Establishment Of Vero And BHK21 Cell Lines With A Stably Expression Of Canine Distemper Virus Cell Receptor SLAM And The Isolation And Authenticate Of Canine Distemper Virus

Canine distemper(CD)is an acute,highly contagious disease.It causative agent is canine distemper virus that infects abroard range of carnivores,and CD has higher morbidity and mortality.Traditional cell lines did not express the CDV receptor protein and were very difficultly to isolate CDV,which severely restricted to study the CDV.Our aim is establish a SLAM-Vero cell line and a SLAM-BHK21 cell line to express a SLAM receptor,and then to compare their sensitivities to the CDV wild strains.Whole genome sequencing and phylogenic analyses of CDV.This study is carried out in the following aspects:? Establishment of SLAM-Vero cell line and SLAM-BHK21 cell lineThe eukaryotic expression vector Pcag-SLAM,which contains the SLAM gene,was transfected into the Vero cells and BHK-21 cells,respectively.Then the cells which harbor the SLAM gene were subsequently screened with G418 combined with end point dilution,RT-PCR,immunofluorescence assay,and western-blotting.The results showed that the SLAM-Vero cells lines and SLAM-BHK21 cells lines were constructed successfully.? Isolation and identification of canine distemper virus.The isolate rate of CDV was explored in SLAM-Vero cell line and SLAM-BHK21 cell line.Comparing the tropism of different tissues to the same cell lines,The isolated CDV have been confirmed by RT-PCR,Indirect Immunofluorescence assay,electron microscopy technique,The results showed that the two strains of CDV were obtained from three CDV positive canine lungs and spleens by using SLAM-Vero cells.While SLAM-BHK21 cell lines,parental Vero cells and parental BHK21 cells failed to isolate the virus..? complete genome sequencing and phylogenetic analyses of canine distemper virus.In this study,The complete genome of one isolate,CDV-SC1706,was determined and The length of complete nucleotide is 15690 bp.Phylogenetic analysis is showed that the CDV-SC1706 isolated displated the highest identity to LN(10)1 strain,and can be classified into Asia-1 genotype.The homologies among SC1706 isolate and CDV strains of Louguantai 1,SX2014 were higher with 98.3% and 98.4 % identity.Show their close genetic relationship.The N gene of SC1706 isolate showed a poor homology of the nucleotide sequence of N gene among SC1706,Onderstepoort vaccine strain,and Shuskiy.There was a 93.5% identity with Onderstepoort vaccine strain,and 93.1% identify with the shuskiy vaccine strain.The lowest homology of 93% was found between SC1706 isolate and CDV3 vaccine strain.This SC1706 wild-type strain had nine N-linked glycosylation sites,which was the same as the strains in the Asia 1 group.