Typing of human enteroviruses by partial sequencing of VP1

Abstract

Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an "unknown" may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.

FIG. 1

RT-PCR amplification of all prototype EV strains with primer pairs 012-011 and 040-011. PCR products were resolved by 1% agarose gel electrophoresis and were visualized by ethidium bromide staining and UV transillumination. (A) CAs, CBs, and PVs amplified with primer pair 012-011. (B) CAs, CBs, and PVs amplified with primer pair 040-011. (C) Echoviruses and numbered EVs amplified with primer pair 012-011. (D) Echoviruses and numbered EVs amplified with primer pair 040-011.

FIG. 2

Distribution of pairwise amino acid identity scores. Peak 1 corresponds to comparisons of homologous strains (same serotype), peak 2 corresponds to comparisons of heterologous strains (different serotype) of the same major phylogenetic cluster, and peak 3 corresponds to comparisons of heterologous strains of different major phylogenetic clusters.