Identification Of Porcine-derived Cellular Receptors For Senecavirus A And Study On The Immunity Properties Of Its Receptor-binding Blocking Polypeptides

Senecavirus A(SVA)is a non-enveloped single-stranded positive-stranded RNA virus,belongs to the genus Senecavirus in the family Picornaviridae.SVA have always been concerned about its ability of cancer treat.At present,Phase I clinical trials for pediatric solid tumors and Phase II clinical trials for small cell lung cancer are in progress.However,pigs are the natural host for SVA,and SVA can cause porcine idiopathic vesicular disease and epidemic transient neonatal losses.The disease did not attract enough attention to pig farms until the outbreak of SVA in Brazil in 2014.At present,the disease has been reported in many countries,including the United States,Vietnam,Thailand,Australia,Colombia and other countries.Since the first outbreak of SVA was reported in Guangdong province in 2015,the disease had been rapidly spread from south to north in China.It is worth to concern about the research on the pathogenic mechanism,immune mechanism,transmission and vaccine to control and prevent SVA infection.In this study,we examined the entry of SVA into PAM-Tang cells(a porcine alveolar macrophage cell line)using EGFP-expressing recombinant SVA as a tool.Anthrax toxin receptor 1(ANTXR1)has been reported to be a receptor for SVA on human cells.In this experiment,PAM-Tang cells were used to investigate whether the porcine anthrax toxin receptor pANTXR1 could mediate SVA entry into cells.The results showed that the infectivity of SVA was attenuated in pANTXR1 knockdown cell lines and enhanced in overexpressing cell lines.The PAM-Tang pANTXR1 knockout cell line(PAM-Tang KO cell line)was constructed,which showed very low susceptibility for SVA infection.pANTXR1 transfection in the PAM-Tang KO cell line enhanced the susceptibility to SVA.In addition,Hela KO cell line(h ANTXR1 knockout Hela cell line)expressed pANTR1 acquired the ability of being infected by SVA.This study also researched the route of SVA enter into PAM-Tang cells,and found that cholesterol played an important role in the process of SVA enter into PAM-Tang cells.In this study,SVA infection properties were investigated using an air-liquid culture system(ALI culture system)of porcine nasal respiratory epithelial cells.ALI culture system is a highly differentiated airway epithelial cell established in vitro,which can simulate the physiological state of airway epithelial cells in vivo.The ALI culture system was used to study the SVA infection characteristics,which could reflect the real situation of SVA infection on porcine respiratory epithelial cells.SVA showed a polarized entry and release characteristic on ALI culture.Western blot and q PCR results showed that SVA mainly entered from basolateral ALI cells,and q PCR results showed that SVA was mainly released from apical compartment.At the same time,SVA infection could induce autophagy in ALI cells.SVA co-localized with the autophagy molecule LC3,and LC3Ⅰ and LC3Ⅱ changed after SVA infected.The autophagy inducer treatment decreased SVA replication in ALI culture system,while the autophagy inhibitor treatment increased SVA replication.The preliminary work of this study has determined that pANTXR1 can mediate SVA entry into pig cells,and the homology is 97% between pANTXR1 and h ANTXR1.At present,the interaction sites between ANTXR1 and SVA have been predicted,and these sites are likely to be the interaction sites between SVA and pANTXR1.The peptides were designed according to the interaction sites of SVA and ANTXR1,and two peptides were screened to have antiviral effects in vitro as the candidate peptides for peptide vaccine named B1 and B2 respectively,and mixed universal T cell epitope T1 Pan DR epitope and T2 natural tetanus sequence(830-844),to preparate peptide vaccine.VP2,T1T2VP2,VP2T1T2 protein were expressed to preparate subunit vaccine.Our results showed that VP2,T1T2VP2 and VP2T1T2 could induce neutralizing antibodies production,while B1,B2,B1+T1+T2 and B2+T1+T2could not.In summary,this study demonstrated that SVA entry into PAM-Tang cells through the pANTXR1 and depend on cholesterol.The infection characteristics of SVA in the air-liquid culture system of respiratory epithelial cells were studied.According to the SVA-ANTXR1 interaction site,peptides were designed and synthesized as the candidate peptides for peptide vaccine and the immune effect was researched.The results are important for our understanding of SVA invasion and transmission mechanisms in natural hosts,and will lay a preliminary foundation for the research of SVA peptide vaccine.