Coxsackievirus A6 (CV-A6) is one of the microorganisms that cause hand, foot, and mouth disease (HFMD) and has emerged as a new threat to HFMD control. Researchers created a pseudovirus luciferase assay (PVLA) for anti-CV-A6 neutralizing antibody (NtAb) quantification in this work after initially developing a single-round pseudovirus infection system for CV-A6. Because cytopathic effect (CPE) is regarded as the gold standard test for anti-enterovirus NtAb identification, comparative research was conducted utilizing 318 clinical blood samples assessed by both PVLA and CPE. 

PVLA had a sensitivity of 94.9% (95% CI of 90.8–97.5%) and a specificity of 92.7% (95% CI of 86.6–96.6%). PVLA and CPE were found to be highly correlated (spearman r = 0.931, P <0.0001) and in good agreement (94.0%, 95% CI between 90.8 and 96.4%), indicating that PVLA could be used as a surrogate assay for anti-CV-A6 NtAb detection and a useful tool for CV-A6 vaccine evaluation and epidemiological surveillance.