Identification and immunogenicity of enterovirus 71 isolates

Objective To identify two enterovirus 71 (EV71) isolates and evaluate their immunogenicity for the purpose of EV71 vaccine development.Methods The morphology of EV71 isolates was determined by electronic microscopy,both of the 087 and 085 isolates were identified by indirect fluorescence assay (IFA) using anti-EV71 monoclonal antibody and by RT-PCR with EV71 specific primers,the genotype was determined by phylogenetic analysis based on VP1 gene sequence.The immunogenicity was evaluated on BALB/c mice by the detection of neutralization antibody and anti-EV71 IgG in the serum with neutralization test and ELISA.Results The round virus particles with 20-30 nm in diameter can be objected by electronic microscopy in the virus infected cells culture which is typical for enterovirus.Both 087 and 085 strains can react with anti-EV71 monoclonal antibody,the positive virus infected cells stained with FITC can he detected by IFA.The 226 bp products were amplified from the total RNA extracted from virus infected cells by RT-PCR with EV71 special primers,not CA16 special primers.The genotype of both strains is CA,which is identical to the other epidemiological EV71 strains in mainland,China in the latest decade.Both strains can induced high level of neutralization antibody before or after inactivation which can neutralize more EV71 strains than themselves,for the same neutralized virus strain,no difference was found between 085 and 087 strains and between live and inactivated group for each strain either,but the ability to neutralize FY-02T strain for the serum from all the immunized groups is lower than other strains significantly(P<0.05).Compared with control,anti-EV71 IgG can be detected in the sera from immunized mice with the subtype IgG1/IgG2a by ELISA,which are not different between 087 and 085 strains but higher in inactivated virus group.Conclusion Both of the two EV71 isolates,085 and 087 strains were identified to be EV71 with genotype C4 and are highly immunogenic.