Reverse Genetics System Of Canine Distemper Virus Strain TM-CC And Recombinant Canine Distemper Attenuated Vaccine Virus Expressing VP2Protein Of Canine Parvovirus

Canine distemper (CD), caused by the canine distemper virus (CDV), is a worldwide infectious disease of dogs and other terrestrial carnivoresand caused often a serious harm to kennel and fur animal breeding industry. In recent decadees, the natural host range of CDV was gradually expanding and comprised almost predominantly carnivores. Therefore, the bisease seriously affects the populations of wild animals.Non-segments negative strand RNA virus (NSNRV) can be recovered entirely from the viral genomic cDNA clone by the reverse genetics, which is a new virological technique. With the development of reverse genetics, it becomes much easy to engineer specific mutations, deletion, modified or insertion of foreign genes into viral genomes. In recent years, the reverse genetics systems of several attenuated CDV vaccine strains have been established in our country, but that of virulent CDV strain is relatively hysteresis. And, it is a major technical obstacle for studying invasion, spread and pathogenesis of CDV. Therefore, it is necessary to develop reverse genetics systems of virulent CDV strains in our county.In this study, we determined the whole genome sequence of CDV TM-CC strain, isolated from the dead tibetan mastiff due to naturally infection, and constructed a full-length plasmid encoding the anti-genome of CDV-TM-CC and the pCI-CDV/NP, pCI-CDV/P and pCI-CDV/L expressing the N, P and L protein, respectively. Meanwhile, we constructed the recombinant plasmid pCAGG-dogSLAM expressing the signaling lymphocyte activation molecules (SLAM) of dog. To generate virus from the plasmids, BSR cells grown in six-well plates were transfectd with the five plasmids by using the calcium phosphate transfection reagent. And, the recombinant virus rCDV-TM-CC was rescued. Compared to the parental strain, the rCDV-TM-CC was almost indistinguishable from the CDV-TM-CC. There are no different in growth properties between rCDV-TM-CC and the parental strain in VerodogSLAM cells. Besides, the rCDV-TM-CC and the parental strain grew to similar levels and reached peak titers of10694TCID50/mL and106.78TCID50/mL, respectively.In order to further investigate the spread, distribution and pathogenesis of CDV in vitro and/or in vivo, the enhanced green fluorescent protein (EGFP) gene was inserted into the non-coding region between H and L gene of TM-CC strain in the pCI-CDV-TM-CC. The resulted recombinant plasmid was designated as pCI-CDV-TM-CC-EGFP. Then, we rescued the recombinant virus rCDV-TM-CC-EGFP expressing EGFP from the plasmid pCI-CDV-TM-CC-EGFP. The rCDV-TM-CC-EGFP was almost indistinguishable from the rCDV-TM-CC. There are no different in growth properties between rCDV-TM-CC-EGFP and rCDV-TM-CC in VerodogSLAM cells. Besides, the rCDV-TM-CC-EGFP grew to similare levels and reached peak titers of106.56TCID50/mL. The recombinant virus has good growth characteristics and genetic stability in VerodogSLAM cells. After serially passages20times in VerodogSLAM cells, the rCDV-TM-CC-EGFP still kept a high level expression of EGFP. Compared with the traditional detection methods of CDV, the detection method of rCDV-TM-CC-EGFP, EGFP as a target protein, is more sensitive, specific and convenient. It provide the technical means for investigating the spread, distribution and pathogenesis of the virulent CDV strain in animals.Canine parvovirus (CPV) disease is an acute and fatal contagious disease caused by CPV, which is a member of the genus Parvovirus of the family Parvoviridae. CPV mainly infects dogs and minks and cause fatal myocarditis, especially in2-3week old pups, and hemorrhagic enteritis. CPV disease is a very serious problem for dog industry and fur animal industry worldwide. For preventing CPV disease, vaccination is the most efficient strategy. Now, inactived vaccine, the most widely used vaccine, still has a few disadvantages, such as ashort duration and a high manufacturing cost. Therefore, it has practical significance to develop safe, effective, low-cost CPV disease vaccine.VP2protein is the major antigen for protective immune responses against CPV infection. In this study, VP2gene of CPV was inseted into the non-coding region between P and M genes of the full-length genome cDNA clone from a attenuatd CDV vaccine strain CDV/R-20/8, serve as a live vaccine vector. On its basis, we generated a recombinant attenuatd CDV vaccine strain, rCDV/R-20/8-CPVVP2, expression CPV VP2protein by the reverse genetics. Expression of CPV VP2protein, in the BSR cells infected by the recombinant virus, was confirmed by indirect immunofluorescence assay (IFA) and western blotting analysis. Compared with the parent strain, the rCDV/R-20/8-CPVVP2has the similar biological characteristics, including high growth titer (105.81TCID50/mL) and excellent adaptability and genetic stability in Vero cells. It is a base for researching a novel and live bivalent vaccine against canine distemper and CPV disease.