EV pairs with >95% identity.

Enteroviruses and rhinoviruses are highly diverse, with over 300 identified types. Reverse transcription-polymerase chain reaction (RT-PCR) assays targeting their VP1, VP4, and partial VP2 (VP4-pVP2) genomic regions are used for detection and identification. The VP4-pVP2 region is particularly sensitive to RT-PCR detection, making it efficient for clinical specimen analysis. However, a standard type identification method using this region is lacking. This study aimed to establish such a method by examining the divergence of VP4-pVP2 amino acid sequences between enterovirus and rhinovirus prototypes. Pairwise analysis of 249 types indicated a 95% threshold for enterovirus intra-species identification but not for rhinovirus prototypes. Protein BLAST search analyses of representative enterovirus prototypes, including EV-A71, EV-D68, CVA6, CVA10, CVA16, and polioviruses (PVs), validated the 95% threshold for typing, with a few exceptions such as PV1-PV2 and CVA6-CVA10, as well as some EV-C types. This study proposes a criterion for typing based on VP4-pVP2 amino acids, which can aid in rapid enterovirus diagnosis during routine clinical or environmental surveillance and emergency outbreaks. Our research confirms the reliability of the suggested VP4-pVP2-based threshold for typing and its potential application in laboratory settings.