Isolation And Sequencing Of The Parainfluenza Virus 5 From Tiger And Pangolan And Its Pathogenicity In Mice

Parainfluenza virus 5(PIV5)is one of the causes of respiratory diseases in animal,and it is distributed worldwide which also became a popular trend in China.The full-length genome of PIV5 is 15,246 nucleotides long and contains seven genes: NP?V/P?M?F?SH?HN?L.PIV5 has many hosts including dogs,tigers,guinea pigs,baboons,etc.The single infection is rare,which often mixed with other pathogens.However,there are few reports about PIV5 infection in the zoo.In the early years,PIV5 antibodies were detected in the serum of tigers.In recent years,PIV5 has been detected and isolated from the lungs of the pandas and the serum of the Amur tiger in our laboratory.However there have been no reports of pangolan infection with PIV5 and other viral infections.In this experiment,the whole gene sequences of PIV5 from Bengal tiger source and pangolin source were determined,homology analysis and genetic evolution analysis of the full-length genome were arried out to explore the evolution of the virus,and the variation of the virus strain.This work can enrich the genomic data information of PIV5 in China and lay the foundation for the prevention and treatment of PIV5 and further research.In this study,one lung sample of the Bengal tiger and 11 samples of lung,liver and kidney of Pangolin were collected from a zoo in Guangdong Province and a wild animal rescue center in Guangdong Province from October 2017 to February 2018.The viruses were isolated and cultured with Vero cells and MDCK cells,and the isolated viruses were identified by RT-PCR,erythrocyte adsorption test and transmission electron microscopy,and named as HMZ(Bengal tiger source)and CAN(Pangolin source).The whole genomes of the isolated viruses were sequenced by segmentation sequencing and uploaded to NCBI with sequence numbers: MH370862(HMZ)and MH362816(CAN).Calculated over the full-genome sequence,HMZ strain shares identities ranging from 97.2% to 99.8% with the sequences of 21 PIV5 isolates downloaded from Gen Bank.The highest nucleotide alignments(99.8%)were observed with the PIV5 1168-1?Mammalian rubulavirus 5 and ZJQ-221 strain,while the lowest alignments(97.2 %)with the D277 strain.Calculated over the full-genome sequence,CAN strain shares identities ranging from97.2% to 99.8% with the sequences of 21 PIV5 isolates downloaded from Gen Bank.The highest nucleotide alignments(99.8%)were observed with the PIV5 1168-1?Mammalian rubulavirus 5 and ZJQ-221 strain,while the lowest alignments(97.2%)with the D277 and08-1990 strain.In the phylogenetic tree,HMZ and CANstrain shared the closed genetic relationship and were in the same branch with Mammalian rubulavirus 5 and ZJQ-221.Their branch is farthest from D277 and 08-1990.In order to investigate the effect of stress on Kunming mice infected with PIV5 CAN strain,Kunming mice were randomly divided into 4 groups: blank control group,stress control group,non-stress infected group and stress infected group.100 ?L(106.36 TCID50)PIV5-CAN was inoculated by nose,and the control group was replaced with an equal amount of DMEM.The results showed that the mice in the infected group had a worse mental state than the control group,and the body weight decreased to varying degrees.In the 8 days of the experiment,the weight of the mice in the blank control group increased by 8.39 % on average;the stress control group increased by 7.57 %;the non-stres infected group increased by 5.47 %;while the stress infected group only increased by 4.05 %.The difference between the infected group and the control group was tested by the statistical software SPSS 17.0 t detection method.The results demonstrated that the blank control group and the stress infected group were significantly different(P<0.05).Through observation of pathological sections,it was found that the alveolar wall of the infected group was thickened,the alveolar epithelial cells proliferated,and accompanied by inflammatory cell infiltration;In the spleen nodules,a large number of lymphocytes were necrosis and decreased,a large number of macrophages were phagocytosis and eosinophilic macrophages increased,suspected macrophage necrosis,and more neutrophil infiltration were in the red pulp and white pulp.This study confirmed that the PIV5 CAN strain is pathogenic to Kunming mice and is more pathogenic under stress conditions.