Analysis Of Complete Genome Sequences And Construction Of The Full-length Genome Clone And Helper Plasmids Of Canine Parainfluenza Attenuated Vaccine Virus

Canine parainfluenza is an acute respiratory infectious disease,caused by the canine parainfluenza virus(CPIV),in dogs and the main clinical symptoms of this disease included fever,cough and running nose.And,CPIV can also cause acute encephalomyelitis and hydrocephalus,which is characterized by posterior limb paralysis and ataxia.Vaccination is an effective way to prevent the spread of CPIV in dogs.Now,the attenuated vaccines are the most widely used canine influenza vaccines.In this study,the complete genome sequence of canine parainfluenza attenuated vaccine virus strain NL/CPI/5 was sequenced and compared with the complete genome sequence of 19 isolated strains from GenBank.These results show that the complete genome of strain NL/CPI/5 includes 15246 nucleotides(nt),which is consistent with 4isolated strains(strain CC-14,strain BC14,strain HeN0718,and strain ZJQ-221),isolated in China in 2014 to 2017.The highest nucleotide identity was observed between strain NL/CPI/5 and strain 1168-1,isolated from Korea in 2009,based on the complete genome sequences.And,the complete genome nucleotide sequence of strain NL/CPI/5has 99.8%,99.1%,99% and 97.1% homology with strain ZJQ-221,strain CC-14,strain BC14 and strain HeN0718,respectively.Comparison of the nucleotide and deduced amino acid sequences revealed that the F protein exhibited 99.7%,99.2%,99.2%,and96.4% nucleotide identity,respectively,and 98.9%,98.6%,98.6%,and 96.2% amino acid sequence identity,respectively,to those of strain ZJQ-221,strain CC-14,strain BC14,and strain HeN0718,respectively.And,the HN protein exhibited 99.5%,99.4%,99.3%,and 97.8% nucleotide identity,respectively,and 98.9%,98.8%,98.6%,and97.5% amino acid sequence identity,respectively,to those of strain ZJQ-221,strain CC-14,strain BC14,and strain HeN0718,respectively.These results suggested that the epidemic strains of CPIV had mutation rate in China,especially F and HN proteins.CPIV infects a wide range of animals,including human,and causes only “kennel cough” in dogs.However,CPIV infects other animals and human with mild or no illness.Therefore,CPIV has the potential to serve as live virus vaccine vector.The development of reverse genetics has provided a powerful tool to create the recombinant CPIV-based vaccines.In this study,we generate a full-length genome cDNA clone plasmid of strainNL/CPI/5,which containing the hammerhead ribozyme sequence(HamRz)was the hammerhead ribozyme sequence(HamRz),the full-length(15,690 nucleotide)cDNA of the strain NL/CPI/5 genome in the antigenomic orientation,and the hepatitis delta virus ribozyme sequence(HdvRz)was inserted between NheI and Not I sites of the plasmid pCI.The resultant plasmid was designated as pCI-CPIV.The cDNA representing the ORF of the GFP or RABV strain ERA G(ERAG)gene was amplified with the primer pair,in which the gene end and gene start sequences of CPIV,the MluI and SalI sites were included.The GFP or ERAG gene was introduced into the CDV genome contained in pCI-CDV through the introduction of MluI and Sal I sites in the P–M noncoding region of the CPIV genome.The resultant plasmid was designated as pCI-CDV-EGFP or pCI-CDV-ERAG.Meanwhile,the open reading frames(ORFs)of the N,P and L genes of the strain NL/CPI/5 were PCR amplified from pCI-CPIV to construct helper plasmids.The amplified N,P,and L genes were inserted in CMV-IE promoter of the plasmid pCI,respectively.The resultant helper plasmids were designated as pCI-CPIVN,pCI-CPIVP and pCI-CPIVL,respectively.To generate virus from the plasmids,BSR cells grown in six-well plates were transfected with the full-length plasmids pCI-CDV-EGFP together with a set of helper plasmids(pCI-CPIVN,pCI-CPIVP and pCI-CPIVL)by using the calcium phosphate transfection kit.At 48 h post-transfection,GFP expression by the transfected cells were observed under a fluorescent microscope.The results indicated that the cells the geneome cDNA of CPIV containing GFP gene was transcribed correctly under the action of CMV-IE promoter,and the transcript RNAs were sheared accurately by the ribozymes,HamRz and HdvRz,which generate the RNA molecule that is identical to the genome RNA of CPIV.Meanwhile,the results indicated that the N,P,and L proteins of CPIV were expressed correctly in the transfected cells.