Evolution Of Canine Parainfluenza Virus And Experimental Study On CPIV DNA Vaccine And Recombinant Canine Adenovirus Type-2 Vaccine

Canine infectious respiratory disease(CIRD), as it was referred to the respiratory illness kennel cough in dogs, The main clinical symptoms included of fever, cough and nose running , which were caused by virus and bacterium,and CPIV was main cause of Canine Infectious Respiratory Disease. Investigation indicate CIRD was one of the main infectious diseases from dogs and was prevalent through world-wide. In order to investigating epidemiological situation of CPIV in our country, hemagglutination inhibition(HI) test and serum neutralizing antibody test were used to research epidemiology of CPIV at some areas of northeast China. The result showed that the positive percentage was 35～52.83%. Fox and raccoon dog from domestic animals were investigated and found that positive rate of CPIV was higher , positive percentage was 34.29～47.83% from fox and 30.95～53.70% from raccoon dog. RT-PCR was used to detected samples collected and 1 were positive from 20.Primers were designed and synthesized to amplify H,F and N gene of CPIV, according to the gene sequence of SV5 from GeneBank. H, F and N gene were subcloned in pMD18-T vector, constructed pMD18-H, pMD18-F and pMD-N vector and sequenced respectively. N of CPIV was compared with N gene sequence of SV5, sequence homology was 99.5% , and were 98.7% and 98.6% with T65, but showed far genetic relationship with Human parainfluenza virus 1, 3, 4 type. H gene nucleotide sequence of FA and FC also shared high sequencing-homology with H (GeneBank NO.BD280413) and showed 99.8% and 99.9%, and were 98.6% and 98.5% with H of W3 isolate strain. F of CPIV was compared with the published sequence for 20 isolates from GenBank, Results indicated that the nucleotides homology of sequence was the highest with F gene of SER from porcine , was 99.8%, and 99.5% and 99.6% with F of BD280413 and AX067835, and belonged to same branch.. F gene of FA and FC was compared with 78524,MIL,CPI+,CPI-,DEN,H221,LN,MEL ,with nucleotide sequence homology range from 97.8% to 98.3% , nucleotide variation range from 0.02% to 2.2%. However, three genes showed lower nucleotide sequencing homology with human and bovine of SV5 and evolution tree of F gene of CPIV indicated F gene was relative conservation. The F protein analysis of CPIV FA and FC established that two strains were extremely similar to porcine SER , there did appear amino acid changes at 160, 457 of FA , two amino acid changes at 22, 370 of FC when compared with SER F gene. It was suggested that FA, FC and SER belonged to same branch. It was also found that there was 9 amino acid changes when compared FA strain with CPIV F from Xinjing strain , 10 differences with 78524,H221 strains, 16 amino acid changes with CPI+,CPI-, and with amino acid variation range from 0.36% to 2.9%. The results demonstrated conservation of CPIV F gene, and F gene can be researched on recombinant vaccine and DNA vaccine as a target geneGenetic vaccines and virus-vectored vaccines were a new vaccine in recent year. In this paper, researching on genetic vaccines and adenovirus vector vaccines. The H and F genes of CPIV FA and CPIV FC strain were sub-cloned into the eukaryotic expression plasmid pVAX1and pCI , which was driven by the CMV promoter, constructed pVAX-H, pVAX-F, pCI-H and pCI-F vector successfully, then Purified plasmids DNA were transfected into MDCK cells by Lipofectamine. proteins expressed were detected by indirect ELISA, it was proved that these proteins showed immunogenicity by Western-blot. The result showed that OD vaule from supernatant was higher than contral. eukaryotic expression products of pVAX-F and pVAX-H could reacted with CPIV anti-serum. The immunogenicity of expressed protein were analyzed by Western-blot. The result indicated that H and F gene of CPIV were expressed successfully, and showed good immunogenicity. The mice were immuned with the expression plasmids. The groups were inoculated with pVAX-F;pVAX-H;pCI-H;pCI-F;pVAX-F+pVAX-H;pCI-H+pCI-F, the two groups with empty vector DNA PVAX1 and pCI, as negative control, and the ninth with attenuated CPIV vaccine as positive control. A total of three inoculations were performed at 15 days intervals. Neutralization and lymphocyte transformation assays were employed to determine the immunological response. As a result, the mice inoculated with these plasmids developed humoral and cellular immune responses against CPIV. The two genes produced stronger immune response. Dogs were inoculated by the gene vaccine for three time and dogs were induced producing immune response.We purified CpG and CpG was used as to inoculating mice with pVAX-F+pVAX-H and pCI-H+ pCI-F respectively. A total of three inoculations were performed at 15 days intervals, neutralization and lymphocyte transformation assays were employed to determine the immunological response, the results showed antibody titer was no obvious different, but lymphocyte proliferation was higher in CpG group than the control group.The CPIV F expression cassette released from pVAX-F plasmid was blunted and ligated into SspI site of pVAXΔE plasmid. The recombinant plasmid was named PVAXΔE-F. The orientation of the F gene expression cassette in PVAXΔE3-F is consistent with the transcript orientation of adenovirus E3 region. PVAXΔE3-F and pPoly2-CAV-2 were digested with NruI/SalI, respectively. The purified NruI/SalI DNA fragment containing the CPIV F expression cassettes was cloned into pPoly2-CAV-2 plasmid to generate recombinant plasmid pPoly2-CAV-2/CPIV-F. The recombinant genome was released from pPoly2-CAV-2/CPIV-F by AscI digestion, and was cotransfected into DK cells with CAV-2 DNA without the Nru I/Sal I segment by Lipofectamine. After three transfections into DK cells with the mixed DNA, the recombinant virus, named CAV-2/CPIV-F, was obtained. The expression of CPIV F gene by CAV-2/CPIV-F in infected DK cells was confirmed by RT-PCR and Western-blot. The specific immune response against CAV-2 and CPIV was induced by CAV-2/CPIV-F in dogs, the SN titers against CPIV was 1:5.6~1:16. It indicated that the F protein was encode by CAV-2/CPIV-F in dogs. In vitro, recombinant virus was good hereditary stability after culturing for 31 passages.