain, which showed relative low antigenic identity with local strains, thus may cause false negative results in CIEP. In this study, home-made CEIP antigen was produced by using the isolated AMDV strain, we also optimized the antigen preparation procedure to achieve the maximum antigen yield. It was found that the established CIEP based on the home-made antigen showed good specificity and sensitivity for virus antibody detection, thus has great potential as an alternative antibody detection method.(4) Development of loop-mediated isothermal amplification(LAMP) for the detection of AMDVWe further developed a simple, specific, rapid and cost effective LAMP method to detect AMDV genome. Results indicated that the six AMDV-specific primers amplified viral genome successfully in LAMP reaction. The amplification product achieved maximum yield at 65°C when the reaction incubated for 45 minutes. Analytical sensitivity test showed that the LAMP assay was 100 times more sensitive than conventional PCR. Importantly, results of LAMP could be visualized directly by naked eye, no specialized equipments and technical expertise are required, suggesting that LAMP is a promising nucleic acid ampli?cation technique for AMDV, especially under field conditions.The current study focused on the Chinese AMDV virus strain, results of this study could be of helpful for AD control in China.