The Non-Neutralizing Antibody Responses Against Various Enteroviruses (EV) Of Major Etiological Agents For HFMD In Adult Population

Infections from hundreds of enterovirus(EV)serotypes and genotypes are ubiquitous in humans,causing a series of diseases including hand,foot,and mouth disease(HFMD).EV71 and CA16 are major etiological agents for HFMD.The neutralizing antibody assay has been the effective serological assay to successfully assess human EV epidemics.The general serological prevalence of EV infections based on ELISA remains unknown.The study is the first to describe the detailed information of the serological prevalence of human non-neutralizing antibody responses against the VP1 of EV-A(CA5,CA10,CA6,CA16,EV71),EV-B(CB3)and EV-C(PV1)viruses,and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on the combination of a NEIBM-based ELISA and a competitive ELISA assay.Part ? Enterovirus VP1 gene synthesis and the prokaryotic expressionThe representative DNA sequences of PV1 and HAV VP1 were synthesized using sequential OE-PCR and were cloned onto expression vector p ET-32 a to construct prokaryotic expression plasmids.Then all the plasmids of p ET-32a-EV71 VP1,p ET-32a-CA16 VP1,p ET-32a-CA5 VP1,p ET-32a-CA6 VP1,p ET-32a-CB3 VP1,p ET-32a-PV1 VP1 and p ET-32a-HAV VP1 were induced to express.The seven proteins were purified using Ni-NTA resin.SDS-PAGE and Wertern blot analysis confirmed the expression of the purified proteins.Part ? The detection and analysis of non-neutralizing antibody responses against various enteroviruses in Shanghai blood donorsIn the present study,the antibody responses against VP1 of the EV-A species(EV71,CA16,CA5 and CA6),of the EV-B species(CB3),of the EV-C species(PV1)and of Hepatovirus genus virus(HAV)were detected and analyzed by a NEIBM(Novel Evolved Immunoglobulin(Ig)-Binding Molecule)-ELISA in August 2013 and May 2014.Each of the 48 samples that were strongly reactive against VP1 of the various viruses was analyzed in a competitive inhibition ELISA.The combined detection and correlation analysis of the anti-VP1 proteins of various enteroviruses by NEIBM-ELISA and competitive ELISA clearly demonstrated that the antibody reactions against VP1 of the various viruses are ubiquitous in adult individuals.The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level,with anti-PV1 VP1 antibodies at the second highest level,and anti-CA5,CA6,CA16 and EV71 VP1 antibodies at a comparatively low level.The lowest level observed was against HAV VP1.All reactions were significantly correlated at different levels,which were approximately proportional to their sequence similarities.Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses.Obvious differences in antibody responses between August 2013 and May 2014 were revealed.Part ? The dynamic monitoring of non-neutralizing antibody responses against various enteroviruses in Shanghai blood donorsTo investigate whether the non-neutralizing antibody response could be used to detect the prevelanceof enteroviruses,the antibody responses against VP1 of the EV-A species(CA10,CA6,CA16,and EV71),of the EV-B species(CB3),and of the EV-C species(PV1)were detected and analyzed by a NEIBM-ELISA and competitive ELISA in 2014.The serological prevalence of anti-VP1 antibodies of serious enteroviruses was demonstrated to show the same level at different times;The correlation of the antibody reactions kept steady at different times;The inhibition activities showed no significant difference beween different times.Unlikely the indexes mentioned above,correlations between the inhibition activitiesare obviously different.The correlations between the inhibition of anti-CA16 VP1 reactions by VP1 of EV71 and CA16 in the samples from April and June 2014 showed the lowest level,with correlation coefficients of less than 0.1,which suggested the prevelance of CA16 in April 2014.The correlations between the inhibition of anti-CB3 VP1 reactions by VP1 of CB3 and the other enteroviruses in the samples from Auguest 2014 reduced consistently,which suggested the prevelance of CB3 in Auguest 2014.The correlations between the inhibition of anti-CA10 VP1 reactions by VP1 of CA10 and the other enteroviruses in the samples from June 2014 reduced consistently,which suggested the prevelance of CA10 in June 2014.The correlations between the inhibition of anti-PV1 VP1 reactions by VP1 of PV1 and the other enteroviruses in the samples from Octomber2014 reduced consistently,which suggested the prevelance of PV1 in Octomber 2014.The detection of anti-VP1 Ig G and Ig M was analyzed to verify the inferencs above.The prevalence of anti-CA16 VP1 Ig G and Ig M in April 2014 was higher than the other times,which showed statistically significant difference;The correlations of anti-CA16 VP1 Ig G and Ig M from April 2014 to Auguest 2014 were higher than the other times;These verified the prevelance of CA16 in April 2014.The correlations of anti-CB3 VP1 Ig G and Ig M from Auguest 2014 to Octomber 2014 showed the highest level,which verified the prevelance of CB3 in Auguest 2014.The correlation of anti-CA10 VP1 Ig G and Ig M in June 2014 showed the highest level,which suggested the prevelance of CA10 or its close relatives in June 2014.The correlation of anti-PV1 VP1 Ig G and Ig M in Octomber 2014 showed the highest level,which suggestedthe prevelance of PV1 or its close relatives in Octomber 2014.On this view,the non-neutralizing antibody responses based on the combination of a NEIBM ELISA and a competitive ELISA assay could be helpful for understanding of the serological prevalence of human antibody responses against the VP1 of enteroviruses and for seroepidemiological surveillance of enteroviruses after further development.