Identification Of EV-A71 Neutralizing Epitope And Study On The Pathogen Spectrum Of Hand, Foot And Mouth Disease Enterovirus In Wuhan Area

Hand,foot and mouth disease(HFMD)is a kind of class C infectious disease caused by a variety of enteroviruses.It mostly occurs in infants and young children,and characterizes with a variety of clinical symptoms,such as fever,oral herpes or skin rashes in hands,feet,buttocks and other parts.Usually,HFMD occurs in children under 5 years old with mild symptoms.In the severe cases,HFMD can lead to serious neurological complications and even death.EV-A71 and CV-A16 are the two main pathogens causing HFMD.In recent years,CV-A6 and CV-A10 have replaced EV-A71 and CV-A16 as the main pathogens of HFMD.The infection of EV-A71 occasionally leads to high fever,neurological complications and fatal pulmonary edema related to brainstem encephalitis.Currently,there is no specific antiviral drug for the treatment of EV-A71.The rapid progress and high mortality of severe patients with EV-A71 infection have brought serious challenges to the specific diagnosis and treatment of EV-A71.Therefore,EV-A71 specific monoclonal antibody is urgently needed to detect the EV-A71 antigen in swab samples of patients with infection at the early stage and treat severe patients.In addition,the elucidation of virus neutralization epitopes and mechanism will be helpful for the development of targeted therapy and vaccine.In neutralization epitope identification.To date,several neutralizing monoclonal antibodies against linear epitopes of EV-A71 has been identified.Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins.Hence,we attempted to screen such neutralizing antibodies against conformational epitopes of EV-A71.Here we describe a neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV-A71 which was mapped using immune escape mutants.In this study,eight immune escape mutants were isolated.Sequence comparison analysis showed that mutations at the position of VP2-159 and VP3-81 abolished the neutralizing activity of mAb 8a4.We simulated the phenotype of the escape mutant strain by using reverse genetics technology to rescue the escape mutant and found that V159 E of VP2 and K81 E of VP3 abolish monoclonal antibody binding and neutralization activity.This is the first report that a conformational epitope is composed of the amino acid residues on both VP2 and VP3.This is the first report that a conformational epitope is composed of the amino acid residues on both VP2 and VP3.In a study on molecular epidemiology,the rectal and throat swabs of hospitalized children were collected from Wuhan Jin Yin Tan Hospital,which were subjected to real-time RT-PCR analysis using Pan-enterovirus probe.The VP1 partial nucleotide sequences were also amplified by reverse transcriptase PCR(RT-PCR)and the phylogenetic characteristics of nucleotide sequences were analyzed by software such as DNAStar7.0,MEGA6.0.Among 1,350 HFMD cases,EVs were identified in 1,137 cases and the overall positive rate was 84.2%.Of the 1,137 cases,eleven different EV serotypes were detected.CV-A6,A10,A16,A2,A4 and EV-A71 accounted for 50.57%,19.88%,11.08%,5.28%,4.66% and 1.50%,respectively.CV-B5,CV-A5,Echo11,CV-A9,CV-B2 and other untypeable EVs accounted for 7.04%.Phylogenetic analysis showed that the representative strains of CV-A6,A10,A16,A2,A4 belonged to D3,C,B1 b,D2 and E2 sub-genotype,respectively.The spectrum of enteroviruses responsible for HFMD epidemic in Wuhan in 2018 was constituted of viruses with various genotypes and CV-A6 was the predominant pathogen.VP1-based phylogenetic analysis for the 5 predominant serotypes revealed that various clades or sub-clades reflected the evolution driven by mutations.The results suggest the complex transmissions of other enteroviruses in Wuhan following EV-A71 vaccine are available.