1 Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
2 Task Force for Child Survival and Development, Atlanta, GA, USA
3 Research Center in Infectious Diseases of the Québec University Hospital Center, Department of Microbiology, Laval University, Québec City, Canada GIV 4G2
Correspondence
Dean D. Erdman
dde1{at}cdc.gov
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ABSTRACT |
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GenBank accession numbers for the reported sequence data: AY485232AY485256.
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INTRODUCTION |
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hMPV has been tentatively assigned to the genus Metapneumovirus (van den Hoogen et al., 2001, 2002
) based upon sequence identity and similar genomic organization to avian pneumovirus (APV). Analyses of hMPV and APV sequences have shown that hMPV shares the closest relationship with APV type C (van den Hoogen et al., 2002
; Bastien et al., 2003
). hMPV, along with APV, HRSV and bovine respiratory syncytial virus (BRSV), belongs to the family Paramyxoviridae, subfamily Pneumovirinae, whose members encode the G protein, a type II mucin-like glycoprotein. The G glycoprotein of HRSV induces a group-specific protective immune response and is associated with enhanced HRSV disease (Sparer et al., 1998
; Tripp et al., 2001
). The G glycoproteins of HRSV and BRSV also participate but do not seem to be essential in virus attachment (Teng et al., 2002
; Schlender et al., 2003
).
The genetic variability of the hMPV G gene has not been fully examined, although limited studies have identified two major lineages of hMPV (Boivin et al., 2002; van den Hoogen et al., 2002
; Peret et al., 2002
; Bastien et al., 2003
). To better assess the genetic variability of the hMPV G gene, we sequenced 25 full-length genes representing both major hMPV lineages from Canadian field isolates obtained over five consecutive epidemic seasons (1997 to 2002).
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METHODS |
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RESULTS |
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Phylogenetic analysis of the G gene ORFs identified two major clusters, designated groups 1 and 2 (Fig. 1). Minor clusters that segregated within each major group were designated subgroups A and B, respectively. Bootstrapping analysis showed strong support for both groups and subgroups. Subgroup 1A ORFs varied in length from 660 (hMPV80-1999 and hMPV83-1997) to 654 nt (hMPV16-2000-16 and hMPV17-2000) as the result of early termination due to a single base substitution at position 654 (CAA
TAA). Subgroups 1B and 2A ORFs were all 711 nt in length. Subgroup 2B sequences were distinguished by a 15 nt in-frame insertion located in a polypurine stretch at nucleotide position 480 and a stop codon at nucleotide positions 694696 which resulted in a 696 nt ORF.
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Potential N-glycosylation acceptor sites (Asn-X-Thr/Ser) were also identified among the hMPV G proteins. Among group 1 proteins, two potential N-glycosylation sites unique to subgroup 1A were identified at amino acid positions 101103 and 153155, and one site unique to subgroup 1B was identified at positions 233235. Among group 2 proteins, one site unique to subgroup A was identified at positions 202204 and four sites unique to subgroup 2B were identified at positions 101103 (also present in subgroup 1A), 169171, 188190 and 215217. One N-glycosylation site located at positions 181183 in the extracellular domain was conserved among group 2 proteins, and one site was conserved among all G protein sequences; however, this site was located in the intracellular domain (positions 3032).
Proline content also varied among the G protein groups and subgroups. Subgroups 1A and 2B had a similar proline content, ranging from 6·4 % to 7·8 %, whereas subgroups 2A and 1B had relatively lower (4·2 to 5·1 %) and higher (8·4 to 9·3 %) proline content, respectively. No N-myristylation sites were identified.
Molecular epidemiology
Cocirculation of hMPV groups and subgroups during a single epidemic season within the same community (Québec City) was observed as previously described (Boivin et al. 2002; Peret et al. 2002
) (Table 1
). Eight of 9 (89 %) hMPV field isolates obtained in years 1997 and 1998 were group 2 viruses and all isolates from the 1998 epidemic period were subgroup 2A. In contrast, 15 of 16 (94 %) isolates obtained after 1998 were group 1 viruses, with most isolates from the 2001 and 2002 epidemic periods subgroup 1B. There was no apparent association between the different virus groups/subgroups and age or clinical presentation of the patients, although too few isolates were examined to adequately address these issues.
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DISCUSSION |
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Sequence variation of the hMPV G gene occurred through nucleotide substitutions, use of alternative transcription termination codons, and insertions that retained the original reading frame. Similar types of change have been described for the G gene of other members of the subfamily Pneumovirinae; use of alternative transcription termination codons has been described for BRSV (Furze et al., 1997) and HRSV (Sullender et al., 1991
; Peret et al., 1998
; Martinez et al., 1999
), and in-frame and frame-shift insertions have been identified with HRSV (Sullender et al., 1991
; Peret et al., 1998
). Like APV, HRSV and BRSV (Johnson et al., 1987
; Juhasz et al., 1994
; Bäyon-Auboyer et al., 2000
; Valarcher et al., 2000
), the nucleotide substitutions resulted in a higher rate of amino acid changes, especially in the ectodomain. This suggests amino acid changes are advantageous, possibly due to immunological pressure.
Comparisons of the hMPV G gene with APV, the virus most closely related to hMPV, showed no significant identity. In contrast, amino acid identities between hMPV and APV N, P, M and F genes ranged from 55 to 78 % for APV A; 51 to 77 % for APV B; and 66 to 88 % for APV C (Bastien et al., 2003).
Despite the genetic variability in the hMPV G protein, the overall structural features varied little. The overall serine/threonine content of the hMPV G protein (32 to 34 %), a predictor of degree of glycosylation, was similar to HRSV (29 to 31 %) (Johnson et al., 1987), and greater than that reported for APV (23·5 to 24·6 %) (Alvarez et al., 2003
). Similar to HRSV groups A and B, the hydrophobicity plots of the predicted hMPV G proteins of both major groups were strikingly similar, despite limited amino acid identity.
Previous studies have identified conserved clusters of cysteine residues in the G glycoprotein of HRSV (reviewed by Collins et al. 2001), APV (Bäyon-Auboyer et al. 2000; Alvarez et al., 2003
) and BRSV (Furze et al., 1997
; Elvander et al., 1998
; Valarcher et al. 2000
), although BRSV G gene isolates lacking the central four cysteine residues have been described (Valarcher et al., 2000
). Cystine nooses have been associated with protein conformation and biological signalling (Lapthorn et al., 1995
). In addition, a CX3C motif has been identified in the HRSV G glycoprotein that mediates chemokine mimicry (Tripp et al., 2001
) and an identical motif was recently identified in APV C (Alvarez et al., 2003
). In contrast, there were no analogous cysteine clusters or other conserved amino acid motifs identified in the G ectodomain of hMPV. The lack of conserved sequences in the ectodomain of the G protein between the two major groups of hMPV may reflect differences in the functional properties of these proteins.
The extensive polymorphism of the hMPV G gene seen in this study raised concerns that sequence variability may have resulted from mutations occurring during virus propagation in cell culture. Although culture isolation and passage of hMPV was necessary in most cases to obtain sufficient viral RNA for sequencing, sequences of two virus strains from subgroup 1B (hMPV228-2002 and hMPV193-2002) were obtained directly from clinical specimens. These sequences were virtually identical to those of other hMPV strains from the same subgroup obtained after multiple passages in cell culture. Moreover, sequences from multiple passages of virus strain hMPV82-1997, which displayed a unique insertion sequence, were identical (data not shown). These observations suggest that isolation and limited passage of hMPV in cell culture does not contribute significantly to nucleotide changes in the G gene, which is consistent with findings for BRSV (Larsen et al., 1998) and HRSV (Cane et al., 1994
).
In contrast to our findings, Biacchesi et al. (2003) recently reported nucleotide heterogeneity among cloned sequences of the SH and G genes of strain hMPV75-1998 (CAN98-75), a virus originally isolated from an immunocompromised child with acute lymphoplastic leukaemia (Peret et al., 2002
; Boivin et al., 2002
; Pelletier et al., 2002
). One possible explanation is that a mixed virus population (or quasispecies), an intrinsic feature of RNA viruses (Elena et al., 2000
), was present in the original clinical specimen or arose during in vitro propagation. Alternatively, nucleotide misincorporations during cloning and sequencing may have occurred. We obtained similar sequences (only two third-position mismatches) for the entire G gene from multiple independent RT-PCR amplifications of hMPV75-1998 and, unlike the sequence reported by Biacchesi et al. (2003)
, the hMPV75-1998 G gene sequences we obtained were nearly identical with sequences from independently isolated hMPV strains circulating during the same epidemic period (hMPV76-1998; hMPV77-1998; hMPV78-1998 and hMPV79-1998).
Community studies of HRSV circulation patterns have documented the temporal displacement of G gene variants of HRSV in successive years, attributing this to changes in the herd immunity of the population in response to antigenic differences between the predominant circulating strains (Cane & Pringle, 1995; Peret et al., 1998
). Although our dataset is limited and geographically restricted, there was a clear indication of a shift between the two major groups of hMPV during the study period, which we speculate may be in response to immune-mediated pressure.
In conclusion, our study documents the extensive polymorphism of the hMPV G gene and confirms its basic features of a type II mucin-like glycoprotein. Long-term studies sampling wider geographical areas will be necessary to provide a more complete picture of the sequence diversity of hMPV. The importance of the G protein variation to the immunobiology of hMPV has yet to be determined and warrants further studies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bastien, N., Normand, S., Taylor, T., Ward, D., Peret, T. C. T., Boivin, G., Anderson, L. J. & Li, Y. (2003). Sequence analysis of the N, P, M and F genes of Canadian human metapneumovirus strains. Virus Res 93, 5162.[CrossRef][Medline]
Bäyon-Auboyer, M.-H., Arnauld, C., Toquin, D. & Eterradossi, N. (2000). Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup. J Gen Virol 81, 27232733.
Biacchesi, S., Skiadopoulos, M. H., Boivin, G., Hanson, C. T., Murphy, B. R., Collins, P. L. & Buchholz, U. J. (2003). Genetic diversity between human metapneumovirus subgroups. Virology 315, 19.[CrossRef][Medline]
Boivin, G., Abed, Y., Pelletier, G. & 6 other authors (2002). Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis 186, 13301334.[CrossRef][Medline]
Boivin, G., De Serres, G., Cote, S., Gilca, R., Abed, Y., Rochette, L., Bergeron, M. G. & Dery, P. (2003). Human metapneumovirus infections in hospitalized children. Emerg Infect Dis 9, 634640.[Medline]
Cane, P. A. & Pringle, C. R. (1995). Evolution of subgroup A respiratory syncytial virus: evidence for progressive accumulation of amino changes in the attachment protein. J Virol 69, 29182925.[Abstract]
Cane, P. A., Matthews, D. A. & Pringle, C. R. (1994). Analysis of respiratory syncytial virus strain variation in successive epidemics in one city. J Clin Microbiol 32, 14.[Abstract]
Elena, S. F., Miralles, R., Cuevas, J. M., Turner, P. E. & Moya, A. (2000). The two faces of mutation: extinction and adaptation in RNA viruses. IUBMB Life 49, 59.[CrossRef][Medline]
Elvander, M., Vilcek, S., Baule, C., Uttenthal, A., Ballagi-Pordany, A. & Belak, S. (1998). Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains. J Gen Virol 79, 29392946.[Abstract]
Falsey, A. R., Erdman, D. D., Anderson, L. J. & Walsh, E. E. (2003). Human metapneumovirus infections in young and elderly adults. J Infect Dis 187, 785790.[CrossRef][Medline]
Furze, J. M., Roberts, S. R., Wertz, G. W. & Taylor, G. (1997). Antigenically distinct G glycoproteins of BRSV strains share a high degree of genetic homogeneity. Virology 231, 4858.[CrossRef][Medline]
Jartti, T., van den Hoogen, B., Garofalo, R. P., Osterhaus, A. D. & Ruuskanen, O. (2002). Metapneumovirus and acute wheezing in children. Lancet 360, 13931394.[CrossRef][Medline]
Johnson, P. R., Spriggs, M. K., Olmsted, R. A. & Collins, P. L. (1987). 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 84, 56255629.[Abstract]
Juhasz, K. & Easton, A. J. (1994). Extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two distinct subgroups. J Gen Virol 75, 28732880.[Abstract]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Lapthorn, A. J., Janes, R. W., Isaacs, N. W. & Wallace, B. A. (1995). Cystine nooses and protein specificity. Nat Struct Biol 2, 266268.[Medline]
Larsen, L. E., Uttenthal, A., Arctander, P. & 6 other authors (1998). Serological and genetic characterization of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaptation and passages in cell culture or calves. Vet Microbiol 62, 265279.[CrossRef][Medline]
Ling, R., Easton, A. J. & Pringle, C. R. (1992). Sequence analysis of the 22K, SH and G genes of turkey rhinotracheitis virus and their intergenic regions reveals a gene order different from that of other pneumoviruses. J Gen Virol 73, 17091715.[Abstract]
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. J Gen Virol 180, 125130.
Pelletier, G., Dery, P., Abed, Y. & Boivin, G. (2002). Respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child. Emerg Infect Dis 8, 976978.[Medline]
Peret, T. C. T., 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. J Gen Virol 79, 22212229.[Abstract]
Peret, T. C. T., Boivin, G., Li, Y., Couillard, M., Humphrey, C., Osterhaus, A. D., Erdman, D. D. & Anderson, L. J. (2002). Characterization of human metapneumoviruses isolated from patients in North America. J Infect Dis 185, 16601663.[CrossRef][Medline]
Schlender, J., Zimmer, G., Herrler, G. & Conzelmann, K. K. (2003). Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection. J Virol 77, 46094616.
Sparer, T. E., Matthews, S., Hussell, T., Rae, A. J., Garcia-Barreno, B., Melero, J. A. & Openshaw, P. J. (1998). Eliminating a region of respiratory syncytial virus attachment protein allows induction of protective immunity without vaccine-enhanced lung eosinophilia. J Exp Med 187, 19211926.
Stockton, J., Stephenson, I., Fleming, D. & Zambon, M. (2002). Human metapneumovirus as a cause of community-acquired respiratory illness. Emerg Infect Dis 8, 897901.[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. J Virol 65, 54255434.[Medline]
Swofford, D. L. (1999). PAUP*. Phylogenetic Analysis using Parsimony (* and Other Methods). Sunderland, MA: Sinauer Associates.
Teng, M. N. & Collins, P. L. (2002). The central conserved cystine noose of the attachment G protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo. J Virol 76, 61646171.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tripp, R. A., Jones, L. P., Haynes, L. M., Zheng, H., Murphy, P. M. & Anderson, L. J. (2001). CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein. Nat Immunol 2, 732738.[CrossRef][Medline]
Valarcher, J. F., Schelcher, F. & Bourhy, H. (2000). Evolution of bovine respiratory syncytial virus. J Virol 74, 1071410728.
van den Hoogen, B. G., de Jong, J. C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R. A. M. & Osterhaus, A. D. M. E. (2001). A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7, 719724.[CrossRef][Medline]
van den Hoogen, B. G., Bestebroer, T. M., Osterhaus, A. D. M. E. & Fouchier, R. A. M. (2002). Analysis of the genomic sequence of a human metapneumovirus. Virology 295, 119132.[CrossRef][Medline]
Received 13 July 2003;
accepted 13 November 2003.