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.

Development and Assay of RNA Transcripts of Enterovirus Species A to D, Rhinovirus Species A to C, and Human Parechovirus: Assessment of Assay Sensitivity and Specificity of Real-Time Screening and Typing Methods

“…This is necessary not only because sequence variability in the 5= UTR is so restricted as to preclude (sero)type identification, but the occurrence of recombination between the 5= UTR and the rest of the genome (41) and the consequent absence of species-specific 5= UTR sequences prevent reliable species identification. A variety of typing assays have been developed, in the case of EV and PeVs, in the VP1 region (5,14,35,36), where sequences have been shown to be highly predictive of EV and HPeV types. Similarly, VP4 sequences provide a reliable indication of HRV types, a more conserved region of the capsid gene that can be amplified with a common set of primers (31,42,50).…”

“…Assays that target the highly conserved 5= untranslated regions (UTRs) of these viruses (typically by real-time PCR) (2,9,18,37,51) therefore need to accommodate naturally occurring sequence variability in this region to ensure equal sensitivity. Similarly, virus species and type identification through amplification and sequencing of coding regions such as VP1 in the case of EVs and HPeV (14,35,36) and VP4 for HRVs (31,42) require often quite degenerate primer sequences to allow effective amplification of these more divergent regions of the genome.…”

Diagnostic moléculaire des infections respiratoires à entérovirus : apport de la PCR et du génotypage pour une meilleure approche de la circulation des souches en Basse-Normandie au cours de l’année 2008

“…Toutefois, ces derniè res anné es, le typage des souches progresse avec principalement l'introduction du gé notypage molé culaire. La plupart des mé thodes de gé notypage sont basé es sur l'amplification et le sé quençage d'une partie du gè ne codant pour la proté ine de capside VP1, ré gion hypervariable codant pour la majorité des é pitopes neutralisants [32][33][34][35]. Peu sensibles, elles sont en gé né ral appliqué es ré trospectivement sur des surnageants de culture ne reflé tant qu'incomplè tement les diffé rents sé rotypes d'EV circulants.…”

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system

“…Agostini and Stoner (1995), Sugimoto et al (1997), Agostini et al (1997), De Santis and Azzi (2000) Identification of different resistance mutations between CSF and blood strains in patients on long-term antiretroviral therapy Cunningham et al (2000), Venturi et al (2000), Cinque et al (2001), Stingele et al (2001) surveillance programmes, but also has diagnostic and prognostic significance: to correctly identify polioviruses, new enterovirus types or variants, enteroviruses responsible of severe infections, or those causing infections in neonates or immunodeficient (Muir et al, 1998). Molecular typing systems for enteroviruses might become a rapid alternative to traditional serotyping, which is timeconsuming, labour-intensive and requires virus isolation in cell culture (Muir et al, 1998;Oberste et al, 1999). DNA sequencing in the CSF has enabled the diagnosis of CNS diseases caused by attenuate vaccine strains rather than wild-type viruses.…”