Genetic Variability of Human Respiratory Syncytial Virus A Strains Circulating in Ontario: A Novel Genotype with a 72 Nucleotide G Gene Duplication

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

Alignment of deduced amino acid sequence of the G protein of RSV-A strains isolated in Ontario during the 2010–2011 winter season.

<p>Alignments are shown relative to the sequences of prototype strain A2 and genotype NA1 strain (AB470478). The AAs shown correspond to positions 201 to 298 of the second hypervariable region of RSV-A strain A2 G protein. The alignment was done by the Clustal W method running within MEGA 5.05. Identical residues are identified as dots. Asterisks indicate the positions of stop codons. The 23 amino acid duplication is enclosed in open boxes. Predicted N-glycosylation sites are shaded in gray. Predicted O-glycosylation sites in RSV-A strain A2 are indicated by small unfilled circles. When compared to the NA1 reference strain, conserved O-glycosylation sites are indicated by black circles.</p

Percentage of nucleotide unpairdness in the region of the 7nt repeat motif at nucleotides 772–778 and 849–850 in RSV-A ON1, rON1 and the reference strain, NA1.

<p>ΔG Gibbs free energy.</p><p>SI-B stem induced backtracking.</p

Parameter estimates, dN/dS, values of log-Likelihood (<i>l</i>), positive selection sites, and Likelihood Ratio Tests (LRT) in the G-gene analysis of RSV-A viruses circulating in Ontario, Canada between November 2010 and February 2011.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032807#s4" target="_blank">Methods</a> for explanation of terms used in parameter estimates column.</p><p>Neutral models (M1a, and M7) were compared with their respective alternative (selection) models (M2a and M8), which allow <i>ω</i>>1. Model comparison can be calculated using 2Δ<i>l</i> = 2 (<i>l</i>₁−<i>l</i>₀)), where <i>l</i>_1 =LRT of alternative model; and <i>l</i>_0 =LRT of null model. Proportion of positively selected sites and their corresponding <i>ω</i>-values in M2a and M8 models are in bold. The significant <i>P</i> values indicated that all analyses find very strong evidence for the selection model.</p>a<p>Positively selected sites using Bayes Empirical Bayes analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032807#pone.0032807-Yang3" target="_blank">[28]</a>. Posterior probability of positively selected sites of M2a model: 50% to 74% (213, 218, 237, 285); 85% to 94% (233, 274); and >95% (232, 250, 251, 253, 265, 266, 273). Posterior probability of positively selected sites of M8 model: 50% to 74% (213, 218, 234, 249, 260, 277, 286); 75% to 84% (237, 285); 85% to 94% (233); and >95% (232, 250, 251, 253, 265, 266, 273, 274, 290).</p

Comparison of predicted viral RNA secondary structures of G gene.

<p>Mfold predicted viral RNA secondary structures for G-gene of A. NA1 (AB470478); B. ON1 (ON67-1210A); C. rON1, a virtual ON1 strain without the 72nt duplication. The boxed figure corresponds to the stem loop structure (SLS) implicated in RdRp pausing at nucleotides 849 and 850. ΔG indicates the minimum free energy values (kilocalories per mole).</p

Phylogenetic trees for Ontario RSV-A nucleotide sequences from (a) the second variable region of the G gene and (b) partial F gene sequences.

<p>Reference strains representing known genotypes are indicated in bold. Isolates of ON1 genotype circulating in Ontario are indicated by a solid square. Isolates belong to genotype GA5 are marked by an open square. Multiple sequences alignment and phylogenetic trees were constructed using Clustal W and neighbour-joining algorithm running within MEGA 5.05 software. Tree topology was supported by bootstrap analysis with 1000 pseudo replicate datasets. Bootstrap values greater than 50 are shown at the branch nodes. The tree was visualized using Dendroscope software, version 2.2.1.17. The scale bar represents the number of nucleotide substitutions per site between close relatives.</p

G protein structural features of RSV-A novel genotype, ON1.

<p>A) Schematic linear representation of the G protein primary structure of the novel ON1 containing a 72 nucleotide insertion. The amino acid sequence between residues 260 and 298 is shown, highlighting the 72 nucleotide segment that has been duplicated (boldface). The amino acid altered by the insertion is marked with a circle and the point of insertion is indicated by an arrow. B) The 72 nucleotide duplication is indicated by 2 horizontal solid lines below the sequences. The 24 amino acid insertion containing the 23 AA duplication is indicated by 2 horizontal lines above the sequences. Numbers corresponding to AAs indicate that the predicted G polypeptide is lengthened to 321 AAs. C) Graphical representation of the predicted G protein of ON1 with central conserved regions and second variable region identified. Duplicated AA sequences are highlighted in boldface. Positively selected sites are marked with a vertical bar under the line.</p