Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010–2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene. Among the 201 consecutive RSV isolates studied, RSV-A (55.7%) was more commonly observed than RSV-B (42.3%). 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication.
References
[1]
Sullender WM, Mufson MA, Prince GA, Anderson LJ, Wertz GW (1998) Antigenic and genetic diversity among the attachment proteins of group A respiratory syncytial viruses that have caused repeat infections in children. The Journal of Infectious Diseases (4): 178. 925–32.
[2]
Beem M (1967) Repeated infections with respiratory syncytial virus. Journal of Immunology 98(6): 1115–22.
[3]
Cane PA (2001) Molecular epidemiology of respiratory syncytial virus. Reviews in Medical Virology 11(2): 103–16.
[4]
Anderson LJ, Hierholzer JC, Tsou C, Hendry RM, Fernie BF, et al. (1985) Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. The Journal of Infectious Diseases 151(4): 626–33.
[5]
Mufson MA, Orvell C, Rafnar B, Norrby E (1985) Two distinct subtypes of human respiratory syncytial virus. J Gen Virol 66: 2111–2124.
[6]
Levine S, Klaiber-Franco R, Paradiso PR (1987) Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 68: 2521–2524.
[7]
Walsh EE, Hruska J (1983) Monoclonal antibodies to respiratory syncytial virus proteins: Identification of the fusion protein. J Virol 47: 171–177.
[8]
Collins PL, Chanock RM, Murphy BR (2001) Fields virology, 4th ed. pp. 1443–1485. Lippincott Williams & Wilkins, Philadelphia, PA.
Peret TC, Hall CB, Schnabel KC, Golub JA, Anderson LJ (1998) Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol 79: 2221–2229.
[11]
Peret TC, Hall CB, Hammond GW, Piedra PA, Storch GA, et al. (2000) Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America. J Infect Dis 181: 1891–1896.
[12]
Venter M, Madhi SA, Tiemessen CT, Schoub BD (2001) Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: Identification of new subgroup A and B genotypes. J Gen Virol 82: 2117–2124.
[13]
Shobugawa Y, Saito R, Sano Y, Zaraket H, Suzuki Y, et al. (2009) Emerging genotypes of human respiratory syncytial virus subgroup A among patients in Japan. J Clin Microb 47: 2475–2482.
[14]
Trento A, Viegas M, Galiano M, Videla C, Carballal G, et al. (2006) Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication. J Virol 80: 975–984.
[15]
Trento A, Galiano M, Videla C, Carballal G, Garcia-Barreno B, et al. (2003) Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. J Gen Virol 84: 3115–3120.
[16]
Sato M, Saito R, Sakai T, Sano Y, Nishikawa M, et al. (2005) Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons. J Clin Microb 43: 36–40.
[17]
Dapat IC, Shobugawa Y, Sano Y, Saito R, Sasaki A, et al. (2010) New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan. J Clin Microb 48: 3423–3427.
[18]
Johnson PR, Spriggs MK, Olmsted RA, Collins PL (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: 5625–5629.
[19]
Lambert DM (1988) Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 164: 458–66.
[20]
Garcia-Beato R, Martinez I, Franci C, Real FX, Garcia-Barreno B, et al. (1996) Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein. Virology 221: 301–309.
[21]
Dewhurst-Maridor G, Simonet V, Bornand JE, Nicod LP, Pache JC (2004) Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus. J Virol Methods 120: 41–49.
[22]
Sullender WM, Sun L, Anderson LJ (1993) Analysis of respiratory syncytial virus genetic variability with amplified cDNAs. J Clin Microb 31: 1224–1231.
[23]
Parveen S, Sullender WM, Fowler K, Lefkowitz EJ, Kapoor SK, et al. (2006) Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India. J Clin Microb 44: 3055–3064.
[24]
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol (In press).
[25]
Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111–120.
[26]
Yang Z (1997) PAML: A program package for phylogenetic analysis by maximum likelihood. Computer Applications in the Biosciences: CABIOS 13: 555–6.
[27]
Yang Z (2007) PAML 4: a program package for phylogenetic analysis by maximum likelihood. Mol Biol Evol 24: 1586–1591.
[28]
Yang Z, Wong WS, Nielsen R (2005) Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol 22: 1107–1118.
[29]
Gupta R, Jung E, Brunak S (2004) Prediction of N-glycosylation sites in human proteins. NetNGlyc 1.0. (NetNGlyc website. Available: http://www.cbs.dtu.dk/services/NetNGlyc/？Accessed on 2012 Feb 07.).
[30]
Julenius K, Molgaard A, Gupta R, Brunak S (2005) Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15: 153–164.
[31]
Zuker M, Mathews DM, Turner DH (1999) Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide in RNA Biochemistry and Biotechnology. Barciszewski J and Clark BFC, editors. NATO ASI Series, Kluwer Academic Publishers, Dordrecht, NL 11–43.
[32]
Wright BE, Reschke DK, Schmidt KH, Reimers JM, Knight W (2003) Predicting mutation frequencies in stem-loop structures of derepressed genes: implications for evolution. Mol Microb 48: 429–441.
[33]
Hall CB, Walsh EE, Schnabel KC, Long CE, McConnochie KM, et al. (1990) Occurrence of groups A and B of respiratory syncytial virus over 15 years: Associated epidemiologic and clinical characteristics in hospitalized and ambulatory children. J Infect Dis 162: 1283–1290.
[34]
Garcia O, Martin M, Dopazo J, Arbiza J, Frabasile S, et al. (1994) Evolutionary pattern of human respiratory syncytial virus (subgroup A): Cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. J Virol 68: 5448–5459.
[35]
Zlateva KT, Lemey P, Moes E, Vandamme AM, Van Ranst M (2005) Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Gen Virol 79: 9157–9167.
[36]
Cane PA, Matthews DA, Pringle CR (1994) Analysis of respiratory syncytial virus strain variation in successive epidemics in one city. J Clin Microb 32: 1–4.
[37]
Kuroiwa Y, Nagai K, Okita L, Yui I, Kase T, et al. (2005) A phylogenetic study of human respiratory syncytial viruses group A and B strains isolated in two cities in Japan from 1980–2002. J Med Virol 76: 241–247.
[38]
Melero JA, Garcia-Barreno B, Martinez I, Pringle CR, Cane PA (1997) Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. J Gen Virol 78: 2411–2418.
[39]
Cane PA, Pringle CR (1995) Evolution of subgroup A respiratory syncytial virus: Evidence for progressive accumulation of amino acid changes in the attachment protein. J Virol 69: 2918–2925.
[40]
Martinez I, Dopazo J, Melero JA (1997) Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants. J Gen Virol 78: 2419–2429.
[41]
Lazar I, Canaan A, Weibel C, Kahn JS (2006) Novel mutations in the respiratory syncytial virus G gene identified in viral isolates from a girl with severe combined immune deficiency treated with intravenous immune globulin. J Clin Virol 37: 168–173.
[42]
Galiano MC, Palomo C, Videla CM, Arbiza J, Melero JA, et al. (2005) Genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001). J Clin Microb 43: 2266–2273.
[43]
Botosso VF, Zanotto PM, Ueda M, Arruda E, Gilio AE, et al. (2009) Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus. PLoS Pathogens 5: e1000254.
[44]
Cane PA (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. J Med Virol 51: 297–304.
[45]
Kirkegaard K, Baltimore D (1986) The mechanism of RNA recombination in poliovirus. Cell 47: 433–443.
[46]
Scott GE, Tarlow O, McCrae MA (1989) Detailed structural analysis of a genome rearrangement in bovine rotavirus. Virus Res 14: 119–127.
[47]
Gault E, Schnepf N, Poncet D, Servant A, Teran S, et al. (2001) A human rotavirus with rearranged genes 7 and 11 encodes a modified NSP3 protein and suggests an additional mechanism for gene rearrangement. J Virol 75: 7305–7314.
[48]
Matthijnssens J, Rahman M, Van Ranst M (2006) Loop model: mechanism to explain partial gene duplications in segmented dsRNA viruses. Biochem Biophys Res Commun 340: 140–144.
[49]
Ballard A, McCrae MA, Desselberger U (1992) Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses. The J Gen Virol 73: 633–638.
[50]
Kojima K, Taniguchi K, Kawagishi-Kobayashi M, Matsuno S, Urasawa S (2000) Rearrangement generated in double genes, NSP1 and NSP3, of viable progenies from a human rotavirus strain. Virus Res 67: 163–171.
[51]
Wilson V, Taylor P, Desselberger U (1988) Crossover regions in foot-and-mouth disease virus (FMDV) recominants correspond to regions of high local secondary structure. Arch Virol 102: 131–139.
[52]
Gorziglia M, Nishikawa K, Fukuhara N (1989) Evidence of duplication and deletion in super short segment 11 of rabbit rotavirus Alabama strain. Virology 170: 587–590.
[53]
Kaguni LS, Clayton DA (1982) Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A 79: 983–987.
[54]
Weaver DT, DePamphilis ML (1982) Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha. J Biol Chem 257: 2075–2086.
[55]
Weaver DT, DePamphilis ML (1984) The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo. J Mol Biol 180: 961–986.
[56]
LaDuca RJ, Fay PJ, Chuang C, McHenry CS, Bambara RA (1983) Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. Biochemistry 22: 5177–5188.
[57]
Bedinger P, Munn M, Alberts BM (1986) Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem 264: 16880–16886.
[58]
Galburt EA, Grill SW, Wiedmann A, Lubkowska L, Choy J, et al. (2007) Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446: 820–823.
[59]
Komissarova N, Kashlev M (1997) Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded. Proc Natl Acad Sci U S A 94: 1755–1760.
[60]
Suo Z, Johnson KA (1998) DNA secondary structure effects on DNA synthesis catalyzed by HIV-1 reverse transcriptase. J Biol Chem 273: 27259–27267.
[61]
Shaevitz JW, Abbondanzieri EA, Landick R, Block SM (2003) Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426: 684–687.
