National Institute for Virology, Private bag X4, 2131 Sandringham, South Africa1
Pediatric Infectious Disease Research Unit, University of the Witwatersrand, South Africa2
Author for correspondence: Marietjie Venter. Fax +27 11 3214212. e-mail Mariav{at}NIV.ac.za
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Most studies on RSV G-protein variability have been based on subgroup A isolates (Cane et al., 1991 , 1999
; Garcia et al., 1994
; Cane & Pringle, 1995a
; Cane, 1997
), with little information available on subgroup B isolates (Sullender et al., 1990
, 1991
; Coggins et al., 1998
; Martinez et al., 1999
), and few studies on both subgroups (Coggins et al., 1998
; Peret et al., 1998
, 2000
; Choi & Lee, 2000
). Our knowledge of the molecular epidemiology of RSV has so far been based mainly on studies done in the developed world with few reports from Africa (Cane et al., 1999
; Roca et al., 2001
). No data are available about the molecular epidemiology over consecutive seasons from sub-Saharan Africa, which has one of the severest HIV/AIDS epidemics in the world (Lepage et al., 1998
).
In this report we examine the molecular epidemiology and evolutionary pattern of RSV over four consecutive epidemics among children in a tertiary academic hospital serving Soweto in South Africa. We report on the co-circulation pattern and genetic variability of both subgroup A and B isolates, and compare the relatedness of RSV isolates from South Africa with genotypes identified thus far from North America as well as RSV sequences from the rest of the world available on the GenBank.
RSV-positive nasal pharyngeal aspirates (NPA) from children (060 months), hospitalized for ALRTI at Chris Hani-Baragwanath hospital (Soweto, South Africa), were stored at -80 °C over four consecutive years (19972000). A total of 225 specimens out of 1187 RSV-positive isolates (18·9%) were selected systematically from every month RSV was isolated (JanuaryNovember, 19972000) for subtyping: 44 from 1997 (42%); 69 from 1998 (22%); 55 from 1999 (15%) and 57 from 2000 (14%). RNA was isolated from NPA as described before (Madhi et al., 2001 ) and subgrouped by RTPCR (Madhi et al., 2001
; Sullender et al., 1993
) followed by an additional nesting step to improve DNA yields for sequencing. Of the 225 isolates, 144 belonged to subgroup A and 81 to subgroup B. During 1997 subgroup A and B co-circulated approximately equally, while subgroup A predominated during 1999 (74%) and 2000 (89%). Subgroup A has been reported to predominate in three out of four seasons in other geographical areas (Hall et al., 1990
). Although subgroup A was clearly dominant in two of the seasons we studied, subgroup B played at least an equally significant role in the other two seasons, suggesting an important role for both subgroups in South African epidemics.
The nucleotide sequence of 270 nucleotides at the C terminus of the G protein gene was determined for 65 subgroup A and 38 subgroup B isolates, spanning each of the four RSV seasons, using fluorescent dye-terminators on an ABI 377 sequencer (Perkin-Elmer Applied Biosystems). This corresponds to nucleotides 649918 of prototype strain A2 (subgroup A) and nucleotides 652921 of prototype strain 18537 (subgroup B) G-protein genes (Johnson et al., 1987 ). Unique sequences were selected from each year (45 subgroup A and 35 subgroup B isolates) for phylogenetic analysis (Fig. 1
). Subgroups were genotyped separately by phylogenetic comparisons with sequences previously assigned to specific genotypes (Peret et al., 1998
, 2000
) and by comparison to various other sequences from around the world (Fig. 1
). Authenticity of new genotypes was tested by performing Blast searches with representatives of each cluster formed by South African sequences and including the closest related sequences identified in GenBank in the phylogenetic analysis. Sequences were arbitrarily considered a genotype if they clustered together with bootstrap values of 70100%. After analysis of our data we further refined these criteria to isolates with a pairwise-distance (p-distance) of less than 0·07 to all other members in the same phylogenetic cluster. One sequence of each of two subgroup A and the two subgroup B clusters of sequences identified in Mozambique in 1999 were also included in the analysis (Roca et al., 2001
), since Mozambique is the closest geographical region to our study site where sequencing data are available.
|
|
These results suggest that SAA1, SAB1, SAB3 and SAB2 are new genotypes of which the three former have so far been identified only in South Africa. South African RSV isolates also clustered with three previously assigned subgroup A and two subgroup B genotypes, confirming the notion that RSV variants can spread worldwide, but also that RSV evolution is dynamic, with new variants appearing in individual communities (Garcia et al., 1994 ; Cane & Pringle, 1992
). Comparison of the Mozambiquen isolates to South African isolates also suggests transfer between communities, as all genotypes identified in Mozambique occurred in the same year in South Africa although at low frequencies. Neither prototype strain A2 nor B1, currently used for the production of experimental vaccines, clustered with any of the South African isolates. The reference sequences (Peret et al., 1998
, 2000
) were useful for assigning the South African isolates to genotypes; however, we suggest that a standardized method for genotyping of RSV would allow more effective comparison of viruses circulating worldwide.
Sequence divergence was higher at the amino acid level than at nucleotide level, confirming previous findings (Sullender et al., 1991 ; Cane & Pringle, 1995a
; Martinez et al., 1999
). The predicted amino acid sequences of isolates in the different genotypes were compared to prototype strains A2 and B1 for subgroup A and B, respectively (Fig. 2
). Genotype-specific amino acid substitutions could be identified, a phenomenon previously described (Peret et al., 1998
). The G-protein of South African isolates (relative to prototype strains A2 and B1) consisted of 297 (SAA1) or 298 (GA7) amino acids, or both (GA2 and GA5), for subgroup A, and 295 (GB4, SAB1 and SAB2) or 292, 295 and 299 (GB3 and SAB3) amino acids for subgroup B. Subgroup A isolates used the UAG stop codon in either nucleotide position 259 or 262. Subgroup B isolates used either the UAA or the UAG stop codons in positions 241, 250 or 262. Changes in stop codon usage are thought to be associated with antigenic variation in RSV escape mutants that recognize strain-specific epitopes (Melero et al., 1997
). The mean proportion of nucleotide substitutions that resulted in amino acid changes among South African isolates was 70·8 and 67% for subgroups A and B, respectively, suggesting positive selection, possibly due to immunological pressure. Collectively, this suggests immune evasion by RSV that could be accomplished through epitope changes. Although the significance of this for the development of effective vaccines remains to be determined, it may be necessary to use more recently isolated strains from different areas for vaccine production.
|
In summary, we have reported the first molecular epidemiological study of RSV over consecutive seasons in Southern Africa and identified four new genotypes. The high percentages of nucleotide changes that resulted in amino acid changes suggest a strong selective pressure in both RSV subgroups. Evidence of different circulation patterns between subgroup A and B viruses over consecutive seasons was found. Subgroup A revealed more variability and displacement of genotypes while subgroup B remained more consistent. These findings provide additional information regarding RSV evolution and strain circulation worldwide and may contribute to future vaccine development.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Cane, P. A. (1997). Analysis of linear epitopes recognised by the primary human antibody response to a variable region of the attachment (G) protein of respiratory syncytial virus. Journal of Medical Virology 51, 297-304.[Medline]
Cane, P. A. & Pringle, C. R. (1992). Molecular epidemiology of respiratory syncytial virus: rapid identification of subgroup A lineages. Journal of Virological Methods 40, 297-306.[Medline]
Cane, P. & Pringle, C. (1995a). Evolution of subgroup A respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein. Journal of Virology 69, 2918-2925.[Abstract]
Cane, P. & Pringle, C. (1995b). Molecular epidemiology of respiratory syncytial virus: a review of the use of reverse transcription-polymerase chain reaction in the analysis of genetic variability. Electrophoresis 16, 329-333.[Medline]
Cane, P., Matthews, D. & Pringle, C. (1991). Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses. Journal of General Virology 72, 2091-2096.[Abstract]
Cane, P. A., Matthews, D. A. & Pringle, C. R. (1994). Analysis of respiratory syncytial virus strain variation in successive epidemics in one city. Journal of Clinical Microbiology 32, 1-4.[Abstract]
Cane, P. A., Weber, M., Sanneh, M., Dackour, R., Pringle, C. R. & Whittle, H. (1999). Molecular epidemiology of respiratory syncytial virus in The Gambia. Epidemiology and Infection 122, 155-160.[Medline]
Choi, E. & Lee, H. (2000). Genetic diversity and molecular epidemiology of the G protein of subgroups A and B of respiratory syncytial viruses isolated over 9 consecutive epidemics in Korea. Journal of Infectious Diseases 181, 1547-1556.[Medline]
Coggins, W. B., Lefkowitz, E. J. & Sullender, W. M. (1998). Genetic variability among group A and group B respiratory syncytial viruses in a childrens hospital. Journal of Clinical Microbiology 36, 3552-3557.
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Department of Genetics, University of Washington, Seattle, WA, USA.
Garcia, O., Martin, M., Dopazo, J., Arbiza, J., Frabasile, S., Russi, J., Hortal, M., Perez-Brena, P., Martinez, I., Garcia-Barreno, B. and others (1994). Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. Journal of Virology 68, 54485459.[Abstract]
Hall, C. B., Walsh, E. E., Schnabel, K. C., Long, C. E., McConnochie, K. M., Hildreth, S. W. & Anderson, L. J. (1990). Occurrence of groups A and B of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children. Journal of Infectious Diseases 162, 1283-1290.[Medline]
Johnson, P. J., Olmsted, R., Prince, G., Murphy, B., Alling, D., Walsh, E. & Collins, P. (1987). Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity. Journal of Virology 61, 3163-3166.[Medline]
Karron, R. A., Buonagurio, D. A., Georgiu, A. F., Whitehead, S. S., Adamus, J. E., Clements-Mann, M. L., Harris, D. O., Randolph, V. B., Udem, S. A., Murphy, B. R. & Sidhu, M. S. (1997). Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proceedings of the National Academy of Sciences, USA 94, 13961-13966.
Kumar, S., Tamura, K., Jakobsen, I. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analyses software. Version 2.0. University Park: Pennsylvania State University.
Lepage, P., Spira, R., Kalibala, S., Pillay, K., Giaquinto, C., Castetbon, K., Osborne, C., Courpotin, C. & Dabis, F. (1998). Care of human immunodeficiency virus-infected children in developing countries. International Working Group on Mother-to-Child Transmission of HIV. Pediatric Infectious Disease Journal 17, 581-586.[Medline]
Madhi, S., Venter, M., Madhi, A., Petersen, K. & Klugman, H. (2001). Differing manifestations of respiratory syncytial virus-associated severe lower respiratory tract infections in human immunodeficiency virus type 1-infected and uninfected children. Pediatric Infectious Disease Journal 20, 164-170.[Medline]
Martinez, I., Valdes, O., Delfraro, A., Arbiza, J., Russi, J. & Melero, J. A. (1999). Evolutionary pattern of the G glycoprotein of human respiratory syncytial viruses from antigenic group B: the use of alternative termination codons and lineage diversification. Journal of General Virology 80, 125-130.[Abstract]
Melero, J. A., Garcia-Barreno, B., Martinez, I., Pringle, C. R. & Cane, P. A. (1997). Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. Journal of General Virology 78, 2411-2418.
Mufson, M., Belshe, R., Orvell, C. & Norrby, E. (1988). Respiratory syncytial virus epidemics: variable dominance of subgroups A and B strains among children, 19811986. Journal of Infectious Diseases 157, 143-148.[Medline]
Peret, T. C., Hall, C. B., Schnabel, K. C., Golub, J. A. & Anderson, L. J. (1998). Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. Journal of General Virology 79, 2221-2229.[Abstract]
Peret, T. C., Hall, C. B., Hammond, G. W., Piedra, P. A., Storch, G. A., Sullender, W. M., Tsou, C. & Anderson, L. J. (2000). Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America. Journal of Infectious Diseases 181, 1891-1896.[Medline]
Roca, A., Loscertales, M. P., Quinto, L., Perez-Brena, P., Vaz, N., Alonso, P. L. & Saiz, J. C. (2001). Genetic variability among group A and B respiratory syncytial viruses in Mozambique: identification of a new cluster of group B isolates. Journal of General Virology 82, 103-111.
Selwyn, B. (1990). The epidemiology of acute respiratory tract infection in young children: comparison of findings from several developing countries. Reviews in Infectious Diseases 12, 870-888.
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Molecular Biology and Evolution 13, 964-969.
Sullender, W. (2000). Respiratory syncytial virus genetic and antigenic diversity. Clinical Microbiology Reviews, Jan., 1-15.
Sullender, W. M., Anderson, K. & Wertz, G. W. (1990). 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 178, 195-203.[Medline]
Sullender, W. M., Mufson, M. A., Anderson, L. J. & Wertz, G. W. (1991). Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses. Journal of Virology 65, 5425-5434.[Medline]
Sullender, W. M., Sun, L. & Anderson, L. J. (1993). Analysis of respiratory syncytial virus genetic variability with amplified cDNAs. Journal of Clinical Microbiology 31, 1224-1231.[Abstract]
Sullender, W. M., Mufson, M. A., Prince, G. A., Anderson, L. J. & Wertz, G. W. (1998). Antigenic and genetic diversity among the attachment proteins of group A respiratory syncytial viruses that have caused repeat infections in children. Journal of Infectious Diseases 178, 925-932.[Medline]
Wertz, G. W., Collins, P. L., Huang, Y., Gruber, C., Levine, S. & Ball, L. A. (1985). Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proceedings of the National Academy of Sciences, USA 82, 4075-4079.[Abstract]
Received 2 March 2001;
accepted 17 May 2001.