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Journal of Clinical Microbiology, March 1998, p. 796-801, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Detection of Respiratory Syncytial Virus A and B and Parainfluenzavirus 3 Sequences in Respiratory Tracts of Infants by a Single PCR with Primers Targeted to the L-Polymerase Gene and Differential Hybridization

Geneviève Eugene-Ruellan,1 François Freymuth,1,* Chokri Bahloul,2 Hassan Badrane,2 Astrid Vabret,1 and Nöel Tordo2

Laboratoire de Virologie Humaine et Moléculaire, Hopital Universitaire Clémenceau, 14033 Caen,1 and Laboratoire des Lyssavirus, Institut Pasteur, 75015 Paris,2 France

Received 30 July 1997/Returned for modification 13 October 1997/Accepted 1 December 1997

A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed to identify the major agents of bronchiolitis in infants: respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3). Two primer sets (P1-P2 and P1-P3) were selected in a conserved region of the polymerase L gene. In infected cell cultures, this method detected RSVA (n = 14), RSVB (n = 13), and PIV3 (n = 13), with the exclusion of PIV1 (n = 4), PIV2 (n = 3), measles virus (n = 6), mumps virus (n = 4), influenza A virus (n = 11), and influenza B virus (n = 4). The differentiation of the amplicons by restriction fragment length polymorphism (RFLP) showed a PvuII site for PIV3 strains and an AvaII site for RSV strains, with RSVA distinguished from RSVB by BglII. The hybridization-EIA, using three internal probes specific for each virus, correlated with the immunofluorescence assay (IFA) and RFLP results. Clinical aspirates from 261 infants hospitalized with bronchiolitis were tested by IFA, viral isolation technique (VIT), and RT-PCR-EIA. RT-PCR-EIA detected RSV sequences in 103 samples (39.4%), and IFA-VIT detected RSV sequences in 109 cases (41.7%). A few samples (2.6%) were IFA-VIT positive but PCR negative, and one sample was RT-PCR-EIA positive only. RT-PCR-EIA detected PIV3 sequences in 14 of the 15 IFA-VIT-positive isolates. The two methods showed very good correlation (96.9%), but RT-PCR-EIA was clearly more efficient in typing, leaving 5% non-A, non-B isolates, while IFA failed to resolve 23% of the isolates. The two methods contradicted each other for <5% of the isolates.

* Corresponding author. Mailing address: Laboratoire de Virologie Humaine et Moléculaire, Hopital Universitaire, avenue G. Clémenceau, 14033 Caen, France. Phone: (33) 02 31 27 25 54. Fax: (33) 02 31 27 25 57. E-mail: freymuth{at}criuc.unicaen.fr.

Journal of Clinical Microbiology, March 1998, p. 796-801, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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