Investigation On The Spike Protein In Invasion And Immune Response Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea（PED）,caused by porcine epidemic diarrhea virus（PEDV）,is an re-emerging porcine viral disease.Vomiting,diarrhea and dehydration are clinical features of PED.PED broken out again in many countries and regions around the world since 2010,and the mortality rate of PED in newborn piglets is as high as 100%.Due to mutation and the lacking of effective vaccines,PED is still epidemic around the world and results in huge economic losses to the pig industry worldwide.PEDV is a single-stranded positive-sense RNA virus with an envelope,belonging to the Nidovirales,Coronaviridae,and Alpha-coronavirus,which has a characteristic“spike”structure of coronaviruses.The S protein is further cleaved into the S1 and S2 protein by proteases.Studies have determined that S protein plays an crucial role in PEDV invasion.Firstly,the S1 protein binds to the receptor:the N-terminus domain（NTD）binds to the sugar and the C-terminus domain（CTD）binds to porcine aminopeptidase N（pAPN）.Then,the fusion peptide（FP）in the S2 protein is exposed via proteases.The FP fuses with cell membrane,and the heptad repeat（HR）1 interacts with HR2 to form a stable fusion core,which brings viral membrane to host cell membrane and completes membrane fusion.In addition,S protein,as a main immunogenic protein of PEDV,can induce production of neutralizing antibodies in the host.Especially,the COE domain,an important neutralization epitope region in the S1-CTD,is the main target for designing PEDV subunit vaccines.Although many studies have been carried out with PEDV S protein,there are still some unresolved questions.For instance,the structure information of S protein is still obscure,which has become an important factor restricting the function research of PEDV S protein.Previous studies showed that pAPN was the receptor for PEDV.However,recent studies have raised controversies on it.Although the structures of some coronavirus fusion core have been reported,there is little information about structure and function of the porcine coronaviruses.In addition,despite some neutralizing antibody epitopes of PEDV S have been identified,it is necessary to further characterize and analyze its epitopes,due to substitution,insertion or deletion in the S1 region of PEDV epidemic strain in recent years.Clarifying these questions will be helpful to understand the pathogenesis of PEDV,and provide new ideas and strategies for prevention and control of the virus,which is necessary for the current severe PED epidemic and urgent requirement for PED prevention and control.Therefore,this paper studied on the above aspects.First,the PEDV S1 subunit,involved in the receptor recognition,was studied.The PEDV S1 protein（21-793 aa）,the S1 truncated protein（S1t,505-629 aa）and the extracellular domain of pAPN（63-963 aa）were prepared.Their functions were verified,and the structures were analyzed through circular dichroism spectroscopy,X-ray crystallography and small angle X-ray scattering.The results showed that the target protein had correct structure folds and biological functions.Then,the interaction between PEDV S1/S1t protein and pAPN was analyzed by gel filtration chromatography,non-denaturing PAGE and surface plasmon resonance.The results showed that there was no interaction between them and further determined pAPN might not be a functional receptor for PEDV.These results support the structural information of PEDV S1 protein and may be helpful to reveal the genuine interaction between PEDV and its host cells.Second,the PEDV S2 fusion core,a key node mediating PEDV membrane fusion,was studied.The target protein was designed and expressed.Circular dichroism spectroscopy showed that the protein had a typicalα-helix structure.The crystal structure of the fusion core main chain was determined by X-ray crystallography.The results showed that HR1 and HR2 formed a six-helix bundle structure.HR1-and HR2-derived peptides were designed and synthesized based on the crystal structure.The virus inhibition experiments showed that the HR2-derived peptide had a significant inhibitory effect on PEDV replication.These results provided the structure information of PEDV S2 protein and new strategies for antiviral research.Finally,the epitopes of COE（CO-64 K equivalent,499-638）,a key neutralizing epitope domain of PEDV S1 subunit,were analyzed.Mice were immunized with PEDV S1t protein and 14 monoclonal antibodies were prepared via immunoperoxidase monolayer assay（IPMA）.Reactivity of monoclonal antibodies against different PEDV strains was different and some monoclonal antibodies could not bind to the PEDV CH/HNHB/2016 strain.Furthermore,a novel linear B cell epitope ⁵⁹²TSLLASACTIDLFGYP⁶⁰⁷ was identified via truncation and peptide scanning.The minimal recognition region of the monoclonal antibodies 4D8F10 and6F3E3 was ⁶⁰⁴FGYP⁶⁰⁷.The 604F,605G and 606Y were key recognition sites.Conservative analysis of this epitope in different PEDV subtypes showed that the⁵⁹²TSLLASACTIDLFGYP⁶⁰⁷ epitope was highly conserved,especially at the key recognition sites.Moreover,monoclonal antibodies 4D8F10 and 6F3E3 were applicable for recognition of the PEDV strains containing the conserved epitope.These results provided new tools and references for the study of the antiviral activity of the novel epitope and the development of diagnostic methods for PEDV.In summary,the role of PEDV S protein in viral invasion and immune response was studied.The structural characteristics of the S1 subunit,which binding to the receptor,were analyzed,and the interaction between the PEDV S1 subunit and pAPN was elucidated.Meanwhile,the crystal structure of the S2 fusion core protein,which mediated the membrane fusion between PEDV and host cells,was determined and the antiviral activity of HR2-derived peptide was verified.In addition,a novel linear B cell epitope was identified in the PEDV COE region,which was highly conserved in the PEDV strains.The above results will provide clues for identification of PEDV functional receptors,the design of antiviral drugs and the development of diagnostic reagents.