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Journal of Clinical Microbiology, December 2002, p. 4554-4560, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4554-4560.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens

Stefania Manzara,1 Michele Muscillo,2 Giuseppina La Rosa,2 Cinzia Marianelli,2 Paola Cattani,1* and Giovanni Fadda1

Institute of Microbiology, Università Cattolica del Sacro Cuore,1 Istituto Superiore di Sanità, Rome, Italy2

Received 8 April 2002/ Accepted 19 August 2002

Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5' noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.

* Corresponding author. Mailing address: Institute of Microbiology, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy. Phone: 39 06 30154336. Fax: 39 06 3051152. E-mail: p_universi{at}libero.it.

Journal of Clinical Microbiology, December 2002, p. 4554-4560, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4554-4560.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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