Evaluation of VP4-VP2 sequencing for molecular typing of human enteroviruses

Abstract

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.

Fig 1. Relationship between 249 EV and RV prototypes based on the VP4-pVP2 amino acid sequence.

The assigned species of Enterovirus alphacoxsackie, E. betacoxsackie, E. coxsackiepol, E. deconjuncti, E. alpharhino, E. betarhino, and E. cerhino prototypes are labeled EV-A, -B, -C, -D, RV-A, -B, and -C, respectively. (A) Phylogenetic analysis. Sequences were aligned using the MUSCLE algorithm. The phylogenetic tree was constructed using the neighbor-joining method with Poisson correction. (B) (left) Matrix of pairwise identity comparison among 249 prototypes. Identity percent is indicated using color-coded boxes; (right) Mean, min, and max values of identity percent for inter- and intra-species comparisons.

Fig 2. Histogram of pairwise identity frequency based on the VP4-pVP2 amino acid sequence.

(A) Comparisons among total EV prototypes. (B) Intra-species comparisons for EVs and RVs are separately presented (EV-A, -B, -C, -D, RV-A, -B, and -C). The proposed 95% threshold is indicated as a dotted line. EV pairs showing >95% identity are listed in Table 1.

Fig 3. Distribution of identities between prototype and matched strains.

Box plots represent the identity percent between the indicated prototypes as a query in the blastp analysis and the top 100 matched references with assigned types. (A) E. alphacoxsackie, (B) E. betacoxsackie, (C) E. coxsackiepol, and (D) E. deconjuncti. Untyped or mislabeled references were excluded from the blastp analysis results.