Complete Genome Sequence Analysis And Establishment Of Reverse Genetic Manipulation System Of Porcine Parainfluenza Virus Type5

Porcine parainfluenza-5 virus(PPIV5)is a non-segmented single-stranded negative-strand RNA virus,belonging to the family Paramyxoviridae,a member of the mumps virus.The virus was first discovered and isolated in monkeys in 1956 and was named monkey virus type 5(SV5),which was officially named(simian parainfluenzavirus 5)on the 1995 International Committee on Virology.The virus was detected in other animals and in cell culture,meaning that the monkey was not the only host of the virus,so the virus was first named(parainfluenza virus 5,PIV5)on the 2009 Virology Classification Committee..The virus was renamed(mammalian rubulavirus 5)at the International Committee of Virology in 2018,and PIV5 will be used herein to refer to the parainfluenza virus.In the preliminary work of this laboratory,the virus was used as a piglet animal regression experiment(detailed data is published separately,not reflected in this article).The results show that although the virus can infect the pig's intestines,it will not cause diarrhea and elevated body temperature.I understand that no virus has reported that the virus causes serious diseases in pigs.Therefore,the purpose of this study was to establish a reverse genetic operating system for PPIV5,which laid the foundation for the delivery of a foreign protein.The isolated PPIV5 virus was named CHNJS-17 strain.According to the sequence published on Gen Bank,we designed 6 pairs of primers covering the genomes of the viruses except the 3' and 5' ends,and amplified them by RT-PCR.The sequences were ligated and transformed into competent cells by the T-vectors,and the plasmids were extracted and sequenced.At the same time,the specific sequences of the 3' and 5' ends of PPIV5 were determined by the RACE technique.Our sequence analysis showed that the strain isolated in our laboratory was 15 246 bp in length,which was consistent with the “six-base principle” of paramyxovirus.The amino acid differences between the fibrin F and HN and the classic strain W3 A were small,98.3% and 97.3%,respectively,and the small hydrophobin(SH)was the most different,91.1%.The CHNJS-17 strain was compared with the 28 PIV5 genome sequences published by NCBI.The phylogenetic tree analysis showed that it was in the same evolutionary branch as the accession numbers KX100034,KY685075,MH370862,MG921602 and MH362816.The analysis showed that it was the highest with 99.8% of MG921602 strain,MH362816 strain,MH370862 strain,KX100034 strain and KC237064 strain,and the lowest homology with KY114804 strain was 97%.It can be seen that the phylogenetic tree division and homology of PIV5 have no correlation with host species.Recombinant poxvirus(MAV-T7)expressing T7 polymerase provides T7 polymerase and is used to rescue negative-strand RNA viruses(NSV).In order to successfully rescue PPIV5,this experiment used it as a transcriptional viral genome.Use.The vector used in this study was the p OK12 vector,which is a low copy vector that can accommodate larger genomes.Add the T7 promoter at the 3' end of the whole genome and three deoxynucleotide Gs that stabilize the T7 promoter,and Hammerhead ribozyme(Ham Rz),and add hepatitis B ribozyme(hepatitis)at the5' end.Delta virus ribozyme,Hdv Rz)and T7 terminator.PIV5 conforms to the "six-base principle" in paramyxovirus,so the addition of Ham Rz and Hdv Rz ensures accurate cleavage of the viral genome and improves the success rate of virus rescue.Since the virus is a negativestrand RNA virus,the auxiliary proteins essential for viral transcription are nucleocapsid protein NP,phosphorylated protein P,and large polymerase protein L.In this study,pc DNA3.1(-)was used.The vector was constructed to construct three helper plasmids for NP,P and L,and the full-length plasmid of the viral genome was co-transfected with hamster kidney cells(BHK21)infected with MAV-T7 in advance at a ratio of 4:2:2:1.Save the virus.In order to distinguish between the rescue strain and the wild strain,the molecular tag which is not present in the parent strain is added to the viral genome,and the molecular marker 4768G-(T)which can distinguish the wild strain and the rescue virus is identified by the identification.Successfully,the rescued PPIV5 was named r PIV5-4768G(T).The rescued r PIV5-4768G(T)was observed by electron microscopy to conform to the appearance of paramyxovirus.The size was different and pleomorphic.The virus was observed in the visual field.The "zipper" virus gene that ruptured and emated,the outer membrane of the virus was densely packed with fibrils,and the indirect immunofluorescence(IFA)results showed that the virus rescued successfully.The one-step growth curve showed that there was a difference between the rescue virus and the parental poison at the 12 th hour.The establishment of the reverse genetic system of r PIV5-4768G(T)with the pathogenic PPIV5 as a parental toxicity can be a good vector for the expression of foreign viral proteins,especially porcine enteroviruses such as porcine epidemic diarrhea virus.(PEDV)The expression of S protein lays the foundation.