| Parainfluenza virus 5(PIV5)is a negative-sense,nonsegmented,single-stranded RNA virus in the genus Rubulavirus of the family Paramyxoviridae.It exists widely in nature,infect many animals include human.It is reported that PIV5 has a high infection rate in pigs,but there are few reports about the pathogeny molecular biology and genetic evolution.In this study,the pathogens were isolated from the samples of intestine tissue of diarrhea pig preserved in our laboratory.The genetic evolution analysis of F gene and full-length genome,the pathogenicity research of PIV5 isolates were carried out,which increased the information of diagnosis,prevention and later vaccine research in PIV5.Five strains were successfully isolated through blind passages in Vero cell.The purified virus suspension was observed by electron microscopy,Paramyxovirus particles were detected.The five strains can agglutinate the red blood cell of cock and the agglutination can be inhibited by standard antiserum of Newcastle disease virus.The isolates were then identified by Illumina next-generation sequencing and RT-PCR,and the results showed that the five strains were p PIV5 strains,which were respectively named HLJ2015/DP1-1/PIV5,HLJ2015/DP2-1/PIV5,SH/2015/122/PIV5,Hu B/YC/2015/PIV5 and JX/2015/1221/PIV5.In order to understand the genetic evolution of PIV5,F gene of five PIV5 isolates were amplified and sequenced by RT-PCR method,the amplified sequences were then analyzed for the genetic evolution.The full-length genome of PIV5 strain SH was sequenced,and the genetic evolution analysis of genome sequence was carried out.A sequence comparison of the F gene among the five isolates revealed 99.6%~99.9% nucleotide identity and 98.9%~99.6% amino-acid identity.Comparison of the five isolates with other PIV5 reference strains showed that they shared the highest sequence similarity with PIV5 strain ZJQ-221 from a lesser panda in China and PIV5 strain 1168-1 from canine in South Korea,the level of sequence identity compared to ZJQ-221 and 1168-1 were ranged from 99.8%~100% and 99.5%~99.8% at the nucleotide level respectively.The results of genome sequence analysis showed that the nucleotide homology between PIV5 strain SH and a prototype PIV5 strain W3 A was 98.7%.The F gene-based phylogenetic tree indicated that five PIV5 strains showed a closely relationship with the PIV5 strain ZJQ-221 and the PIV5 strain 1168-1,formed a single clade.Phylogenetic tree based on genome indicated that PIV5 strain SH showed a closely relationship with the PIV5 strain 1168-1and three Chinese PIV5 strains(PIV5 HMZ,PIV5 SR,PIV5 ZJQ-221)isolated from panthera tigris tigris,panthera tigris altaica and lesser panda,respectively.Five strains formed a single clade,differ from the PIV5 W3 A.Phylogenetic tree based on NP,F and HN genes was then built,and the results were basically the same as phylogenetic tree based on genome.In order to know the pathogenicity of PIV5,BALB/c mice were infected with PIV5 strain SH by artificial infected way.The results showed that PIV5 is non-pathogenic to BALB/c mice.In this study,five strains of PIV5 were isolated from intestine tissue samples of pig.The results of sequence analysis showed that the genome structure of PIV5 is stable,rarely difference was found between different PIV5 strains,animal studies showed that PIV5 strain SH failed to establish illness,indicating that PIV5 is non-pathogenic to BALB/c mice. |
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