Background/Objectives: Enterovirus 71 (EV71) and coxsackieviruses A16 (CA16), A10 (CA10), and A6 (CA6) are the primary pathogens that cause hand, foot, and mouth disease (HFMD). Currently, many manufacturers are developing bivalent, trivalent, and tetravalent vaccines that target these antigens. Cell-based neutralization assay (CBNA), the gold standard for detecting neutralizing antibodies (NtAbs), which are used as indicators of HFMD vaccine efficacy, has several limitations. We aimed to develop a novel assay for detecting NtAbs against a quadrivalent HFMD vaccine. Methods: We developed a four-color pseudovirus-based neutralization assay (PBNA), utilizing fluorescent reporter genes, to rapidly evaluate neutralizing antibodies against EV71, CA16, CA10, and CA6 in multivalent vaccines and compared it with CBNA. Results: PBNA could rapidly and simultaneously detect NtAbs against the four serotypes and required lesser amounts of sera compared to CBNA. A good consistency in determining NtAb titers was observed for PBNA and CBNA. Conclusions: PBNA provides a robust tool for evaluating the efficacy of multivalent HFMD vaccines and conducting seroepidemiological studies.

Enterovirus A71 (EVA71) is a major pathogen that causes hand, foot, and mouth disease (HFMD). Although the symptoms of HFMD can be self-limiting, severe meningitis, encephalitis, myocarditis, and acute flaccid paralysis may occur. Upon EVA71 infection, the host cells deploy an intricate network of factors to orchestrate cellular responses and maintain cellular homeostasis. However, the virus has evolved various strategies to avoid unfavorable host restrictions and to establish a productive infection process. As response regimens are correlated with disease symptoms, exploring the interactions between the virus and host contributes to understanding the pathogenesis and underlying mechanisms of infection. In this review, we summarized the recent research progress related to pro-viral factors during EVA71 infection and discussed the underlying mechanisms employed by EVA71 to facilitate virion production. These insights may help identify antiviral therapeutic candidates and support vaccine development.