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

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

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

Sensitivity.

<p>Absorbance plot of twelve sera from patients clinically diagnosed with MCV (India n = 10; UK n = 2; control group of 0–1 year old individuals n = 17).</p

German seroprevalence.

<p>Distribution of anti-MC084S antibodies in a German population tested by direct binding ELISA (A) Serological responses to MCV antigen MC084 in a German population (n = 289; ages 0–40 years) expressed as the δODU value of an individual serum sample. The horizontal bar within each group represents the median absorbance measurement. (B) Percent seropositivities in different age groups after cut–off of 0.36 (i) 0–1 years (4.5%), (ii) 2–5 years (25%), (iii) 6–10 years (23.4%), 11–20 years (12.5%), and 21–40 years (13.5%).</p

Protein purification.

<p>Characterisation of over expressed recombinant fusion protein GST-MC084S and FPLC purified recombinant MC084S protein by SDS-PAGE and Western Blot. M: Molecular weight markers expressed in kDa. (A) Over-expressed 40 kDa Recombinant GST-MC084S fusion protein separated in a 4–12% Bis-Tris gel. (B) FPLC purified 14 kDa protein separated in a 15% Bis-Arylamide gel. Both gels were stained with Coomassie Brilliant Blue R-250. (C) GST-MC084S fusion protein after transfer to nitrocellulose (D) FPLC purified MC084S. The membranes were probed with Strep MAB-Classic HRP conjugate (IBA-lifesciences). Arrow heads indicate the locations of proteins.</p