DETECTION OF RESPIRATORY SYNCYTIAL VIRUS-A AND VIRUS-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

A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-P CR-EIA) has been developed to identify the major agents of bronchiolit is in infants: respiratory syncytial viruses A and B (RSVA and RSVB) a nd 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 inf luenza B virus (n = 4). The differentiation of the amplicons by restri ction fragment length polymorphism (RFLP) showed a PvuII site for PIV3 strains and an AvaII site for RSV strains, with RSVA distinguished fr om RSVB by BglII. The hybridization-EIA, using three internal probes s pecific for each virus, correlated with the immunofluorescence assay ( IFA) and RFLP results, Clinical aspirates from 261 infants hospitalize d with bronchiolitis were tested by IFA, viral isolation technique (VI T), 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 1 4 of the 15 IFA-VIT-positive isolates, The two methods showed very goo d correlation (96.9%), but RT-PCR-EIA was clearly more efficient in ty ping, 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.