Study On Isolation, Identification Of Aleutian Disease Virus And Construction Of Five Nucleic Acid Vaccines And Immunogenicity

Aleutian disease(AD), also called plasmacytosis, is a chronic viral infection of minks that causes autoimmune disorders gradually leading to immune failure. The pathogenic mechanism of AD led to prevent the disease become a worldwide problem. The vaccine of AD has not been developed until now, which are significant economic burdens on mink farming. Therefore, on the basis of isolation and identification of ADV, the whole genome sequencing of isolated strains were analysed. The whole gene nucleic acid vaccine was constructed by amino acid mutation of candidate strains. Based on the previous work of the lab, the ADV NS1 gene nucleic acid vaccine, ADV NS1 truncated gene nucleic acid vaccine, ADV VP2 gene nucleic acid vaccine and ADV VP2 truncated gene nucleic acid vaccine were truncated. The mouse and minks were immunized with five nucleic acid vaccine, the immunogenicity of five nucleic acid vaccine was analysised. The study would make a exploration for the Aleutian disease vaccine.In this study, the oxidation peroxidase monolayer assay was used to detect ADV isolates in the kidneys of dead minks with suspected ADV infection sampled from northern China. The five strains of ADV(ADV-DL124, ADV-DL125, ADV-JL3, ADV-QD2, ADV-QD3) were isolated, whose median tissue culture infective doses(TCID50) were 105.7 TCID50/mL, 105.0 TCID50/mL, 104.6 TCID50/mL, 105.2 TCID50/mL, and 104.1 TCID50/mL, respectively. According to the gene sequence published in GenBank of ADV, 12 pairs of primers were designed. The whole genome sequencing of five isolate virus was tested by 12 pairs of primers. The results revealed that the sequence lengths of the five strains were 4476 bp, 4538 bp, 4572 bp, 4569 bp and 4566 bp, respectively. The result of nucleotide homology between the five isolated AD strains and the European or American strains(ADV-G, ADV-U, ADV-SL3) showed that the homology between ADV-SL3 and ADV-QD3 was the lowest, which was 92.8% and the homology between ADV-U and ADV-JL3 was highest, which 96.8%.Based on the result of mink toxicity test, ADV-DL125 isolate was used in this experiment for its prominent symptoms and obvious visceral pathological changes. The nucleic acid vaccine pcDNA3.1-ADV was constructed by Overlap-extension PCR, and site-directed mutation of the amino acids sites 352, 395 and 534 was conducted. At the same time, according to the study on the immune effect of ADV virus truncated gene, the four nucleic acid vaccines were structured(pcDNA3.1-VP2, pcDNA3.1-VP2-D, pcDNA3.1-NS1 and pcDNA3.1-NS1-D). Five nucleic acid vaccines were tested by indirect immunofluorescence technique and Western-blot, and result showed that the nucleic acid vaccines had good reactionogenicity. The mice were immunized with recombinant plasmids, the changes of the mice at the level of humoral and cellular immunity were detected, and the result was showed that the recombinant plasmid could stimulate the body to produce humoral immune response and enhance the level of body cell immune response.The minks were immunized with five nucleic acid vaccine, or both with pcDNA3.1-NS1 nucleic acid vaccine and pcDNA3.1-VP2 nucleic acid vaccine, and were challenged with ADV。 The result showed that the minks were immunized with pcDNA3.1-ADV nucleic acid vaccine or pcDNA3.1-VP2+NS1 nucleic acid vaccine which could cause the body to produce ADV antibodies, and the content of CD8+ cells in peripheral blood lymphocytes was lower. Afer the virus challenge test, the minks immunized with pcDNA3.1-ADV nucleic acid vaccine or pcDNA3.1-VP2+NS1 nucleic acid vaccine which the content of gamma globulin and CIC in serum was lower compared with other group. The pcDNA3.1-ADV nucleic acid vaccine may be a good candidate for ADV vaccine.