Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1993 May;31(5):1224–1231. doi: 10.1128/jcm.31.5.1224-1231.1993

Analysis of respiratory syncytial virus genetic variability with amplified cDNAs.

W M Sullender 1, L Sun 1, L J Anderson 1
PMCID: PMC262908  PMID: 8099086

Abstract

Antigenic and genetic heterogeneities exist within the two major antigenic groups of respiratory syncytial (RS) virus. We developed a polymerase chain reaction (PCR)-based assay that not only differentiates the two RS virus groups but allows distinctions within groups on the basis of changes in the nucleotide sequences, as revealed by restriction fragment analysis. In this assay, viral RNA served as a template for cDNA synthesis with extension from a synthetic oligonucleotide primer complementary to bases 164 to 186 in the F protein mRNA. For PCR amplification, two group-specific 5' primers were added. The two primers corresponded to the G protein mRNA sequence of group B (bases 10 to 30) or group A (bases 247 to 267) RS virus. Agarose gel electrophoresis readily discriminated the 1.1-kb group B and the 0.9-kb group A virus amplification products. All 47 viruses tested were assigned to the same group by both PCR and monoclonal antibody reaction pattern analysis. Restriction fragment analysis of the amplified DNAs revealed 12 restriction patterns for group A viruses and 7 restriction patterns for group B viruses, while the monoclonal antibody reaction patterns revealed seven patterns for group A viruses and 3 patterns for group B viruses. Most viruses with the same monoclonal antibody reaction patterns had different restriction patterns, and some viruses with the same restriction patterns had different monoclonal antibody reaction patterns. Thus, the results of the PCR assay concurred with the monoclonal antibody reaction pattern analysis for group classification of RS viruses, while the restriction fragment analysis identified greater diversity within groups than was seen with the monoclonal antibody analysis.

Full text

PDF
1224

Images in this article

1226Image
on p.1226
1226Image
on p.1226
1228Image
on p.1228
1228Image
on p.1228

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akerlind B., Norrby E., Orvell C., Mufson M. A. Respiratory syncytial virus: heterogeneity of subgroup B strains. J Gen Virol. 1988 Sep;69(Pt 9):2145–2154. doi: 10.1099/0022-1317-69-9-2145. [DOI] [PubMed] [Google Scholar]
  2. Anderson L. J., Hendry R. M., Pierik L. T., Tsou C., McIntosh K. Multicenter study of strains of respiratory syncytial virus. J Infect Dis. 1991 Apr;163(4):687–692. doi: 10.1093/infdis/163.4.687. [DOI] [PubMed] [Google Scholar]
  3. Anderson L. J., Hierholzer J. C., Tsou C., Hendry R. M., Fernie B. F., Stone Y., McIntosh K. Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. J Infect Dis. 1985 Apr;151(4):626–633. doi: 10.1093/infdis/151.4.626. [DOI] [PubMed] [Google Scholar]
  4. Cane P. A., Matthews D. A., Pringle C. R. Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses. J Gen Virol. 1991 Sep;72(Pt 9):2091–2096. doi: 10.1099/0022-1317-72-9-2091. [DOI] [PubMed] [Google Scholar]
  5. Cane P. A., Pringle C. R. Respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (SH gene) and restriction mapping (N gene). J Gen Virol. 1991 Feb;72(Pt 2):349–357. doi: 10.1099/0022-1317-72-2-349. [DOI] [PubMed] [Google Scholar]
  6. Collins P. L., Huang Y. T., Wertz G. W. Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. J Virol. 1984 Feb;49(2):572–578. doi: 10.1128/jvi.49.2.572-578.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cristina J., López J. A., Albó C., García-Barreno B., García J., Melero J. A., Portela A. Analysis of genetic variability in human respiratory syncytial virus by the RNase A mismatch cleavage method: subtype divergence and heterogeneity. Virology. 1990 Jan;174(1):126–134. doi: 10.1016/0042-6822(90)90061-u. [DOI] [PubMed] [Google Scholar]
  8. Ferre F., Garduno F. Preparation of crude cell extract suitable for amplification of RNA by the polymerase chain reaction. Nucleic Acids Res. 1989 Mar 11;17(5):2141–2141. doi: 10.1093/nar/17.5.2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Finger R., Anderson L. J., Dicker R. C., Harrison B., Doan R., Downing A., Corey L. Epidemic infections caused by respiratory syncytial virus in institutionalized young adults. J Infect Dis. 1987 Jun;155(6):1335–1339. doi: 10.1093/infdis/155.6.1335. [DOI] [PubMed] [Google Scholar]
  10. Hendry R. M., Burns J. C., Walsh E. E., Graham B. S., Wright P. F., Hemming V. G., Rodriguez W. J., Kim H. W., Prince G. A., McIntosh K. Strain-specific serum antibody responses in infants undergoing primary infection with respiratory syncytial virus. J Infect Dis. 1988 Apr;157(4):640–647. doi: 10.1093/infdis/157.4.640. [DOI] [PubMed] [Google Scholar]
  11. Hendry R. M., Pierik L. T., McIntosh K. Prevalence of respiratory syncytial virus subgroups over six consecutive outbreaks: 1981-1987. J Infect Dis. 1989 Aug;160(2):185–190. doi: 10.1093/infdis/160.2.185. [DOI] [PubMed] [Google Scholar]
  12. Hendry R. M., Talis A. L., Godfrey E., Anderson L. J., Fernie B. F., McIntosh K. Concurrent circulation of antigenically distinct strains of respiratory syncytial virus during community outbreaks. J Infect Dis. 1986 Feb;153(2):291–297. doi: 10.1093/infdis/153.2.291. [DOI] [PubMed] [Google Scholar]
  13. Johnson P. R., Collins P. L. The A and B subgroups of human respiratory syncytial virus: comparison of intergenic and gene-overlap sequences. J Gen Virol. 1988 Nov;69(Pt 11):2901–2906. doi: 10.1099/0022-1317-69-11-2901. [DOI] [PubMed] [Google Scholar]
  14. Johnson P. R., Collins P. L. The fusion glycoproteins of human respiratory syncytial virus of subgroups A and B: sequence conservation provides a structural basis for antigenic relatedness. J Gen Virol. 1988 Oct;69(Pt 10):2623–2628. doi: 10.1099/0022-1317-69-10-2623. [DOI] [PubMed] [Google Scholar]
  15. Johnson P. R., Jr, Olmsted R. A., Prince G. A., Murphy B. R., Alling D. W., Walsh E. E., Collins P. L. Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity. J Virol. 1987 Oct;61(10):3163–3166. doi: 10.1128/jvi.61.10.3163-3166.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson P. R., Spriggs M. K., Olmsted R. A., Collins P. L. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5625–5629. doi: 10.1073/pnas.84.16.5625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Morgan L. A., Routledge E. G., Willcocks M. M., Samson A. C., Scott R., Toms G. L. Strain variation of respiratory syncytial virus. J Gen Virol. 1987 Nov;68(Pt 11):2781–2788. doi: 10.1099/0022-1317-68-11-2781. [DOI] [PubMed] [Google Scholar]
  18. Mufson M. A., Belshe R. B., Orvell C., Norrby E. Subgroup characteristics of respiratory syncytial virus strains recovered from children with two consecutive infections. J Clin Microbiol. 1987 Aug;25(8):1535–1539. doi: 10.1128/jcm.25.8.1535-1539.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mufson M. A., Orvell C., Rafnar B., Norrby E. Two distinct subtypes of human respiratory syncytial virus. J Gen Virol. 1985 Oct;66(Pt 10):2111–2124. doi: 10.1099/0022-1317-66-10-2111. [DOI] [PubMed] [Google Scholar]
  20. Paton A. W., Paton J. C., Lawrence A. J., Goldwater P. N., Harris R. J. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification. J Clin Microbiol. 1992 Apr;30(4):901–904. doi: 10.1128/jcm.30.4.901-904.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  22. Storch G. A., Anderson L. J., Park C. S., Tsou C., Dohner D. E. Antigenic and genomic diversity within group A respiratory syncytial virus. J Infect Dis. 1991 Apr;163(4):858–861. doi: 10.1093/infdis/163.4.858. [DOI] [PubMed] [Google Scholar]
  23. Storch G. A., Park C. S., Dohner D. E. RNA fingerprinting of respiratory syncytial virus using ribonuclease protection. Application to molecular epidemiology. J Clin Invest. 1989 Jun;83(6):1894–1902. doi: 10.1172/JCI114096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Storch G. A., Park C. S. Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus. J Med Virol. 1987 Aug;22(4):345–356. doi: 10.1002/jmv.1890220407. [DOI] [PubMed] [Google Scholar]
  25. Stott E. J., Taylor G., Ball L. A., Anderson K., Young K. K., King A. M., Wertz G. W. Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus. J Virol. 1987 Dec;61(12):3855–3861. doi: 10.1128/jvi.61.12.3855-3861.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sullender W. M., Anderson K., Wertz G. W. The respiratory syncytial virus subgroup B attachment glycoprotein: analysis of sequence, expression from a recombinant vector, and evaluation as an immunogen against homologous and heterologous subgroup virus challenge. Virology. 1990 Sep;178(1):195–203. doi: 10.1016/0042-6822(90)90394-7. [DOI] [PubMed] [Google Scholar]
  27. Sullender W. M., Anderson L. J., Anderson K., Wertz G. W. Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay. J Clin Microbiol. 1990 Aug;28(8):1683–1687. doi: 10.1128/jcm.28.8.1683-1687.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sullender W. M., Mufson M. A., Anderson L. J., Wertz G. W. Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses. J Virol. 1991 Oct;65(10):5425–5434. doi: 10.1128/jvi.65.10.5425-5434.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sullender W. M., Wertz G. W. Synthetic oligonucleotide probes differentiate respiratory syncytial virus subgroups in a nucleic acid hybridization assay. J Clin Microbiol. 1991 Jun;29(6):1255–1257. doi: 10.1128/jcm.29.6.1255-1257.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tsutsumi H., Onuma M., Suga K., Honjo T., Chiba Y., Chiba S., Ogra P. L. Occurrence of respiratory syncytial virus subgroup A and B strains in Japan, 1980 to 1987. J Clin Microbiol. 1988 Jun;26(6):1171–1174. doi: 10.1128/jcm.26.6.1171-1174.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Walpita P., Mufson M. A., Stanek R. J., Pfeifer D., Connor J. D. Distinguishing between respiratory syncytial virus subgroups by protein profile analysis. J Clin Microbiol. 1992 Apr;30(4):1030–1032. doi: 10.1128/jcm.30.4.1030-1032.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walsh E. E., Hall C. B., Schlesinger J. J., Brandriss M. W., Hildreth S., Paradiso P. Comparison of antigenic sites of subtype-specific respiratory syncytial virus attachment proteins. J Gen Virol. 1989 Nov;70(Pt 11):2953–2961. doi: 10.1099/0022-1317-70-11-2953. [DOI] [PubMed] [Google Scholar]
  33. Waris M. Pattern of respiratory syncytial virus epidemics in Finland: two-year cycles with alternating prevalence of groups A and B. J Infect Dis. 1991 Mar;163(3):464–469. doi: 10.1093/infdis/163.3.464. [DOI] [PubMed] [Google Scholar]
  34. Wertz G. W., Collins P. L., Huang Y., Gruber C., Levine S., Ball L. A. Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4075–4079. doi: 10.1073/pnas.82.12.4075. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES