Efficacy Of Synthetic-Peptide Candidate Vaccines Against Serotype-A FMDV In Cattle And Analysis Of The Interaction Between FMDV Proteins And Host Cellular Proteins

Foot-and-mouth disease(FMD)is one of the most contagious and economically important viral diseases affecting cloven-hoofed animals,including ruminants,swine,and certain wild ungulates.The etiological agent,foot-and-mouth disease virus(FMDV),belongs to the genus Aphthovirus of the family Picornaviridae.Although a comprehensive control strategy including quarantine,surveillance,and vaccination has been properly used,FMD still occurred in china and other parts of the world.An urgent need exists for developing a safe and effective vaccine that prevents animals from becoming infected or progressing to FMD.VP1 is the major structural and most immunogenic protein of FMDV.It can elicit the neutralizing antibodies as there are many critical determinants on it and has a role in virus attachment.This indicates that VP1 is an attractive target for the production of new genetically engineered vaccines against FMD.VP1 is recognized as a key factor of the FMDV life cycle,and exhibited its multiple functions via interactions with various host and viral proteins.The nonstructural proteins are involved in FMDV replication,and inhibit the host immune response.The 2B protein has been implicated responsible for cell membrane rearrangements and function in FMDV infection,release and autophagy.Despite a recent increase in studies of the proteins of FMDV,knowledge of their cellular targets and the functions of virus-host interactions remain limited,this knowledge gap significantly limits further understanding of FMDV biology and the rational development of more safe and effective vaccines.In this study,we constructed an efficacious synthetic-peptide vaccine against serotype A FMDV for use in cattle.We explored the interacting partners of VP1 in host cells and the effects of the interaction on the FMDV life cycle.And we investigated the roles of 2B in virus-host interactions.The results of our study provide empirical data for facilitating the development of novel vaccine against FMD,and further our understanding of the possible cellular mechanisms involved in FMDV infection.The results were shown as follows:1.We designed three synthetic-peptide vaccines,59 to 87 aa in size,based on immunogenic epitopes in the VP1,3A,and 3D proteins of the A/HuBWH/CHA/2009 strain of the FMDV,corresponding to amino acid positions 129 to 169 of VP1,21 to 35 of 3A,and 346 to 370 of 3D.The efficacies of the vaccines were evaluated in cattle and guinea pigs challenged with serotype-A FMDV.All of the vaccines elicited the production of virus-neutralizing antibodies.The PB peptide,which contained sequences corresponding to positions 129 to 169 of VP1 and 346 to 370 of 3D,demonstrated the highest levels of immunogenicity and immunoprotection against FMDV.Two doses of 50 ?g of the synthetic PB peptide vaccine provided 100% protection against FMDV infection in guinea pigs,and a single dose of 100 ?g provided 60% protection in cattle.These findings provide empirical data for facilitating the development of synthetic-peptide vaccines against FMD.2.We investigated the roles of VP1 in virus-host interactions by constructing a cDNA library obtained from FMDV-infected swine tissues,and used a split-ubiquitin based yeast two-hybrid system to identify host proteins that interacted with VP1.We found that VP1 interacted with amino acids 329~572 in the C-terminal region of the zyxin,which containing three LIM-domains.The VP1-zyxin interaction was confirmed by the coimmunoprecipitation of VP1 and zyxin and the colocalization of VP1 and zyxin in HEK293 T cells.We also found that the expression of zyxin was upregulated in FMDV-infected BHK-21 cells,and that the overexpression of zyxin promoted FMDV replication,whereas the small interfering RNA-mediated knockdown of zyxin expression suppressed the production of infectious virions.This is the first report of the role of cellular zyxin in FMDV replication.Our findings provide important insight into mechanisms of FMDV-host interactions and their roles in virus replication.3.To gain insight into the role of the 2B protein in the FMDV life cycle,we performed the yeast two-hybrid assay using FMDV protein 2B as the bait and the constructed swine cDNA library as the prey to screen the 2B-interacting proteins.The amino acids 208~437 in the C-terminal region of the eEF1 G subunit of eukaryotic elongation factor 1,which is essential for protein synthesis,was identified to be a specific host binding partner of FMDV 2B.The interaction between 2B and eEF1 G was confirmed by co-immunoprecipitaion and confocal assays.To determine whether eEF1 G modulates FMDV replication,we evaluated the replication of FMDV in cells in which eEF1 G expression was upregulated or downregulated.The results of the qRT-PCR analysis and the virus titer assay showed that the overexpression of eEF1 G promoted FMDV replication,whereas the knockdown of eEF1 G expression suppressed the replication of FMDV.This finding is consistent with the upregulation of eEF1 G expression after FMDV infection.The results of our study provided important information regarding the mechanisms of FMDV replication and the interactions between the virus and eEF1G.