Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis

Stool samples from sixteen cases of children with meningitis originating fr om four different and geographically isolated parts of Greece were investig ated for enteroviruses. The conventional method of cell culture in four dif ferent cell lines was initially used for the isolation of enteroviruses. Th e results showed a cytopathic effect (CPE) in all cases after two, or even more successive passages in only one cell line (RD), although a less-than-s atisfactory CPE was obtained in many cases. Seroneutralization with RIVM mi xed hyperimmune antisera followed and the isolates were typed as Coxsackie B viruses. The method of RT-PCR with enterovirus-specific primers targeted to the highly conserved 5 ' -UTR of the genome was initially used for the d etection of enteroviruses from the inoculated cell cultures. A positive RT- PCR result was obtained for all of the clinical samples rapidly and accurat ely and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformatio n Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed f irst of all that the isolates had an identical restriction pattern with Cox sackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological associ ation of the isolates. The speed of the molecular methodology that was used in comparison with the conventional methods and its possible significance for the description of virus evolution and circulation in the populations i s discussed. (C) 2001 Wiley-Liss, Inc.