Genetic variability among Group A and B respiratory syncytial virus isolates from a large referral hospital in New Delhi, India

Abstract

Respiratory syncytial virus (RSV) is an important childhood pathogen of acute lower respiratory infections in developed and developing countries. The molecular epidemiology of RSV in India is largely unknown. The present study was undertaken to standardize and evaluate reverse transcription-PCR (RT-PCR) for the rapid and simultaneous detection of RSV groups A and B in clinical samples and to study intragroup genetic variability. RT-PCR was evaluated by comparing the results of seminested RT-PCR with centrifugation-enhanced cultures on 200 nasopharyngeal aspirates from children with acute lower respiratory infections. RSV was isolated in 34 nasopharyngeal aspirates by centrifugation-enhanced cultures and identified in 45 samples by RT-PCR. In 15 samples RSV was identified by seminested RT-PCR alone and in four by centrifugation-enhanced cultures alone. Of the 45 samples positive for RSV by nested PCR, 15 belonged to group A, 29 to group B, and one sample suggested a mixed infection. Group B RSV predominated in both years of the 2-year study. Genetic variability within RSV groups was studied by restriction fragment analysis of 35 PCR products. Among both group A and group B RSV, two different composite patterns were observed. Thus, RSV was found to be a major pathogen of acute lower respiratory tract infections in India, as it was detected in 24.5% of children by RT-PCR. RT-PCR provides a sensitive method for detection and typing of RSV group A and B viruses in clinical samples as well as a means to study intragroup variations. However, a higher sensitivity of detection of RSV in clinical samples can be obtained by its combination with additional techniques, such as virus cultivation.

FIG. 1.

(A) Agarose gel showing the results of external RT-PCR on clinical samples (nasopharyngeal aspirates). lane M, φX174 HaeIII digest (molecular weight marker); lane 1, negative control; lane 2, positive control; lanes 3 to 7, clinical samples. A band of 1.1 kb is observed in lanes 2, 3, 5, and 7. (B) Agarose gel showing the results of seminested PCR on clinical samples lane M, φX174 HaeIII digest (molecular weight marker); lane 1, negative control; lane 2, group A positive control; lane 3, group B positive control; lanes 3 to 9, clinical samples. A band of 0.9 kb of group A RSV is seen in lanes 2, 8, and 9, and a band of 0.78 kb of group B RSV is seen in lanes 3, 5 and 6. No bands were seen in lanes 4, 7, and 10.

FIG. 2.

Agarose gel showing restriction enzyme pattern of group A RSV with RsaI, HincII, and PstI enzymes. Panel A (digestion with RsaI): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.9-kb uncut amplicon and digested amplicon of positive control of group A, respectively, lanes 3 to 6, digested amplicons of group A sample strains. The ra1 pattern was seen in lanes 2 to 5, and the ra2 pattern was seen in lane 6. Panel B (digestion with HincII): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.9-kb uncut amplicon and digested amplicon of positive control of group A, respectively; lanes 3 to 5, digested amplicons of group A sample strains. The ha1 pattern was seen in lanes 2 to 5. Panel C (digestion with PstI): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.9-kb uncut amplicon and digested amplicon of positive control of group A, respectively; lanes 3 to 6, digested amplicons of group A sample strains. The pa1 pattern was seen in lanes 2 to 5, and the pa2 pattern was seen in lane 6.

FIG. 3.

Agarose gel showing restriction enzyme pattern of group B RSV with RsaI, HincII, PstI, and AluI. Panel A (digestion with RsaI): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.78-kb uncut amplicon and digested amplicon of positive control of group B, respectively; lanes 3 and 4, digested amplicons of group B sample strains. The rb1 pattern was seen in lanes 2 to 4. Panel B (digestion with HincII): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.78-kb uncut amplicon and digested amplicon of positive control of group B, respectively; lane 3, digested amplicons of group B sample strain. The hb1 pattern (uncut) was seen in lanes 2 and 3. Panel C (digestion with PstI): lane M, φX174 HaeIII digest (molecular weight marker); lanes 1 and 2, 0.78-kb uncut amplicon and digested amplicon of positive control of group B, respectively; lane 3, digested amplicons of group B sample strain. The pb1 pattern (uncut) was seen in lanes 2 and 3. Panel D (digestion with AluI): lane M, φX174 HaeIII digest (molecular weight marker), lanes 1 and 2, 0.78-kb uncut amplicon and digested amplicon of positive control of group B, respectively; lanes 3 and 4, digested amplicons of group B sample strains. The ab1 pattern was seen in lanes 2 and 4, and the ab2 (uncut) pattern was seen in lane 3.

FIG. 4.

Schematic diagram of restriction enzyme patterns of amplicons of seminested PCR. Lane M, øX174 molecular weight markers; ua, undigested amplicon of group A RSV; ra1, restriction endonuclease pattern of group A strains with RsaI; ra2, restriction endonuclease pattern of group A strains with RsaI; ha1, restriction endonuclease pattern of group A strains with HincII; pa1, restriction endonuclease pattern of group A strains with PstI; pa2, restriction endonuclease pattern of group A strains with PstI; ub, undigested amplicon of group B RSV; rb1, restriction endonuclease pattern of group B strains with RsaI; hb1, restriction endonuclease pattern of group B strains with HincII; pb1, restriction endonuclease pattern of group B strains with PstI; ab1, restriction endonuclease pattern of group B strains with AluI; ab2, restriction endonuclease pattern of group B strains with AluI.