RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus type 71 strains from Asia

A specific and sensitive method based on RT-PCR was developed to detect ent erovirus 71 (EV71) from patients with hand, foot and mouth disease, myocard itis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from c onserved parts of the VP1 capsid gene were designed on the basis of good co rrelation with sequences of EV71 strains. These primers successfully amplif ied 44 strains of EV71 including 34 strains isolated from Singapore in 1997 and 1998, eight strains from Malaysia isolated in 1997 and 1998, one Japan ese strain and the neurovirulent strain EV71/7423/MS/87, RT-PCR of 30 strai ns of other enteroviruses including coxsackievirus A and B, and echoviruses failed to give any positive amplicons. Hence, RT-PCR with these primers sh owed 100% correlation with serotyping. Direct sequencing of the RT-PCR prod ucts of 20 EV71 strains revealed a distinct cluster with two major subgroup s, thus enabling genetic typing of the viruses. The genetic heterogeneity o f these strains culminated in amino acid substitutions within the VPI, VP2 and VP3 regions. The sequencing of a 2.9 kb fragment comprising the capsid region and the major part of 5' UTR of two Singapore strains revealed that they belonged to a group distinct from the prototype EV71/BrCr strain and t he EV71/7423/MS/87 strain. The dendrogram generated from 341 bp fragments w ithin the VP1 region revealed that the strains of Singapore, Malaysia and T aiwan belong to two entirely different EV71 genogroups, distinct from the t hree genogroups identified in another recent study. (C) 2000 Elsevier Scien ce B.V, All rights reserved.