Development Of A Novel Enterovirus71and Coxsackievirus A16Neutralization Test For Measurement Of Neutralizing Antibody Titer

Objective The conventional serological methods for evaluation of EV71andCVA16neutralizing antibodies based on inhibition of cytopathic effect (CPE) whichneeds operating live virus, so a safe and objective pseudovirus assay for detecting EV71or CVA16neutralizing antibodies was developed, which is fast and efficient. MethodsEV71or CVA16pseudovirus containing firefly luciferase gene in place of the capsidgene P1in its genome was generated. Pseudovirus was produced by cotransfection of293T cells with three plasmids:1, the T7RNA polymerase expression plasmid(pcDNA3.1-T7RNA Pol/pcDNA3.1-5’NTR-P1);2, the chimeric pT7-CVA16-luc orpT7-EV71-luc replicon plasmid (a reversely transcribed full length viral genomeencoding firefly luciferase gene in place of the P1region);3, the viral capsid expressionplasmid (VR1012-EGFP-2A-P1). These generated EV71or CVA16pseudoviruses caninfect Vero or RD cells and mediate the luciferase gene expression, which is a singleround of infection. Several sera were compared by pseudoparticle neutralization assaywith the conventional serological methods. Results Successfully generated the EV71and CVA16pseudoviruses carrying luciferase gene, which could mediate luciferaseexpression in Vero or RD cells after infection. Neutralizing antibody titers measured bypseudovirus methods with those obtained by the conventional serological methods,demonstrated a highly significant correlation between the two methods. ConclusionDeveolped a safe and objective pseudovirus assay for detecting EV71and CVA16neutralizing antibodies. This method can be used for detecting Neutralizing Antibodiesof other picornaviruses like HAV, and will give approach to determine whether there iscross-reactivity among different subgenogroup of EV71and CVA16.