Whole Gene Sequence Analysis Of Parainfluenza Virus 5 From Different Sources And Its Pathogenicity To Mice

Parainfluenza virus 5,also known as canine parainfluenza virus,is the main cause that cause canine,cat and other animal respiratory diseases,which caused serious losses to China's dog industry and even other aquaculture industry.The parainfluenza virus 5 is a member of paramyxovirus family of the mumps virus.It is a RNA virus with a total length of 15246bp.It consists of seven genes:NP,V/P,M,F,SH,HN,L.According to the serological survey,the virus was a worldwide distribution,and a wide range of hosts,dogs,rats,cattle,pigs and other animals infected with this virus are reported.The parainfluenza virus 5 in this study were isolated from red panda,Siberian tiger and South China tiger,which were provided by some zoos in Guangdong.Before that,the red panda can infect Parainfluenza virus 5 have not been reported,tiger could infect with this virus has reported,but no in-depth research,related sequences couldn't find in Genbank.Therefore,it is of great significance to carry out whole-gene amplification of the isolated red panda source and the tiger source parainfluenza virus 5,which can be used to understand the genetic evolution,prevention and treatment of parainfluenza virus 5 and provides the reference for the research of the vaccine.In this study,14 samples of suspected parainfluenza virus 5?PIV5?infection were collected from 2014 to 2016?4 for pandas,4 African lions,1 Siberian tiger,1 South China tiger,4 ring-tailed lemurs?.After grinding,centrifugation,filtration and other treatment,The virus was inoculated into Vero cells for virus isolation and culture.The virus was harvested and identified as parainfluenza virus 5.Three strains of parainfluenza virus 5were successfully isolated and named as ZJQ-221?red panda?,PIV5-SR?Siberian tiger?and PIV5-SC?South China tiger?,the positive rate was 21.4%?due to the inconvenience of sampling in the zoo,the number of samples is limited?.Three kinds of virus solutions were inoculated to Vero cells,spread to the third generation after Vero cells can make vacuoles,pull the net phenomenon.Observed by transmission electron microscopy,the virus solution was round,with capsule,diameter of 50²00nm virus particles.12 pairs of specific primers were designed to amplify the whole gene of the panda and Siberian tiger isolates,and the expected size bands were obtained.Sequencing,cutting,splicing to get two complete whole gene sequences.The results showed that the nucleotide sequence similarity of the two strains was 97.2%⁹9.8%and 97.1%⁹9.7%,respectively,and the similarity of the deduced amino acid sequences was 97.2%⁹8.8%and97.7%⁹9.5%,respectively.Both strains were the most homologous to the 1168-1 isolates isolated from the Korean dog in 2009,which were 99.8%and 99.7%,respectively,and the nucleotide sequences were 34 and 43,respectively,relative to 1168-1 Mutation,and deduced amino acid sequence similarity were up to 98.8%and 99.5%,respectively,11 and25 changes.Establish a genetic evolutionary tree,we know that both viruses were closely related to the isolates of Korean Canine source isolate 1168-1,and were most distant from the United Kingdom Homo sapiens isolates PIV5-LN and RQ.The pathogenicity of the virus was studied in Kunming mice.The mice were inoculated with 25?L and 50?L of 10⁶ TCID₅₀/100?L of PIV5-SR virus solution,25?L and50?L of 10⁶ TCID₅₀/100?L of ZJQ-221 virus solution,inoculated with 25?L and 50?L DMEM medium as a control.The mice were infected with hair disorder and mental state.According to the weight change of mice,the virus had inhibitory effect on the growth of mice,and the inhibitory effect was obvious.The mice were sacrificed after 8 days of poisoning,check the organs and found that the lungs have bleeding,interstitial widening.RT-PCR showed that each organ contains parainfluenza virus 5,indicating that the virus is infected with mice.Take the lungs for pathological sections,found alveolar wall thickening,inflammatory cell infiltration.This study shows that the red panda source and the northeastern tiger source parainfluenza virus 5 have a certain pathogenicity to Kunming mice.