Construction Of Molecular Clone And The Minigenome Of Canine Parainfluenza Virus

Canine parainf luenza virus(CPIV) is the primary cause of self-lim ited tracheobronchitis of dogs, namely cough. Clinically, alone infection of CPIV causes nonfatal diseases, only characterized by mild respiratory symptoms, and this is not very common; The most cases are mixed infection with other pathogens, being aggravated and increasing the risk of death. CPIV was first found and isolated at 1967, then relevant reports have been found around the world, and prevalence of the virus has widely spread in the world and in our country. In the development of all kinds of virus vaccine research es, the most signif icant scientific progress is reverse genetic technology. There are many studies about human parainf luenza virus and Bovine parainfluenza virus type3, but no reports about genetic engineering vaccine of CPIV until now. As the maturity of reverse genetic technology of negative strand RNA virus, parainf luenza virus-vectored live vaccines have potentially broad application,the research results of HPIV and BPI V3 can also be used for reference by studies of CPIV.The object of this experiment is to construct infectious molecular clone and the minigenome of of CPIV. The RT PCR products of gene fragments of HRB- V strain of CPIV were cloned from NCBI Gen Bank library using the primers based on the PIV5 1168-1 strain sequence analyzed with computer, then cloned into p HW2000 vector, sequenced and analyzed knowing the genome sequence, and compared with sequences of PIV5 reference strains deposited in Gen Bank. The isolate genome was 15246 nucleotides in length including 5 UTR, 7 ORFS and 3 UTR. Fusion(F) gene, haemagglutinin(HN) gene and complete genome analysis demonstrated that HRB- V is closely related with reference strains CPI-\CPI+. The nucleotide sequence homology of F and HN gene with the reference strains was between 96.5%-98.8% and 98.1%-99.5% and amino acid sequence homology was between 94.2%-98% and 97%-99.3%, respectively. Above all suggested the virus strain of HRB- V had little differences and close genetic relationships with other PIV5 isolates.The RT PCR products of gene fragments NP、P and L were cloned into pCI-neo vector using the primers based on the HRB- V strain, constructed there eukaryotic expression plasm id, named helper plasmid. Us ing the reverse gene of eGFP replace the entire coding region only keep leader and trailer, which has close relation with the ability of replicat ion, transcription and virus particle packing cloned into pVAX1 vector, constructing the minigenome of CPIV, that can be used to analyze and identify the helper plam ids. The successful expression of NP, P and L plasm ids laid the foundation for the further rescue of CPIV.This study succeeded in construction of molecular clone and complete genome analys is of canine parainf luenza virus. Then recombinant plasmids of pCI-NP, pCI-P and pCI-L which was identified by minigenome was obtained. In summary, all of these studies laid the base for further development CPIV reverse genetics system.