Functional Study On 3C Protein Of Seneca Virus A

Senecavirus A(SVA)belongs to the genus Senecavirus,family Picornaviridae.SVA infection is the cause of sows vesicular disease and neonatal piglets acute death syndrome.The clinical symptoms are vesicular lesions,anorexia,claudication and acute death of neonatal piglets.The earlier strain,SVV-001,was non-pathogenic to pigs and has been used in human cancer research due to its oncolytic properties.Over time,there is growing evidence that SVA infection is associated with porcine idiopathic vesicular disease.Since 2015,outbreaks of SVA have occurred in many pig producing countries,more than 70%pig farms have suffered huge economic losses.The mortality rate of newborn piglets is as high as 30%-70%,posing a serious threat to the healthy and sustainable development of the global pig industry.SVA is a newly emerging porcine viral infectious disease.The research on the transmission route and pathogenesis of SVA is not sufficient,and there is no available commercial vaccine or effective drug,which needs to be paid close attention to.Like other picornavirus 3C proteins,SVA 3C has a conservative catalytic cassette.SVA 3C is primarily responsible for the hydrolysis of viral polyproteins and the assembly of virions,which is essential for viral replication.3C proteins are responsible for 8 of the 11 cleavage sites of poly proteins,including VP2/VP3,VP3/VP1,VP1/2A,2B/2C,2C/3A,3A/3B,3B/3C and 3C/3D.In addition,3C protein also plays an important role in antagonizing type I interferon,avoiding host innate immune response and inducing apoptosis of host cells.In this study,the monoclonal antibody of SVA 3C protein was prepared for the first time,which provided a powerful immunological tool for the functional study of viral protein and the establishment of antibody detection methods.Preliminary analysis of amino acid residues of 3C protein showed that the 3C(H202A)mutant significantly reduced its ability to induce apoptosis.PIV5 was used as the vaccine vector to express SVA P12A3C protein.It was observed that SVA P12A3C polypeptide was correctly processed into a single protein after infecting cells with the recombinant virus.Virus-like particles of SVA were also observed under electron microscope,which provided a theoretical basis for the pathogenesis of SVA and vaccine development.1.Preparation of monoclonal antibody against Senecavirus A3C proteinIn this study,SVA 3C protein was expressed by prokaryotic expression system and BALB/c mice were immunized.Then,two monoclonal antibodies(mAbs)against 3C protein,12-G9 and 23-H2,were screened by indirect immunofluorescence assay(IFA).IFA and Western blot results showed that the two mAbs could specifically recognize SVA 3C protein without cross-reaction with other picornaviruses.The reactivity of the two mAbs against different SVA strains was further evaluated by serological experiments.The results showed that both two mAbs could recognize 7 SVA strains.Subcellular localization of SVA 3C protein was performed by confocal microscopy.It was found that the 3C protein was partially localized to mitochondria.The successful preparation of monoclonal antibody against SVA 3C protein provides a powerful tool for the study of the structure and function of viral proteins and the development of new diagnostic reagents.2.Functional analysis of Senecavirus A 3C proteinSVA 3C protease induces apoptosis and cleaves host cell proteins in the late stage of viral infection,showing cytotoxicity.In order to obtain less toxic mutant of SVA 3C protease,sitedirected mutation of some amino acids in 3C gene was carried out in this study.It was found that the sites H48,H78,H178,C160,L85,L100,L132 and L148 were critical to the replication of the virus and could not be successfully saved after the mutation.The Western blot results of eukaryotic plasmid transfection showed that the three mutants of L105P,L148P and H202A retained the 3C protease cleavage activity and increased the expression of 3C protein in host cells by reducing apoptosis.By Hoechst staining,Western blot and flow cytometry analysis,it was found that these three mutants reduced the apoptosis induced by 3C protein to varying degrees.Among them,the 3C(H202A)mutant most significantly reduced the apoptosis of host cells.These findings provide a theoretical reference for the pathogenic mechanism of SVA,vaccine research and development,and the function of viral proteins.3.Role of 3C protein in virus-like particlesIn this study,a recombinant PIV5 infectious clone plasmid expressing SVA capsid protein polypeptide P1 and 3C protease gene was constructed and the virus was saved.IFA results showed that two recombinant PIV5 viruses,rPIV5-SVA-P12A3C(wt)and rPIV5-SVAP12A3C(H202A),were successfully obtained.Western blot results showed that single SVA VP3 and 3C proteins were detected in the recombinant virus-infected cell samples,suggesting that VLP assembly may have occurred.Transmission electron microscopy results showed that SVA VLP with a size of 25-30nm was formed.This study verified the feasibility of PIV5 as a carrier of SVA VLP vaccine,and provided a new strategy for the development of new SVA vaccine.