| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 100 === Enterovirus 71 (EV71) is a positive single-stranded RNA virus of the Picornaviridae family. It can cause mild disease such as fever, hand, foot, and mouth disease (HFMD), and herpangina, but some infections are associated with neurological complications like aseptic meningitis, encephalitis and acute flaccid paralysis. Several outbreaks were reported worldwide since it had been isolated firstly in California in 1969 known as EV71 genotype A. Studies on EV71 epidemiology showed that genotype B3-B5 and C2-C5 have caused large outbreaks in the Asia-Pacific region since 1997. Some evidences indicate that neutralizing antibody play a protective role in EV71 infection. Previous reports revealed that the genotypic shift with the changes of antigenicity by using human antisera against the various subgenotypes of EV71. In this study, we hypothesized the antigenic difference may result from the genetic variations of EV71. First, vaccine strain-immunized rabbits’ antisera were used to determine the antigenic variations among different EV71 genotypes by in vitro micro-neutralization-ELISA assay. Second, we applied healthy human and patients’ antisera against EV71 genotype B4, B5 and C2 to further investigate the viral antigenicity. The results showed that the antisera titers against genotype B4 were at least fourfold increase compared to those against genotype B5 or C2, suggesting genotype B4 had the different antigenicity from genotypes B5 or C2. Amino acid sequences comparison of genotypes B4 and B5 revealed three substitutions, position 98, 145, and 164, on capsid proteins VP1. In addition, substitutions at position 145 and 164 in VP1 were found between genotypes B4 and C2. Site-directed mutagenesis of genotype B4 or B5 virus with position 98 or 164 mutation in VP1 altered neutralizing antibody titers significantly. Besides, double mutant virus of VP1 98 or 164 along with 145 also affect the antibody titers. Antisera against mutant virus with C2 or B4 VP1 protein replacement showed neutralizing antibody titers similar to those against C2 or B4 virus, respectively, indicating that VP1 protein is the immunodominant neutralizing region. To visualize the antigenic variation of these viruses, the antigenic map was constructed. We found that the amino acid position 98 and 164 in VP1 are critical to EV71 antigenic properties and position 145 also plays a role. The results will help to understand the antigenicity of EV71 and EV71 vaccinology.
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