Identification Of Porcine PIV5 Strains,Preparation Of Monoclonal Antibody And Construction Of Virus Infectious Clone

Parainfluenza virus 5(PIV5)is a single negative strand RNA virus,which belongs to Paramyxoviridae,Paramyxoviridae and mumps virus.Since it was isolated from monkey kidney cells in 1950s,it has been isolated from humans,dogs,pigs,cattle and other mammals.PIV5 is a recessive infection in nature,and most animals have no serious clinical symptoms.Although PIV5 has been discovered for many years,the analysis of codon usage patterns of PIV5 genome is still blank.In view of this,the whole genome sequences of more than 40 PIV5 strains were collected from GenBank,and a series of codon and nucleotide analysis were performed.In recent years,there are many reports about PIV5 isolates in China.There are many hosts,including procine,bovine,canine,horses,tigers,lesser panda,pangolins and other mammals.However,there are few studies on the molecular biological characteristics and genetic evolution of PIV5.Thus,we prepared monoclonal antibodies against PIV5 NP protein,sequenced the whole genome of four procine PIV5(pPIV5)strains preserved in the laboratory,and found that they contain SH gene,which is unique to pPIV5 strains from China,while SH can't be expressed normally due to mutation of pPIV5 strains from other countries.Further studies on biological characteristics showed that the Chinese porcine PIV5 strain grew well on IPEC-J2 and PIG-PAM cells,and almost didn't produce cell pathological changes.In this study,a full-length infectious clone of pPIV5 was constructed for the first time,and its SH gene was replaced by EGFP gene.A stable PIV5 rescue platform was established,which laid a foundation for the research of PIV5 as a genetic engineering vaccine vector and the study of virus gene function.Through preliminary research on the recombinant virus,it is found that PIV5 may antagonize the expression of IRF3 through SH to play a role in resisting host cell apoptosis.1.Sequence analysis and evolutionary analysis of PIV5 strainFour pPIV5 strains(HLJ2015/DP1-1/PIV5,HLJ2015/DP2-1/PIV5,HuB/YC/2015/PIV5,JX/2015/1221/PIV5)stored in the laboratory were infected with Vero cells,respectively.After RT-PCR and IFA identification,the full length of the four strains was amplified and cloned into pMDl 9-T vector to complete the whole genome sequencing(GenBank accession number:MT890696,MT890697,MT890699,MT890700).Sequence analysis showed that the genome length of the four PIV5 strains were 15246 nt,following the "six base principle",and their structures were arranged according to 3'UTR-NP-V/P-M-F-SH-HN-L-5'UTR.The results of homology analysis showed that the nucleotide homology of the four strains was 99.9%?100.0%,which was the closest to that of SH-1202 strain from China.It further enriched the genomic data of PIV5 in China and provided reference for epidemiological monitoring of PIV5 in China.The result of homology analysis showed that the nucleotide homology among the 4 pPIV5 strains was 99.9%?100.0%,which was the closest to the SH-1202 strain from procine of China,which further enriched the domestic PIV5 genome data and provided reference materials for domestic PIV5 epidemiological monitoring.Codon preference research is often used to study the evolutionary relationship between different strains and the genomic characteristics of viruses.Therefore,more than 40 PIV5 full genome sequences were collected,and the codon usage patterns of available PIV5-ORF sequences were analyzed by bioinformatics software.The results showed that PIV5-ORF was rich in AU sequences,and there was a preference for the use of nucleotides.The codon usage patterns of PIV5-ORF is affected by natural selection,mutation pressure and dinucleotide abundance.These results further clarify the genomic characteristics of PIV5 and provide a reference for the study of PIV5 evolution.2.Growth characteristics of different PIV5 strains and preparation of monoclonal antibodiesFour pPIV5 strains involved in this research,as well as the BC-14 strain from cattle,ZJQ-221 strain from lesser panda,and SH-1202 strain from procine preserved in the laboratory were infected with MDBK,MDCK,Vero,IEPC-J2,LLC-PK1,PIG-PAM,PK-15,ST,ST-R,ST IRF3 KO,ST MX1 KO and ST RIG-I KO cell lines(the last nine cell lines are procine cell lines).Compared with pPIV5 strains reported in other countries,Chinese pPIV5 strains can express the SH gene and grow well on IPEC-J2 and PIG-PAM cells.The titers of the pPIV5 strains on the innate immune gene-deficient cell lines are higher than that on the non-deleted cell lines,suggesting that the pPIV5 strains have a weaker ability to antagonize the interferon of the porcine cell lines.The NP gene of PIV5 is highly conserved and stable,and can be detected in the early stage of virus infection.Therefore,in this study,NP protein with good hydrophilicity was selected,and the truncated NP protein was expressed by prokaryotic expression system.High purity and high concentration NP recombinant protein was obtained through a series of purification steps.After immunizing mice with the purified protein,spleen lymphocytes of mice with strong IFA positive results were selected to fuse with hybridoma cells.Then through positive screening and subcloning,a hybridoma cell line that can stably secrete a monoclonal antibody(McAb)specific to the PIV5 NP protein is obtained.The results of follow-up experiments showed that the ascites McAb could specifically recognize PIV5 NP protein,which was suitable for IFA,Western blot and other experiments.The preparation of monoclonal antibody against PIV5 NP protein not only provides convenience for the identification of PIV5,but also lays a foundation for the follow-up study of PIV5 molecular biology.3.Construction of infectious clone of PIV5 and identification of recombinant virusIn this study,a full-length infectious clone of pPIV5 was constructed by enzyme digestion and homologous recombination.Its SH gene was replaced by EGFP gene.Under the action of powerful T7 promoter and helper plasmids,the virus was rescued in BHK-21 cells.Thus,a platform that could stably rescue PIV5 was established.By studying the biological characteristics of the recombinant virus,it was found that the growth characteristics of the SH deleted strain and the parent strain were similar,indicating that SH is an excellent insertion site for foreign genes.Further studies have shown that the expression level of TNF-? in ST cells of the full-length infectious cloned virus was significantly increased after the SH gene was deleted.