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

Abstract

Stool samples from sixteen cases of children with meningitis originating from four different and geographically isolated parts of Greece were investigated for enteroviruses. The conventional method of cell culture in four different cell lines was initially used for the isolation of enteroviruses. The 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-satisfactory CPE was obtained in many cases. Seroneutralization with RIVM mixed 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 detection of enteroviruses from the inoculated cell cultures. A positive RT-PCR result was obtained for all of the clinical samples rapidly and accurately and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformation Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed first of all that the isolates had an identical restriction pattern with Coxsackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological association 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 is discussed.

Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

“…These primers yielded amplicons that were 435 bp long; they were adjusted to a concentration of 10 pmol/l in sterile distilled water and were stored at Ϫ20°C (11,32,33,34).…”

PCR–SSCP: A Method for the Molecular Analysis of Genetic Diseases

“…Moreover, the subtraction of mitochondrial cytochrom C oxidase subunit I [74], identification of an enterovirus in aseptic meningitis [75], the diagnosis of fungal infections [76] and the investigation of ribosomal DNA [77] are some more significant examples of its contribution over understanding the above cited diseases.…”

Nucleotide Analysis and Phylogenetic Study of the Homology Boundaries of Coxsackie A and B Viruses

“…The Reverse Transcription of the extracted RNAs was performed using two different protocols as previously described [5] and three different portions of the produced c-DNAs were then amplified using three different primer pairs ( Table 2). The UC53-UG52 primer set [5,[20][21][22][23][24][25][26][27] was selected for the affirmation of viral presence in the samples, whereas the two other sets of primers (292-222/EUC2-EUG3a,b,c) provided the amplicons of the genomic fragments that were, subsequently, sequenced. These included a 350 bp amplicon of the 5¢ moiety of the VP1 capsid gene [6] and a 1452 bp sequence product encompassing the 3¢ end of VP1, the full length of 2A and 2B genes and the 5¢ end of 2C [7].…”