Study On Isolation, Identification, Major Gene Cloning And Immunity Of Mink Canine Distemper Virus

Canine distemper (CD), caused by canine distemper virus (CDV) which belongs to the Morbillivirusgenus of the Paramyxoviridae virus family, is a acute and highly contagious disease causing considerabledamages to the fur animal farming industry, threatening wildlife conservation and zoo animal. With thechanges in the environment and CDV adaptation to prevailing factors, CDV natural host's range expandsincessantly. In addition, the new CDV variants leading to the incidence and mortality rate of CD infectedanimals are increasing, and prevalent and clinical symptoms developed are more and more complicated atthe present time. Therefore, its harmful effects become more serious. In particular, when giant panda andmacaque and so forth quadrumana are infected CDV, CDV pathogenesis became protrusion. Latestresearch confirms that CDV can infect human prosoma osteoclast and that indicates CDV natural hostextends the possibility that it could infect human beings. It may be the second viral infection diseasepassing from dog to human beings.Due to worldwide distribution of CDV and the widespread natural host and huge cross infection,eliminating CDV is almost impossible. However, a vaccine can be used as an active immunity to controlCD. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling theoccurrence of CD. In order to develop a new kind of safe and efficient CDV genetically engineeredvaccine, we have conducted the following works:1. Isolation and Identification of Canine Distemper Virus from MinkA strain of Canine Distemper Virus was isolated and identified systemically from the liver of a deadmink suspected of having Canine Distemper, at a mink farm in Qingdao, Shandong Province. The resultsindicated that typical and disciplinary CDV cell pathological changes appeared and the physical andchemical characteristics were similar to that of CDV after the virus strain had inoculated Vero cell. Thecytopathogenic effect can be interdicted by Rabbit anti-CDV positive serum. Rotundity and ellipseeosinophil inclusion body can be seen in cytoplasm during inclusion body examination. Indirectimmunofluorescent assay showed that BHK-21 cell inoculated the CDV resulting in idiosyncratic brightgreen fluorescence and that green fluorescence did not appeared in the normal cell. The total RNAextracted from Vero cell culture inoculated with the virus were amplified with Reversetranscription-polymerase chain reaction(RT-PCR), and two objective segments (324bp and 1053bp) wereobtained Thus, the virus strain was proved to be CDV and named as CDVSJ.2. Cloning and Sequencing Analysis on hemagglutinin (H) genes of Isolated Canine Distemper Virus StrainH gene of CDVSJ strain was amplified with RT-PCR, and the purified RT-PCR products werelinked with pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombinedcloning plasmid pMD-18T-CDVH was created and sequenced. Sequencing result indicated that H genesection of CDVSJ strain was of a 1596bp fragment encoding 532 amino acids, which contained 5potential N-glycosylation positions located in 76Aa～78Aa, 318Aa～320Aa, 349Aa～351Aa, 383Aa～385Aa, 514Aa～516Aa; and also contained 12 cysteine residues located at 66, 81, 115, 211,223, 304,309, 317, 417, 493, 502, 529 positions. Compared with the nucleotide sequences of 01-2689, 007Lm,2544Han95, 25259, A75/17, American dog, black leopard, Liud, CDTaichung, Convac, Danish dog,Danish mink, DK91D,Greenlandic dog, Hamatsu, Javelina, KDK-1, LP, Onderstepoort, Snyder Hill,Tanu96, TN, Ueno and Yanaka strains, the homology is 90.7%, 91.0%, 91.9%, 92.2%, 92.0%, 91.2%,91.8%, 91.9%, 90.9%, 96.9%, 92.0%, 92.4%, 92.0%, 92.1%, 90.9%, 91.8%, 91.1%, 92.5%, 96.6%,98.6%, 91.1%, 91.1%, 91.0% and 91.0%, respectively. Derivative amino acids homology are 89.9%,90.1%, 91.4%, 90.2%, 91.9%, 91.4%, 91.9%, 91.7%, 91.2%, 95.1%, 91.4%, 91.9%, 91.6%, 91.4%,90.4%, 91.2%, 90.8%, 91.4%, 94.6%, 97.0%, 90.8%, 90.6%, 91.0% and 90.8%, respectively. Comparedwith Onderstepoort, Convac, Hamatsu, A75/17 and TN strains, CDVSJ strain contained 5 potentialN-glycosylation positions according to derivative amino acids sequence analysis. There were two moreN-glycosylation position at 318Aa～320Aaand383Aa～385Aa compared with the Onderstepoort vaccinestrain. One N-glycosylation positions, 530Aa～532Aa, were lacking compared with Convac and A75/17strains. Three N-glycosylation positions, 236Aa～238Aa, 511Aa～513Aa and 530Aa～532Aa, werelacking compared with Hamatsu strains.Two N-glycosylation position, 236Aa～238Aa and 511Aa～513Aa was lacking compared with TN strain. Clustering analysis results showed that CDVSJ strain isclosely related to the Snyder Hill strain. Sequence homologies are a close comparison to Onderstepoortand Convac, which form one group. However, the kinship between them and standard virulent strainHamatsu is far away.3. Cloning and Sequence Analysis of fusion (F) Gene of Canine Distemper Virus StrainF gene of CDVSJ strain was amplified with RT-PCR and the purified RT-PCR product was linkedto pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombined cloningplasmid pMD-18T-CDVF was built and sequenced. Sequencing results indicated that F gene section ofCDVSJ strain was of a 1053bp fragment encoding 351 amino acids of which 9 were cysteine residues.Compared with the nucleotide sequences of 01-2601, 2544-Han95, 5804P, 25259, A75-17, DOGDK91C,ONP, PDV-2 and 98-2646strains, homology are 95.4%, 93.8%, 93.9%, 94.3%, 94.5%, 93.9%, 97.3%,94.7% and 94.1%, respectively. Derivative amino acids homology are 97.4%, 96.6%, 97.7%, 98.0%,97.4%, 97.4%, 97.2%, 98.0% and 96.6%, respectively. Antigen index comparative results showed that there are great differences in 40-50th amino acids between isolated CDVSJ and vaccine Onderstepoortand the virulent A75-17 strain.Compared with Onderstepoort, A75/17 and PDV-2 strains, the number andposition of cysteine residues of F protein in each strain was the same, being 9. That determines that CDVF gene is a comparative conservative structural protein gene.4. Eukaryotic expression of Canine Distemper Virus F geneCloned F gene segment in pMD-18T simple vector was re-cloned to pcDNA3.1 (+) eukaryonexpression vector plasmid, forming eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF successfully. Eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF were transfected into BHK-21 cell by using lipoplast mediate method and has beenproved with ELISA, indirect fluorescent antibody technique and RT-PCR detection that the objectivegene was successfully expressed in mammalian cell.5. Immunization test of Eukaryon recombinant expression plasmidIn order to study immunity characteristics of Eukaryon recombinant expression plasmid, Eukaryonrecombinant expression plasmid pcDNA3.1-F and pcDNA3.1-SF were injected intramuscularly intoJapanese albino rabbit. Meanwhile, pcDNA3.1 (+), normal saline negative control and CDV vaccinepositive were used as control. ELISA results showed that specificity antibody to CDV was produced inthe Japanese albino rabbit. Moreover antibody level increased with increasing immunizing times anddoses. The humoral immunity level of recombinant plasmid group was significantly higher than those ofnormal saline and pcDNA3.1 (+) control groups, however, but lower than that of attenuated vaccinegroup. Lymphocyte transformation test results showed that recombinant plasmid immunity groupproduced cellular immunity, but was lower than that of the attenuated vaccine group.A strain of CDV was isolated from the liver of the suspect CD dead mink, and H and F gene ofCDVSJ strain were cloned and sequenced, providing useful information for molecular biological study ofthe virus. The recombined eukaryotic expression plasmid of CDV F protein gene was successfullyexpressed in the body of laboratory animal, producing both humoral and cell immunity. The results of thestudy provide fundamental information for the further study on the protective function of CDV H and Fgene, for the development of new type and efficient diagnostic reagent for diagnosis and prevention ofCD and DNA vaccine.