Canine Distemper Virus Molecular Epidemiological Investigation From Foxes, Minks, Raccoon Dogs And Construction Of The Completed CDNA Of CDV3Vaccine Strain

Canine distemper virus (CDV) is the pathogen of multisystemic infectious disease affecting all carnivorous animals with high incidence and mortality.Vaccine is an effective prevention measures, but the outbreak of CDV still be popular in many countries throughout the world, suggesting that new changes about the pathogenesis of CDV may occur. Therefore, the following research were main about molecular epidemiology and reverse genetic operations of CDV.Hemagglutinin protein gene (H) was considered to be the important factor of the CDV mutation, to study the molecular epidemiology variation of CDV in recent years, Seven field CDV strains were collected from infected minks, foxes, raccoon dogs in different provinces of China in2011～2013, the H genes were cloned and sequenced. In the analysis, the homologies between nucleotide and deduced amino acid sequences of H gene of the seven strains were98.7%～100.0%and98.4%～100.0%respectively. However, to these of representative strains of vaccines were90.8%～91.2%and90.5%～90.9%respectively. Phylogenetic analysis showed that all the strains were characterized as Asia-1genotype. Three strains of mink and fox from Shandong province (SD (12)1, SD (12)2, SD(12)3) had an amino acid substitution from Y to H at position549which located in the H protein receptor (SLAM) binding region. Unlike the other strains, two mink strains (SD (12)1,SD (12)3) had an additional potential N-glycosylation site (NTR) caused by an amino acid mutation of I→N at position542. These findings indicate that a degree of CDV adaption to these non-dog hosts (mink, fox).To build reverse genetic operation platform of CDV3vaccine strains, RT-PCR method was applied to get five fragments that cover the full length of CD V3vaccine strain genome, through the directional cloning technology to clone it to modified the eukaryotic expression vector pCI, a full length cDNA plasmid (pCI-rCDV3) were constructed. Further, the help plasmids pCI-CDV3-N, pCI-CDV3-P, pCI-CDV3-L were accquired by directional cloning N, P, L gene of CDV3into pCI vector. The result of sequencing, enzyme digestion and immunofluorescence assay of the plasmids show that the full length cDNA clone plasmid of CDV3build successful, help plasmids can express the help proteins. After the initial exploration of rescuing CDV3, this study lay the foundation of further research on the reverse genetic operation of CDV3.