1999 Workshop Abstracts | Virus Evolution Home Page | Plant Biology Home Page

Identification and classification of human picornaviruses using capsid sequence data

M. Steven Oberste, K Maher, M.R. Flemister, G. Marchetti, D.R. Kilpatrick, and Mark A. Pallansch
Centers for Disease Control and Prevention, Atlanta, GA, USA

Members of the family Picornaviridae, primarily the rhinoviruses and enteroviruses, are the most common viruses that infect humans and also commonly infect a wide variety of mammalian species. Picornaviruses (over 200 serotypes in nine genera) have traditionally been classified by antigenic type based on a serum neutralization assay, often by using overlapping pools of type-specific antisera. The virus antigenicity is the most significant viral property related to immunity from disease, but is technically difficult and time-consuming to assay. We have developed generic VP1 RT-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other human and animal picornaviruses. We have used RT-PCR and amplicon sequencing to identify enteroviruses that were difficult to type by neutralization. Amplicon sequences were compared to a database of all available picornavirus VP1 sequences, including all human enteroviruses, four animal enteroviruses, six human rhinoviruses, and the published sequences of viruses in all other picornavirus genera. In this scheme, a VP1 sequence identity score of at least 75% to any EV prototype strain indicates that the isolate is of the homologous serotype, provided that the second-highest identity score (next closest serotype) is less than 70% (Figure 1). A high score of between 70% and 75% or a second-highest score greater than 70% indicates a tentative identification that must be confirmed by other means, whereas a high score of less than 70% indicates that the sequence of the isolate does not match any sequence in the database. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. In retrospective studies and special sequencing studies, 5-10% of isolates were found to have been mistyped using standard antiserum pools. However, the molecular type was always confirmed as correct using monospecific sera, suggesting that molecular serotyping is more accurate than conventional antigenic methods. Molecular methods have also identified at least four candidate new picornavirus serotypes. RT-PCR coupled with amplicon sequencing is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.

Abstract - Presented at the Virus Evolution Workshop
Ardmore, OK
October 21 - 24th, 1999

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e-mail: mroossinck@noble.org

Dr. Marilyn Roossinck
Plant Biology Division
The Noble Foundation
P.O. Box 2180
Ardmore, OK 73402

phone: 580 224-6630