Phylogenetic tree of RSV A/RSV B strains and reference sequences of identified genotypes.

<p>Phylogenetic trees for RSV A (A) and RSV B (B) strains were constructed with maximum-likelihood method with 1,000 bootstrap replicates using MEGA 6 software. RSV strains from Heidelberg/Germany are indicated by “•HD” followed by their strain identification number. Number of identical strains is indicated in brackets after the strain identifier. Reference strains representing known genotypes were retrieved from GenBank and included in the tree (labels include accession number). The genotype assignment is shown on the right by brackets. Prototype strains (M11486 for subgroup A and M17213 for subgroup B) were used as an outgroup. Bootstrap values greater than 70% are indicated at the branch nodes. The scale bar represents the number of nucleotide substitutions per site. cl.  =  cluster.</p

Basic and clinical characteristics of RSV positive children by genotype.

<p>*RSV A genotype GA5 was not included in this table as this genotype was only present in one patient. In total, 112 of 134 RSV positive patients could be sequenced and a genotype could be determined.</p>#<p>Hospital stay was only calculated for patients who stayed at least 24 hours in hospital.</p><p>SD =  standard deviation; RTI = respiratory tract infection, RSV =  Respiratory Syncytial Virus.</p><p>Basic and clinical characteristics of RSV positive children by genotype.</p

Alignment of deduced amino acid sequences of the second variable region of the G protein of RSV-B strains isolated in Heidelberg/Germany during 2012–2013 winter season.

<p>Alignments are shown relative to the sequence of a prototype BA strain (GenBank accession number AY333364). Alignment of sequences was performed using the Clustal W 1.6 method via MEGA 6 software. The amino acid positions correspond to positions 210 to 315 of the G protein of the BA strain. Identical residues are indicated by dots, asterisks indicate the position of stop codons. Number of identical strains is indicated in brackets after the strain identifier in the left column. Boxes frame the 20 amino acid duplication. Gray shading highlights predicted N-glycosylation sites. Open circles indicate predicted O-glycosylation sites of the prototype BA strain; potential O-glycosylation sites of Heidelberg strains are indicated by black dots. Genotypes are shown on the right by brackets.</p

Alignment of deduced amino acid sequences of RSV-A strains.

<p>A) Alignment of RSV-A genotype NA1 and GA5 are shown relative to the sequence of prototype strain A2 (GenBank accession number M11486). Alignment of sequences was performed using the Clustal W 1.6 method via MEGA 6 software. The amino acid positions correspond to positions 210 to 298 of the G protein of strain A2. Identical residues are indicated by dots, asterisks indicate the position of stop codons. Number of identical strains is indicated in brackets after the strain identifier in the left column. Gray shading highlights predicted N-glycosylation sites. Unfilled circles indicate predicted O-glycosylation sites of the prototype strain A2; potential O-glycosylation sites of Heidelberg strains are indicated by black dots. The genotype assignment is shown on the right by brackets. B) Alignments are shown relative to the sequence of ON1 strain first described in Canada (GenBank accession number JN257693). Alignment of sequences was performed using the Clustal W 1.6 method via MEGA 6 software. The amino acid positions correspond to positions 210 to 298 of the G protein of the prototype strain A2. Identical residues are indicated by dots, asterisks indicate the position of stop codons. Boxes frame the 23 amino acid duplicated region of the 24 amino acid insertion. Gray shading highlights predicted N-glycosylation sites. Unfilled circles indicate predicted O-glycosylation sites of the Canadian reference ON1 strain; potential O-glycosylation sites of Heidelberg strains are indicated by black dots. On the right hand site, GenBank and Heidelberg strains are labeled with the country and time of occurrence (month/year). ¹ Sequences were published in GenBank only. HD =  Heidelberg; WUE =  Wuerzburg, cl. =  cluster.</p

Weekly/monthly distribution of subgroup RSV A/RSV B in children ≤2 years with acute RTI in Heidelberg/Germany, winter season 2012/13.

<p>Weekly/monthly distribution of subgroup RSV A/RSV B in children ≤2 years with acute RTI in Heidelberg/Germany, winter season 2012/13.</p

Bioinformatics.

<p>(A) Transmembrane plot (TMHMM Server v.2.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Krogh1" target="_blank">[25]</a> of mc084 amino acids 1–318; (B) hydropathy plot of MC084 protein with predicted high hydrophilic/antigenic regions indicated by black boxes. The full length ORF (MC084 1–318; predicted molecular weight 34.2 kD; shown on top) was cloned into vRB12 using specific primers tailed with restriction enzyme sites <i>BamHI</i>-<i>HindIII</i>) and C-terminal StrepII epitope tag. The resulting plasmid p319 was sequenced and the recombinant vaccinia virus v319 isolated on BSC-1 cells using the plaqueless mutant system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Blasco1" target="_blank">[26]</a>. N- and C-terminal (in yellow) truncations were subcloned from the original full length MCV gene into pGEX-2TK for overexpression in <i>E. coli</i> BL21 (RIL⁺). TMHMM was used to determine transmembrane regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Blasco1" target="_blank">[26]</a> whereas the Kyte-Doolittle plot was used to identify hydrophilic regions with predicted high antigenicity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088734#pone.0088734-Kyte1" target="_blank">[27]</a>.</p

pGEX 2TK construct.

<p>(A) Schematic of recombinant plasmid p332 with a MC084 specific insert of 107 amino acids (V123-R230); predicted molecular weight 14 kD (B) Schematic of fusion protein of GST (green), followed by Thrombin kinase site (red), MC084 V123-R230 (grey), and strep II tag (blue); predicted antigenic site (black) (C) Western blot giving 40 kD GST fusion protein GST-MC084S (V123-R230) detected using Strep MAB-Classic HRP conjugate (IBA-lifesciences). Vector NTI (vNTI) was used to produce virtual molecules and schematic diagrams of constructs prior to molecular cloning (InforMax, Inc).</p

HaCaT Immunofluorescence.

<p>(A) HaCaT cell culture infected with recombinant vaccinia virus expressing MC084S (v319). Reactivity of high titre human serum HDV0901071 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). (B) HaCaT cell culture infected with recombinant vaccinia virus expressing MC084S (v319). Reactivity of low titre human serum HDV0900040 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). (C) Mock infected cells. Reactivity of high titre human serum HDV0901071 and secondary antibody AlexaFluor 488 (Green) goat anti-human IgG (H+L). Nuclei are stained with DAPI (Hoechst) and shown in blue. Samples were analysed for fluorescence emission properties by using confocal scanning laser microscopy Leica TCS SP2 AOBS.</p

Summary of seroprevalences in German and UK populations.

<p>*SLE – Systemic Lupus Erythematosus.</p>†<p>Autoimm. – General autoimmune conditions.</p>#<p>PPMS – Primary progressive multiple sclerosis.</p><p>^{<>\scale 80%\raster="rg1"\<>}RRMS – Relapsing remitting multiple sclerosis.</p

Tissue stain details.

<p>Microscopy (4×) of a Molluscum contagiosum lesion section (17315/11) stained with MC patient positive serum (CF2012-1) and haematoxylin-eosin counterstain (upper left hand corner). Three insets showing details at various magnifications [inset 1-(10×), inset 2-(20×) and inset 3-(20×)].</p