Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

IgM-ELISA on 300 ng of DENV-2 Ewt (A) and DENV 1–4 Equad mixture (B) with different DENV, WNV and negative (NEG) sera (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Measurements were performed in duplicates in at least two independent experiments.</p

Alignment of the amino acid sequences containing the fusion loop domain from E proteins of DENV 1–4, WNV, JEV, YFV, TBEV and the four point mutations of the QUAD proteins.

<p>Amino acids numbering: 1 is start of the E protein.</p

Comparison of different antigens for the detection of DENV IgG.

<p>Sera positive for IgG against DENV, WNV, TBEV or negative control sera were analyzed with the Panbio Indirect IgG ELISA (black), the DENV-2 Ewt protein (light gray, lined) or the DENV1-4 Equad mix (dark grey, lined). The absolute absorbance is indicated. Cut-Off values for the Panbio test were obtained by calculation of the internal standard of the manufacturer; these are indicated at the right and only refer to this test (horizontal bars: DENV-positive results with an OD-value higher than 1.1*cut-off, equivocal results having an OD-value between 1.1*cut-off and 0.9*cut-off, negative results with an OD-value lower than 0.9*cut-off).</p

Sperm transfected with E6/E7 plasmid penetrate oocytes.

<p><b>a</b>. Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. <b>b</b>. PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). <b>c</b>. Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. <b>d</b>. Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.</p

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.</p

HPV interacts with syndecan-1 on human sperm.

<p><b>a</b>. Cytofluorimetric analysis with mouse anti-human CD138 (syndecan-1), FITC conjugated on sperm before and after treatment with Heparinase-III. Samples without anti CD138 were used as negative controls. <b>b</b>. Immunofluorescence analysis (confocal fluorescence microscope) for syndecan-1 (CD138, FITC conjugated of sperm before and after treatment with Heparinase-III. Upper panel: negative control (omission of CD138); central panel: syndecan-1; lower panel: syndecan-1 after Heparinase-III treatment. <b>c</b>. Cytofluorimetric analysis of sperm exposed to HPV16 L1 capsid protein, treated or not with Heparinase-III and analyzed with mouse monoclonal HPV16 L1 antibody. Samples incubated with secondary phycoerythrin anti-mouse antibody alone were used as negative controls. <b>d</b>. Immunofluorescence analysis for HPV L1 capsid protein (confocal fluorescence microscope) in sperm exposed to HPV16 L1 treated or not with Heparinase-III and analyzed with mouse monoclonal antibody anti HPV16 L1 and DAPI. <b>e</b>. Colocalization analysis of HPV L1 capsid protein and syndecan-1 by immunofluorescence (confocal fluorescence microscope) of sperm exposed to HPV16 L1 treated or not with Heparinase-III and analyzed with mouse anti-human CD138 (syndecan-1) monoclonal antibody, FITC conjugated and mouse monoclonal antibody anti HPV16 L1 and DAPI.</p

Detection and localization of HPV in human sperm.

<p><b>a</b>. Fluorescence in situ hybridization (fluorescence microscope) for HPV DNA on sperm from a patient with HPV16 in semen. Infected and noninfected sperm are shown. Red: HPV DNA (Texas red); blue: nuclear staining (DAPI). <b>b</b>. Immunofluorescence (confocal fluorescence microscope) for HPV16 capsid protein L1 on sperm from a control (left) and a patient with HPV16 in semen (right). Upper panel, L1 antibody; central panel, L1 antibody and Pisum Sativum (acrosome); lower panel, L1 antibody and Pisum Sativum after induction of the acrosome reaction. Red: HPV16 L1; green: Pisum Sativum; blue: nuclear staining (DAPI). <b>c</b>. PCR for HPV E7 gene from sperm DNA. Lane M: DNA marker (100 bp); 1: negative control (no template); 2: positive control (sperm transfected with recombinant plasmid pIRES2-AcGFP1-E6E7); 3: sperm from a patient with HPV16 in semen; 4: sperm from a control subject.</p

Oocytes penetrated by sperm transfected with HPV E6/E7 express HPV E6/E7 genes.

<p><b>a</b>. PCR for E6 gene from single oocyte penetrated by transfected sperm. Lane M: DNA marker (100 bp); 1: positive control (pIRES2-AcGFP1-E6E7 plasmid); 2: single oocyte penetrated by sperm transfected with recombinant plasmid; 3: negative control (no template); 4: single oocyte penetrated by sperm transfected only with Lipofectamine 2000. <b>b</b>. GFP-E6 expression in oocyte penetrated by control sperm. <b>c</b>. GFP-E6 expression in oocyte penetrated by transfected sperm. <b>d</b>. RT-PCR for E6 gene from single oocyte penetrated by transfected sperm. Lane M: DNA markers (100 bp); 1: negative control (no template for Reverse Transcription); 2: negative control (no template for PCR); 3: single oocyte with green fluorescence penetrated by transfected sperm; 4: single oocyte without green fluorescence penetrated by control sperm.</p