Research On The Molecule Evolution And B Cell Epitope Screening Of Enterovirus C96

Objective: Hand foot and mouth disease(HFMD)is an infectious disease that mostly occurs in infants and young children.It is widely prevalent in the Asia-Pacific region and there are several outbreaks have occurred.However,there is no specific treatment for HFMD at the moment and broad-spectrum antiviral therapy is still the main clinical treatment.There are no approved HFMD vaccines for the market except 3 vaccines specially designed for EV71.EV71 and CA16 are the main pathogens that can cause HFMD.However,in recent years,the incidence of HFMD caused by infections of non-EV71 and non-CA16 is on the rise,such as EVC96.EVC96 belongs to the group C of enterovirus,and the infection of EVC96 can lead to HFMD,acute flaccid paralysis,acute asthenic myelitis,acute hemorrhagic conjunctivitis and other diseases.In recent years,relevant cases have been reported in many areas at home and abroad.,and it has potential risk of endangering the security of public health.In this study we analyzed the molecular characteristics of EVC96,explored its epidemic regularity and variation trend in mainland China,provided basic data for early warning,diagnosis and treatment of newer enterovirus infections in China.We also predicted and screened B-cell linear epitopes of EVC96,which laid a foundation for the establishment of EVC96 diagnostic methods and the development of epitope vaccine of EVC96.Methods:1.The full-length sequence and VP1 region sequence of EVC96 genome were obtained.Except for the strain A240 of EVC96 isolates is from Guangdong CDC and P86 sequenced by our research group,all of them were from GenBank.Then constructed the phylogenetic tree with MEGA7.0,and analyzed the homology by DNAMAN software.Using Simplot3.5.1 and RDP4.9 software to analyze the reorganization of EVC96 to further explore the evolution trend of EVC96 in mainland China.2.Predict the B cell epitope of EVC96 by using 4 online prediction server with different algorithms,DNASTAR software and SWISSMODEL server.3.Synthesize the candidate epitope polypeptide of EVC96 B cells.The purity was determined by HPLC-MS and the reactivity was determined by indirect ELISA.4.The epitope polypeptide of EVC96 B cells was cross-reacted with 10 common pathogenic polyclonal antibodies of HFMD.The synthesized peptide was subjected to immunize rabbit after linked to KLH protein to obtain polyclonal antibodies,and then cross-reacted with 10 common pathogens of HFMD.The conservative analysis of the epitope polypeptide sequence was carried out with EVC96 Chinese isolates and prototypes of 10 common HFMD pathogens.Results:1.Analyze molecular characteristics of EVC96 genome,full-length VP1 and partial VP1 sequence worldwide,construct phylogenetic tree of VP1 region.EVC96 isolates from China can be divided into three gene clusters,Cluster 1-3,and all Chinese EVC96 isolates belong to the same branch.The genetic distance between Cluster 3 and Cluster 2 was 0.258,and between Cluster 1 was 0.241,between Cluster 2 and Cluster 1 was 0.261,within Cluster 3 was 0.187,within Cluster 2 was 0.242 and within Cluster 1 was 0.036.According to the full-length VP1 sequence,EVC96 isolates from China can be divided into 6 genotypes and 8 genotypes according to partial VP1 sequence.2.According to the analysis of gene characteristics,the delegate strain A240 of EVC96 was selected for analysis.Simplot analysis showed that A240 had high similarity with EVC96 prototype in P1 region,but lower similarity in other regions.In 2B,2C and 3C region,the highest similarity was found between CA24 prototype Joseph and A240,the highest similarity was found between CA27 prototype G-12 at 5?end of 3D region.Bootscan analysis showed that A240 might recombine with EV99 prototype strain BAN00-10461 in 5?-UTR region,in 2B,2C,3A and 5?end of 3C regions might recombine with CA24 prototype strain,at 5'end of 3C region shows possibility of small fragment recombination with CA19 prototype strain,and in 3D region fragment recombination occurred with CA11 prototype strain Belgium-1 and PV2 prototype MEF-1.By using RDP4.9 we found two typical recombination events in A240 through seven algorithms.The parents were EV109 prototype strain,CA19 prototype strain and Shenzhen isolate KR919804.1 and CA11 prototype strain.Previous studies found that KF495604,a Guangdong isolate belonging to genotype III,was highly similar to EVC96 prototype strain in P1 region,and that CA24 and EVC102 might be recombined in P2 and P3 region.And in P3 region,it might be recombined with HQ415759.1,a 2009 EVC96 Shandong isolate belonging to genotype II,while HQ415759.1 and EV102 prototype strain might be recombined in 3C region.3.9 candidate B cell epitopes of EVC96 were predicted and screened.P9(aa282-304:VPYNGTGVDIKEGTLTPITPVNS,VP1)reacted positively with EVC96 antiserum,but not with EVC96 negative serum,and did not react with 10 other common HFMD enterovirus antisera.The polypeptide antiserum of P9 did not react with the antigens of 10 common HFMD enteroviruses.The titer of the polyclonal antibody obtained by immunizing rabbits with KLH-P9 conjugated polypeptide was over 1:256 000,and the polyclonal antibody did not react with the whole virus antigen of 10 common pathogens of HFMD.The P9 polypeptide sequence was highly conserved in EVC96 Chinese isolates,but not in the corresponding regions of 10 other common pathogenic prototypes of HFMD.Conclusion:1.Chinese EVC96 isolates has the same origin,large variation and has transmit for a long time in China:All Chinese isolates of EVC96 are located in the same branch of phylogenetic tree and can be divided into multiple genotypes.The genetic distances between and within genotypes are large,and there are inter-type and intra-type recombination.2.P9 is a specific B-cell epitope of EVC96 with antigenicity and immunogenicity,which lays a foundation for the establishment of EVC96 diagnostic method and the development of epitope vaccine for EVC96.