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

Abstract

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.

FIG. 1

Sequences deduced from the alignment of the functional motifs A and C in the L gene of RSVA, PIV3, Sendai virus (SEND), PIV2, measles virus (MEAS), mumps virus (MUMP), simian virus (SV5), and NDV. The upper line shows the amino acid sequences of the motifs (uppercase letters, conserved residues; lowercase letters, variable residues). The corresponding nucleotide sequences are aligned with reference to a consensus (CONS). Their positions are given with reference to the initiation codon of each L protein (₁ATG). The structure of P3 is given with reference to P2. Dashes indicate conserved nucleotides.