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    ATCC dengue virus type 2 denv2 th 36 strain
    Characteristics of cell lines used and their responses to salivary gland extract treatment regarding <t> DENV2 </t> infectivity enhancement and cell migration
    Dengue Virus Type 2 Denv2 Th 36 Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/dengue+virus+type+2+denv2+th+36+strain/Dengue+virus+type+2/pmc03911723-99-1-12
    Average 99 stars, based on 242 article reviews
    dengue virus type 2 denv2 th 36 strain - by Bioz Stars, 2026-09
    99/100 stars

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    1) Product Images from "Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host"

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    Journal: Journal of Virology

    doi: 10.1128/JVI.02235-13

    Characteristics of cell lines used and their responses to salivary gland extract treatment regarding DENV2 infectivity enhancement and cell migration
    Figure Legend Snippet: Characteristics of cell lines used and their responses to salivary gland extract treatment regarding DENV2 infectivity enhancement and cell migration

    Techniques Used: Infection, Migration

    Mosquito saliva temporally enhances dengue virus vRNA in mouse embryonic fibroblasts. (A) NIH 3T3 MEFs were either left untreated (gray squares) or were treated (black diamonds) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Total RNA was extracted, and relative DENV2 vRNA values were normalized to 250 ng of input RNA by RT-qPCR. (B) NIH 3T3 MEFs were untreated (black squares) or treated (gray squares) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Cells were detached from wells by trypsin treatment, and cells were counted by light microscopy. Fold change between SGE-treated and untreated time points are indicated for both panels. Data represent averages from three independent experiments, including standard errors of the means. Student's t tests were performed for individual time points to assess statistical significance.
    Figure Legend Snippet: Mosquito saliva temporally enhances dengue virus vRNA in mouse embryonic fibroblasts. (A) NIH 3T3 MEFs were either left untreated (gray squares) or were treated (black diamonds) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Total RNA was extracted, and relative DENV2 vRNA values were normalized to 250 ng of input RNA by RT-qPCR. (B) NIH 3T3 MEFs were untreated (black squares) or treated (gray squares) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Cells were detached from wells by trypsin treatment, and cells were counted by light microscopy. Fold change between SGE-treated and untreated time points are indicated for both panels. Data represent averages from three independent experiments, including standard errors of the means. Student's t tests were performed for individual time points to assess statistical significance.

    Techniques Used: Virus, Incubation, Quantitative RT-PCR, Light Microscopy

    Mosquito saliva enhances dengue virus infectivity in mouse embryonic fibroblasts. NIH 3T3 MEFs (A) and RAW 264.7 macrophage cells (B) were treated with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. Total RNA was extracted, and the copy numbers of DENV2 vRNA were normalized to 250 ng of input RNA by RT-qPCR. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. A mock-infected control was included. Unbound virus was washed from cells, and cells were fixed with 4% paraformaldehyde and stained with anti-DENV2 envelope antibody (red) and DAPI (blue). (D) NIH 3T3 MEFs were treated with 0, 0.5, 1.0, and 2.0 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 2 and 18 h. Focus-forming immunoperoxidase assays were performed on Vero cells using NIH 3T3 MEF supernatants in a 6-well format. (E) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Cells were stained for DENV2 NS1 using an immunoperoxidase assay, and NS1-positive cells were counted 18 hpi. These data were not normalized to the total amount of cells on the plate. (F) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.0 saliva equivalents (SE) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Total RNA was extracted, and expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. Data represent the averages from at least six independent experiments, including standard errors of the means. Student's t tests were performed on samples treated with the highest concentration of SGE to assess statistical significance.
    Figure Legend Snippet: Mosquito saliva enhances dengue virus infectivity in mouse embryonic fibroblasts. NIH 3T3 MEFs (A) and RAW 264.7 macrophage cells (B) were treated with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. Total RNA was extracted, and the copy numbers of DENV2 vRNA were normalized to 250 ng of input RNA by RT-qPCR. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. A mock-infected control was included. Unbound virus was washed from cells, and cells were fixed with 4% paraformaldehyde and stained with anti-DENV2 envelope antibody (red) and DAPI (blue). (D) NIH 3T3 MEFs were treated with 0, 0.5, 1.0, and 2.0 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 2 and 18 h. Focus-forming immunoperoxidase assays were performed on Vero cells using NIH 3T3 MEF supernatants in a 6-well format. (E) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Cells were stained for DENV2 NS1 using an immunoperoxidase assay, and NS1-positive cells were counted 18 hpi. These data were not normalized to the total amount of cells on the plate. (F) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.0 saliva equivalents (SE) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Total RNA was extracted, and expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. Data represent the averages from at least six independent experiments, including standard errors of the means. Student's t tests were performed on samples treated with the highest concentration of SGE to assess statistical significance.

    Techniques Used: Virus, Infection, Incubation, Quantitative RT-PCR, Control, Staining, Expressing, Concentration Assay

    Relative RT-qPCR data from SGE treatments of mouse embryonic fibroblasts followed by infection with multiple dengue virus serotypes and isolates a
    Figure Legend Snippet: Relative RT-qPCR data from SGE treatments of mouse embryonic fibroblasts followed by infection with multiple dengue virus serotypes and isolates a

    Techniques Used: Infection, Virus

    SGE enhances virus attachment to exposed glycosaminoglycans and induces cell migration. (A) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then allowed to incubate for 4 h at 37°C before observation by phase-contrast light microscopy. (B) NIH 3T3 MEFs were treated with 1.0 SGE for 0, 5, 15, 30, and 45 min. Equal volumes of protein lysates were loaded onto SDS-PAGE gels, protein was transferred to PVDF membranes, and the expression of multiple proteins was analyzed by Western blotting. Laminin protein (LP), laminin receptor (LR), fibronectin (F), integrin (Int), collagen type I (Coll), endo180 (Endo), and actin were used. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min and inoculated with DENV2 at an MOI of 1 for 1 h. Unbound virus was washed from cells, and cells were fixed with paraformaldehyde and stained for DENV2 envelope (red) and DAPI (blue). (D) DENV2 was untreated (−) or pretreated with 100 μg/ml heparan sulfate (HS), chondroitin sulfate A (CS-A), and chondroitin sulfate B (CS-B) for 30 min at room temperature. NIH 3T3 MEFs were then treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with glycosaminoglycan-treated or untreated DENV2 at an MOI of 1 for 1 h at 37°C. Unbound virus was removed, and then cells were allowed to incubate for 18 h. Total RNA was extracted, and DENV2 vRNA and GAPDH expression was analyzed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) ECM material was isolated on cell culture plates by washing off monolayers of NIH 3T3 MEF cells with 10 mM EDTA solution in PBS for 10 min at 37°C. Wells were either treated with buffer alone (PBS) or treated with 2.0 SGE for 10 min at 37°C. Wells were then inoculated with untreated or CS-B-treated DENV2 for 1 h at 37°C. Unbound virus was removed, total RNA was extracted, and DENV2 vRNA expression was analyzed by RT-qPCR and normalized per well. (F and G) MEFs were seeded onto either untreated (−) or poly-d-lysine-treated (poly-d-lysine) polystyrene cell culture plates and treated with buffer alone (PBS) or with 1.0 SGE and then inoculated with DENV2 at an MOI of 1 for 1 h. (F) Cell migration was observed by light microscopy. (G) Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. (H) Total RNA was extracted from MEFs suspended in collagen matrices, and the expression of DENV2 vRNA was assessed by RT-qPCR 18 hpi and normalized to grams of collagen. Data represent the averages from more than three independent experiments. Student's t tests were performed to analyze statistical significance.
    Figure Legend Snippet: SGE enhances virus attachment to exposed glycosaminoglycans and induces cell migration. (A) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then allowed to incubate for 4 h at 37°C before observation by phase-contrast light microscopy. (B) NIH 3T3 MEFs were treated with 1.0 SGE for 0, 5, 15, 30, and 45 min. Equal volumes of protein lysates were loaded onto SDS-PAGE gels, protein was transferred to PVDF membranes, and the expression of multiple proteins was analyzed by Western blotting. Laminin protein (LP), laminin receptor (LR), fibronectin (F), integrin (Int), collagen type I (Coll), endo180 (Endo), and actin were used. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min and inoculated with DENV2 at an MOI of 1 for 1 h. Unbound virus was washed from cells, and cells were fixed with paraformaldehyde and stained for DENV2 envelope (red) and DAPI (blue). (D) DENV2 was untreated (−) or pretreated with 100 μg/ml heparan sulfate (HS), chondroitin sulfate A (CS-A), and chondroitin sulfate B (CS-B) for 30 min at room temperature. NIH 3T3 MEFs were then treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with glycosaminoglycan-treated or untreated DENV2 at an MOI of 1 for 1 h at 37°C. Unbound virus was removed, and then cells were allowed to incubate for 18 h. Total RNA was extracted, and DENV2 vRNA and GAPDH expression was analyzed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) ECM material was isolated on cell culture plates by washing off monolayers of NIH 3T3 MEF cells with 10 mM EDTA solution in PBS for 10 min at 37°C. Wells were either treated with buffer alone (PBS) or treated with 2.0 SGE for 10 min at 37°C. Wells were then inoculated with untreated or CS-B-treated DENV2 for 1 h at 37°C. Unbound virus was removed, total RNA was extracted, and DENV2 vRNA expression was analyzed by RT-qPCR and normalized per well. (F and G) MEFs were seeded onto either untreated (−) or poly-d-lysine-treated (poly-d-lysine) polystyrene cell culture plates and treated with buffer alone (PBS) or with 1.0 SGE and then inoculated with DENV2 at an MOI of 1 for 1 h. (F) Cell migration was observed by light microscopy. (G) Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. (H) Total RNA was extracted from MEFs suspended in collagen matrices, and the expression of DENV2 vRNA was assessed by RT-qPCR 18 hpi and normalized to grams of collagen. Data represent the averages from more than three independent experiments. Student's t tests were performed to analyze statistical significance.

    Techniques Used: Virus, Migration, Light Microscopy, SDS Page, Expressing, Western Blot, Staining, Quantitative RT-PCR, Isolation, Cell Culture

    SGE-mediated enhancement of dengue virus infectivity is inhibited by Pefabloc SC in vitro. (A and B) NIH 3T3 MEFs were treated with buffer (PBS), pretreated with 1.0 SGE, or pretreated with 1.0 boiled SGE (Boiled), followed by inoculation with DENV2 at an MOI of 0.1 for 1 h. (A) Cells were observed by light microscopy 18 hpi (B) and then total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (C) SGEs were prepared and individually treated with the protease inhibitors antipain-2HCl (Anti), bestatin (Bes), chymostatin (Chy), E-64 (E64), leupeptin (Leu), pepstatin (Pep), phosphoramidon (Pho), pefabloc SC (Pef), and aprotinin (Apr). MEFs were either treated with buffer alone (PBS) or treated with 1.0 SGE with and without protease inhibitors for 10 min and inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed. DENV2 vRNA was normalized to GAPDH by RT-qPCR. (D) MEFs were treated with Pef or a protease inhibitor cocktail containing Pef (PI) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) Prior to extracting total RNA as described for panel C, cells were observed by phase-contrast light microscopy. Data represent averages from at least three independent experiments. Student's t tests were performed to assess statistical significance when appropriate.
    Figure Legend Snippet: SGE-mediated enhancement of dengue virus infectivity is inhibited by Pefabloc SC in vitro. (A and B) NIH 3T3 MEFs were treated with buffer (PBS), pretreated with 1.0 SGE, or pretreated with 1.0 boiled SGE (Boiled), followed by inoculation with DENV2 at an MOI of 0.1 for 1 h. (A) Cells were observed by light microscopy 18 hpi (B) and then total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (C) SGEs were prepared and individually treated with the protease inhibitors antipain-2HCl (Anti), bestatin (Bes), chymostatin (Chy), E-64 (E64), leupeptin (Leu), pepstatin (Pep), phosphoramidon (Pho), pefabloc SC (Pef), and aprotinin (Apr). MEFs were either treated with buffer alone (PBS) or treated with 1.0 SGE with and without protease inhibitors for 10 min and inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed. DENV2 vRNA was normalized to GAPDH by RT-qPCR. (D) MEFs were treated with Pef or a protease inhibitor cocktail containing Pef (PI) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) Prior to extracting total RNA as described for panel C, cells were observed by phase-contrast light microscopy. Data represent averages from at least three independent experiments. Student's t tests were performed to assess statistical significance when appropriate.

    Techniques Used: Virus, Infection, In Vitro, Light Microscopy, Expressing, Quantitative RT-PCR, Protease Inhibitor

    Pefabloc SC inhibits SGE-mediated enhancement of dengue and West Nile viruses in vivo. Four groups of 10 IFNAR−/− C57BL/6 mice were inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE and 1% DMSO (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+P+S). Twenty-four hpi, footpads (A), lymph nodes (B), blood (C), and spleens (D) were harvested into RLT buffer, and RNA was extracted for RT-qPCR analysis. DENV vRNA was normalized to total RNA per reaction. (E) C57BL/6 mice were inoculated in a single rear footpad (7 per group) with 500 PFU WNV and 1% DMSO (W) or with 1 mM Pefabloc SC in DMSO (W+P). Relative viral load was assessed in blood by RT-qPCR 24 hpi. (F) Three groups of 10 C57BL/6 mice were inoculated in rear footpads with 500 PFU WNV and 1% DMSO (W), with 1.0 C. tarsalis SGE and 1% DMSO (W+S), or with both 1.0 C. tarsalis SGE and 1 mM Pefabloc SC in DMSO (W+P+S). Relative viral load was assessed in footpads by RT-qPCR 24 hpi. Each symbol represents one mouse. The dotted line represents the background threshold. Mann-Whitney analysis was performed to assess statistical significance between groups. Data represent one experiment.
    Figure Legend Snippet: Pefabloc SC inhibits SGE-mediated enhancement of dengue and West Nile viruses in vivo. Four groups of 10 IFNAR−/− C57BL/6 mice were inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE and 1% DMSO (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+P+S). Twenty-four hpi, footpads (A), lymph nodes (B), blood (C), and spleens (D) were harvested into RLT buffer, and RNA was extracted for RT-qPCR analysis. DENV vRNA was normalized to total RNA per reaction. (E) C57BL/6 mice were inoculated in a single rear footpad (7 per group) with 500 PFU WNV and 1% DMSO (W) or with 1 mM Pefabloc SC in DMSO (W+P). Relative viral load was assessed in blood by RT-qPCR 24 hpi. (F) Three groups of 10 C57BL/6 mice were inoculated in rear footpads with 500 PFU WNV and 1% DMSO (W), with 1.0 C. tarsalis SGE and 1% DMSO (W+S), or with both 1.0 C. tarsalis SGE and 1 mM Pefabloc SC in DMSO (W+P+S). Relative viral load was assessed in footpads by RT-qPCR 24 hpi. Each symbol represents one mouse. The dotted line represents the background threshold. Mann-Whitney analysis was performed to assess statistical significance between groups. Data represent one experiment.

    Techniques Used: In Vivo, Quantitative RT-PCR, MANN-WHITNEY

    RT-qPCR analysis of TH2/TH1 cytokine expression in footpads and lymph nodes from IFNAR−/− C57BL/6 mice infected with DENV2 and treated with SGE and Pefabloc SC. Relative expression of IL-4 (A and B), IL-5 (C and D), IL-6 (E and F), IL-10 (G and H), IFN-γ (I and J), and TNF-α (K and L) was examined in footpads and draining lymph nodes 24 hpi in mice inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of either DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+S+P). Relative cytokine levels were normalized to GAPDH. Each dot represents a single mouse. Mann-Whitney tests were performed between groups to assess statistical significance. Data represent one experiment.
    Figure Legend Snippet: RT-qPCR analysis of TH2/TH1 cytokine expression in footpads and lymph nodes from IFNAR−/− C57BL/6 mice infected with DENV2 and treated with SGE and Pefabloc SC. Relative expression of IL-4 (A and B), IL-5 (C and D), IL-6 (E and F), IL-10 (G and H), IFN-γ (I and J), and TNF-α (K and L) was examined in footpads and draining lymph nodes 24 hpi in mice inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of either DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+S+P). Relative cytokine levels were normalized to GAPDH. Each dot represents a single mouse. Mann-Whitney tests were performed between groups to assess statistical significance. Data represent one experiment.

    Techniques Used: Quantitative RT-PCR, Expressing, Infection, MANN-WHITNEY

    Identification of CLIPA3 as a component involved in SGE-mediated infectivity enhancement. (A) HPLC trace showing protein concentrations in 80 100-μl fractions after reverse-phase HPLC fractionation of 100 A. aegypti SGE. (B) Ten μl of each HPLC fraction was diluted in 90 μl PBS and used as SGE for in vitro SGE-mediated infectivity enhancement assays. Eighteen hpi, RNA was harvested and RT-qPCR was performed. Relative levels of DENV2 were normalized to GAPDH. (C) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti head (H), salivary gland (SG), midgut (MG), ovary (O), and carcass (CA) tissues. Relative levels of CLIPA3 were normalized to actin. (D) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti salivary glands 7 days after microinjection with 500 ng control or CLIPA3 siRNA. Relative levels of CLIPA3 were normalized to actin. Each dot represents 3 pooled salivary glands. (E) In vitro SGE-mediated infectivity enhancement assay using buffer alone (PBS), 1.0 SGE from control siRNA-treated mosquitoes (Control), or 1.0 SGE from CLIPA3 siRNA-treated mosquitoes. Relative levels of DENV2 were normalized to GAPDH. Data represent averages from at least 6 independent experiments. (F) Light microscopy images of NIH 3T3 MEFs shown in panel E 6 h post-SGE treatment. Student's t tests were performed to assess statistical significance when appropriate.
    Figure Legend Snippet: Identification of CLIPA3 as a component involved in SGE-mediated infectivity enhancement. (A) HPLC trace showing protein concentrations in 80 100-μl fractions after reverse-phase HPLC fractionation of 100 A. aegypti SGE. (B) Ten μl of each HPLC fraction was diluted in 90 μl PBS and used as SGE for in vitro SGE-mediated infectivity enhancement assays. Eighteen hpi, RNA was harvested and RT-qPCR was performed. Relative levels of DENV2 were normalized to GAPDH. (C) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti head (H), salivary gland (SG), midgut (MG), ovary (O), and carcass (CA) tissues. Relative levels of CLIPA3 were normalized to actin. (D) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti salivary glands 7 days after microinjection with 500 ng control or CLIPA3 siRNA. Relative levels of CLIPA3 were normalized to actin. Each dot represents 3 pooled salivary glands. (E) In vitro SGE-mediated infectivity enhancement assay using buffer alone (PBS), 1.0 SGE from control siRNA-treated mosquitoes (Control), or 1.0 SGE from CLIPA3 siRNA-treated mosquitoes. Relative levels of DENV2 were normalized to GAPDH. Data represent averages from at least 6 independent experiments. (F) Light microscopy images of NIH 3T3 MEFs shown in panel E 6 h post-SGE treatment. Student's t tests were performed to assess statistical significance when appropriate.

    Techniques Used: Infection, Fractionation, In Vitro, Quantitative RT-PCR, Expressing, Microinjection, Control, Light Microscopy

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    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host
    Article Snippet: Mouse-adapted dengue virus type 1 (DENV1) Hawaii strain was obtained from the ATCC (VR1254). .. Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345). ..



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    dengue virus type 2 denv2 th 36 strain  (ATCC)
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    ATCC dengue virus type 2 denv2 th 36 strain
    Characteristics of cell lines used and their responses to salivary gland extract treatment regarding <t> DENV2 </t> infectivity enhancement and cell migration
    Dengue Virus Type 2 Denv2 Th 36 Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/dengue+virus+type+2+denv2+th+36+strain/Dengue+virus+type+2/pmc03911723-99-1-12
    Average 99 stars, based on 1 article reviews
    dengue virus type 2 denv2 th 36 strain - by Bioz Stars, 2026-09
    99/100 stars
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    Characteristics of cell lines used and their responses to salivary gland extract treatment regarding DENV2 infectivity enhancement and cell migration

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Characteristics of cell lines used and their responses to salivary gland extract treatment regarding DENV2 infectivity enhancement and cell migration

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Infection, Migration

    Mosquito saliva temporally enhances dengue virus vRNA in mouse embryonic fibroblasts. (A) NIH 3T3 MEFs were either left untreated (gray squares) or were treated (black diamonds) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Total RNA was extracted, and relative DENV2 vRNA values were normalized to 250 ng of input RNA by RT-qPCR. (B) NIH 3T3 MEFs were untreated (black squares) or treated (gray squares) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Cells were detached from wells by trypsin treatment, and cells were counted by light microscopy. Fold change between SGE-treated and untreated time points are indicated for both panels. Data represent averages from three independent experiments, including standard errors of the means. Student's t tests were performed for individual time points to assess statistical significance.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Mosquito saliva temporally enhances dengue virus vRNA in mouse embryonic fibroblasts. (A) NIH 3T3 MEFs were either left untreated (gray squares) or were treated (black diamonds) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Total RNA was extracted, and relative DENV2 vRNA values were normalized to 250 ng of input RNA by RT-qPCR. (B) NIH 3T3 MEFs were untreated (black squares) or treated (gray squares) with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 1, 2, 6, 12, and 18 h. Cells were detached from wells by trypsin treatment, and cells were counted by light microscopy. Fold change between SGE-treated and untreated time points are indicated for both panels. Data represent averages from three independent experiments, including standard errors of the means. Student's t tests were performed for individual time points to assess statistical significance.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Virus, Incubation, Quantitative RT-PCR, Light Microscopy

    Mosquito saliva enhances dengue virus infectivity in mouse embryonic fibroblasts. NIH 3T3 MEFs (A) and RAW 264.7 macrophage cells (B) were treated with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. Total RNA was extracted, and the copy numbers of DENV2 vRNA were normalized to 250 ng of input RNA by RT-qPCR. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. A mock-infected control was included. Unbound virus was washed from cells, and cells were fixed with 4% paraformaldehyde and stained with anti-DENV2 envelope antibody (red) and DAPI (blue). (D) NIH 3T3 MEFs were treated with 0, 0.5, 1.0, and 2.0 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 2 and 18 h. Focus-forming immunoperoxidase assays were performed on Vero cells using NIH 3T3 MEF supernatants in a 6-well format. (E) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Cells were stained for DENV2 NS1 using an immunoperoxidase assay, and NS1-positive cells were counted 18 hpi. These data were not normalized to the total amount of cells on the plate. (F) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.0 saliva equivalents (SE) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Total RNA was extracted, and expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. Data represent the averages from at least six independent experiments, including standard errors of the means. Student's t tests were performed on samples treated with the highest concentration of SGE to assess statistical significance.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Mosquito saliva enhances dengue virus infectivity in mouse embryonic fibroblasts. NIH 3T3 MEFs (A) and RAW 264.7 macrophage cells (B) were treated with SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. Total RNA was extracted, and the copy numbers of DENV2 vRNA were normalized to 250 ng of input RNA by RT-qPCR. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 18 h. A mock-infected control was included. Unbound virus was washed from cells, and cells were fixed with 4% paraformaldehyde and stained with anti-DENV2 envelope antibody (red) and DAPI (blue). (D) NIH 3T3 MEFs were treated with 0, 0.5, 1.0, and 2.0 SGE for 10 min, inoculated with DENV2 at an MOI of 0.1 for 1 h, and then incubated for 2 and 18 h. Focus-forming immunoperoxidase assays were performed on Vero cells using NIH 3T3 MEF supernatants in a 6-well format. (E) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Cells were stained for DENV2 NS1 using an immunoperoxidase assay, and NS1-positive cells were counted 18 hpi. These data were not normalized to the total amount of cells on the plate. (F) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.0 saliva equivalents (SE) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Total RNA was extracted, and expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. Data represent the averages from at least six independent experiments, including standard errors of the means. Student's t tests were performed on samples treated with the highest concentration of SGE to assess statistical significance.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Virus, Infection, Incubation, Quantitative RT-PCR, Control, Staining, Expressing, Concentration Assay

    Relative RT-qPCR data from SGE treatments of mouse embryonic fibroblasts followed by infection with multiple dengue virus serotypes and isolates a

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Relative RT-qPCR data from SGE treatments of mouse embryonic fibroblasts followed by infection with multiple dengue virus serotypes and isolates a

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Infection, Virus

    SGE enhances virus attachment to exposed glycosaminoglycans and induces cell migration. (A) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then allowed to incubate for 4 h at 37°C before observation by phase-contrast light microscopy. (B) NIH 3T3 MEFs were treated with 1.0 SGE for 0, 5, 15, 30, and 45 min. Equal volumes of protein lysates were loaded onto SDS-PAGE gels, protein was transferred to PVDF membranes, and the expression of multiple proteins was analyzed by Western blotting. Laminin protein (LP), laminin receptor (LR), fibronectin (F), integrin (Int), collagen type I (Coll), endo180 (Endo), and actin were used. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min and inoculated with DENV2 at an MOI of 1 for 1 h. Unbound virus was washed from cells, and cells were fixed with paraformaldehyde and stained for DENV2 envelope (red) and DAPI (blue). (D) DENV2 was untreated (−) or pretreated with 100 μg/ml heparan sulfate (HS), chondroitin sulfate A (CS-A), and chondroitin sulfate B (CS-B) for 30 min at room temperature. NIH 3T3 MEFs were then treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with glycosaminoglycan-treated or untreated DENV2 at an MOI of 1 for 1 h at 37°C. Unbound virus was removed, and then cells were allowed to incubate for 18 h. Total RNA was extracted, and DENV2 vRNA and GAPDH expression was analyzed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) ECM material was isolated on cell culture plates by washing off monolayers of NIH 3T3 MEF cells with 10 mM EDTA solution in PBS for 10 min at 37°C. Wells were either treated with buffer alone (PBS) or treated with 2.0 SGE for 10 min at 37°C. Wells were then inoculated with untreated or CS-B-treated DENV2 for 1 h at 37°C. Unbound virus was removed, total RNA was extracted, and DENV2 vRNA expression was analyzed by RT-qPCR and normalized per well. (F and G) MEFs were seeded onto either untreated (−) or poly-d-lysine-treated (poly-d-lysine) polystyrene cell culture plates and treated with buffer alone (PBS) or with 1.0 SGE and then inoculated with DENV2 at an MOI of 1 for 1 h. (F) Cell migration was observed by light microscopy. (G) Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. (H) Total RNA was extracted from MEFs suspended in collagen matrices, and the expression of DENV2 vRNA was assessed by RT-qPCR 18 hpi and normalized to grams of collagen. Data represent the averages from more than three independent experiments. Student's t tests were performed to analyze statistical significance.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: SGE enhances virus attachment to exposed glycosaminoglycans and induces cell migration. (A) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then allowed to incubate for 4 h at 37°C before observation by phase-contrast light microscopy. (B) NIH 3T3 MEFs were treated with 1.0 SGE for 0, 5, 15, 30, and 45 min. Equal volumes of protein lysates were loaded onto SDS-PAGE gels, protein was transferred to PVDF membranes, and the expression of multiple proteins was analyzed by Western blotting. Laminin protein (LP), laminin receptor (LR), fibronectin (F), integrin (Int), collagen type I (Coll), endo180 (Endo), and actin were used. (C) NIH 3T3 MEFs were treated with buffer alone (PBS) or with 2.5 SGE for 10 min and inoculated with DENV2 at an MOI of 1 for 1 h. Unbound virus was washed from cells, and cells were fixed with paraformaldehyde and stained for DENV2 envelope (red) and DAPI (blue). (D) DENV2 was untreated (−) or pretreated with 100 μg/ml heparan sulfate (HS), chondroitin sulfate A (CS-A), and chondroitin sulfate B (CS-B) for 30 min at room temperature. NIH 3T3 MEFs were then treated with buffer alone (PBS) or with 1.0 SGE for 10 min and then inoculated with glycosaminoglycan-treated or untreated DENV2 at an MOI of 1 for 1 h at 37°C. Unbound virus was removed, and then cells were allowed to incubate for 18 h. Total RNA was extracted, and DENV2 vRNA and GAPDH expression was analyzed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) ECM material was isolated on cell culture plates by washing off monolayers of NIH 3T3 MEF cells with 10 mM EDTA solution in PBS for 10 min at 37°C. Wells were either treated with buffer alone (PBS) or treated with 2.0 SGE for 10 min at 37°C. Wells were then inoculated with untreated or CS-B-treated DENV2 for 1 h at 37°C. Unbound virus was removed, total RNA was extracted, and DENV2 vRNA expression was analyzed by RT-qPCR and normalized per well. (F and G) MEFs were seeded onto either untreated (−) or poly-d-lysine-treated (poly-d-lysine) polystyrene cell culture plates and treated with buffer alone (PBS) or with 1.0 SGE and then inoculated with DENV2 at an MOI of 1 for 1 h. (F) Cell migration was observed by light microscopy. (G) Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR 18 hpi. DENV2 vRNA was normalized to GAPDH. (H) Total RNA was extracted from MEFs suspended in collagen matrices, and the expression of DENV2 vRNA was assessed by RT-qPCR 18 hpi and normalized to grams of collagen. Data represent the averages from more than three independent experiments. Student's t tests were performed to analyze statistical significance.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Virus, Migration, Light Microscopy, SDS Page, Expressing, Western Blot, Staining, Quantitative RT-PCR, Isolation, Cell Culture

    SGE-mediated enhancement of dengue virus infectivity is inhibited by Pefabloc SC in vitro. (A and B) NIH 3T3 MEFs were treated with buffer (PBS), pretreated with 1.0 SGE, or pretreated with 1.0 boiled SGE (Boiled), followed by inoculation with DENV2 at an MOI of 0.1 for 1 h. (A) Cells were observed by light microscopy 18 hpi (B) and then total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (C) SGEs were prepared and individually treated with the protease inhibitors antipain-2HCl (Anti), bestatin (Bes), chymostatin (Chy), E-64 (E64), leupeptin (Leu), pepstatin (Pep), phosphoramidon (Pho), pefabloc SC (Pef), and aprotinin (Apr). MEFs were either treated with buffer alone (PBS) or treated with 1.0 SGE with and without protease inhibitors for 10 min and inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed. DENV2 vRNA was normalized to GAPDH by RT-qPCR. (D) MEFs were treated with Pef or a protease inhibitor cocktail containing Pef (PI) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) Prior to extracting total RNA as described for panel C, cells were observed by phase-contrast light microscopy. Data represent averages from at least three independent experiments. Student's t tests were performed to assess statistical significance when appropriate.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: SGE-mediated enhancement of dengue virus infectivity is inhibited by Pefabloc SC in vitro. (A and B) NIH 3T3 MEFs were treated with buffer (PBS), pretreated with 1.0 SGE, or pretreated with 1.0 boiled SGE (Boiled), followed by inoculation with DENV2 at an MOI of 0.1 for 1 h. (A) Cells were observed by light microscopy 18 hpi (B) and then total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (C) SGEs were prepared and individually treated with the protease inhibitors antipain-2HCl (Anti), bestatin (Bes), chymostatin (Chy), E-64 (E64), leupeptin (Leu), pepstatin (Pep), phosphoramidon (Pho), pefabloc SC (Pef), and aprotinin (Apr). MEFs were either treated with buffer alone (PBS) or treated with 1.0 SGE with and without protease inhibitors for 10 min and inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed. DENV2 vRNA was normalized to GAPDH by RT-qPCR. (D) MEFs were treated with Pef or a protease inhibitor cocktail containing Pef (PI) for 10 min and then inoculated with DENV2 at an MOI of 0.1 for 1 h. Unbound virus was removed, and cells were allowed to incubate for 18 h. Total RNA was extracted, and the expression of DENV2 vRNA and GAPDH was assessed by RT-qPCR. DENV2 vRNA was normalized to GAPDH. (E) Prior to extracting total RNA as described for panel C, cells were observed by phase-contrast light microscopy. Data represent averages from at least three independent experiments. Student's t tests were performed to assess statistical significance when appropriate.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Virus, Infection, In Vitro, Light Microscopy, Expressing, Quantitative RT-PCR, Protease Inhibitor

    Pefabloc SC inhibits SGE-mediated enhancement of dengue and West Nile viruses in vivo. Four groups of 10 IFNAR−/− C57BL/6 mice were inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE and 1% DMSO (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+P+S). Twenty-four hpi, footpads (A), lymph nodes (B), blood (C), and spleens (D) were harvested into RLT buffer, and RNA was extracted for RT-qPCR analysis. DENV vRNA was normalized to total RNA per reaction. (E) C57BL/6 mice were inoculated in a single rear footpad (7 per group) with 500 PFU WNV and 1% DMSO (W) or with 1 mM Pefabloc SC in DMSO (W+P). Relative viral load was assessed in blood by RT-qPCR 24 hpi. (F) Three groups of 10 C57BL/6 mice were inoculated in rear footpads with 500 PFU WNV and 1% DMSO (W), with 1.0 C. tarsalis SGE and 1% DMSO (W+S), or with both 1.0 C. tarsalis SGE and 1 mM Pefabloc SC in DMSO (W+P+S). Relative viral load was assessed in footpads by RT-qPCR 24 hpi. Each symbol represents one mouse. The dotted line represents the background threshold. Mann-Whitney analysis was performed to assess statistical significance between groups. Data represent one experiment.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Pefabloc SC inhibits SGE-mediated enhancement of dengue and West Nile viruses in vivo. Four groups of 10 IFNAR−/− C57BL/6 mice were inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE and 1% DMSO (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+P+S). Twenty-four hpi, footpads (A), lymph nodes (B), blood (C), and spleens (D) were harvested into RLT buffer, and RNA was extracted for RT-qPCR analysis. DENV vRNA was normalized to total RNA per reaction. (E) C57BL/6 mice were inoculated in a single rear footpad (7 per group) with 500 PFU WNV and 1% DMSO (W) or with 1 mM Pefabloc SC in DMSO (W+P). Relative viral load was assessed in blood by RT-qPCR 24 hpi. (F) Three groups of 10 C57BL/6 mice were inoculated in rear footpads with 500 PFU WNV and 1% DMSO (W), with 1.0 C. tarsalis SGE and 1% DMSO (W+S), or with both 1.0 C. tarsalis SGE and 1 mM Pefabloc SC in DMSO (W+P+S). Relative viral load was assessed in footpads by RT-qPCR 24 hpi. Each symbol represents one mouse. The dotted line represents the background threshold. Mann-Whitney analysis was performed to assess statistical significance between groups. Data represent one experiment.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: In Vivo, Quantitative RT-PCR, MANN-WHITNEY

    RT-qPCR analysis of TH2/TH1 cytokine expression in footpads and lymph nodes from IFNAR−/− C57BL/6 mice infected with DENV2 and treated with SGE and Pefabloc SC. Relative expression of IL-4 (A and B), IL-5 (C and D), IL-6 (E and F), IL-10 (G and H), IFN-γ (I and J), and TNF-α (K and L) was examined in footpads and draining lymph nodes 24 hpi in mice inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of either DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+S+P). Relative cytokine levels were normalized to GAPDH. Each dot represents a single mouse. Mann-Whitney tests were performed between groups to assess statistical significance. Data represent one experiment.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: RT-qPCR analysis of TH2/TH1 cytokine expression in footpads and lymph nodes from IFNAR−/− C57BL/6 mice infected with DENV2 and treated with SGE and Pefabloc SC. Relative expression of IL-4 (A and B), IL-5 (C and D), IL-6 (E and F), IL-10 (G and H), IFN-γ (I and J), and TNF-α (K and L) was examined in footpads and draining lymph nodes 24 hpi in mice inoculated into a single rear footpad with 20 μl containing 107 genome equivalents (GE) of either DENV2 and 1% DMSO (D), 107 GE DENV2 with 1 mM Pefabloc SC in DMSO (D+P), 107 GE DENV2 with 1.0 SGE (D+S), or 107 GE with 1 mM Pefabloc SC in DMSO and 1.0 SGE (D+S+P). Relative cytokine levels were normalized to GAPDH. Each dot represents a single mouse. Mann-Whitney tests were performed between groups to assess statistical significance. Data represent one experiment.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Quantitative RT-PCR, Expressing, Infection, MANN-WHITNEY

    Identification of CLIPA3 as a component involved in SGE-mediated infectivity enhancement. (A) HPLC trace showing protein concentrations in 80 100-μl fractions after reverse-phase HPLC fractionation of 100 A. aegypti SGE. (B) Ten μl of each HPLC fraction was diluted in 90 μl PBS and used as SGE for in vitro SGE-mediated infectivity enhancement assays. Eighteen hpi, RNA was harvested and RT-qPCR was performed. Relative levels of DENV2 were normalized to GAPDH. (C) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti head (H), salivary gland (SG), midgut (MG), ovary (O), and carcass (CA) tissues. Relative levels of CLIPA3 were normalized to actin. (D) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti salivary glands 7 days after microinjection with 500 ng control or CLIPA3 siRNA. Relative levels of CLIPA3 were normalized to actin. Each dot represents 3 pooled salivary glands. (E) In vitro SGE-mediated infectivity enhancement assay using buffer alone (PBS), 1.0 SGE from control siRNA-treated mosquitoes (Control), or 1.0 SGE from CLIPA3 siRNA-treated mosquitoes. Relative levels of DENV2 were normalized to GAPDH. Data represent averages from at least 6 independent experiments. (F) Light microscopy images of NIH 3T3 MEFs shown in panel E 6 h post-SGE treatment. Student's t tests were performed to assess statistical significance when appropriate.

    Journal: Journal of Virology

    Article Title: Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    doi: 10.1128/JVI.02235-13

    Figure Lengend Snippet: Identification of CLIPA3 as a component involved in SGE-mediated infectivity enhancement. (A) HPLC trace showing protein concentrations in 80 100-μl fractions after reverse-phase HPLC fractionation of 100 A. aegypti SGE. (B) Ten μl of each HPLC fraction was diluted in 90 μl PBS and used as SGE for in vitro SGE-mediated infectivity enhancement assays. Eighteen hpi, RNA was harvested and RT-qPCR was performed. Relative levels of DENV2 were normalized to GAPDH. (C) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti head (H), salivary gland (SG), midgut (MG), ovary (O), and carcass (CA) tissues. Relative levels of CLIPA3 were normalized to actin. (D) RT-qPCR assay measuring relative expression of CLIPA3 in female A. aegypti salivary glands 7 days after microinjection with 500 ng control or CLIPA3 siRNA. Relative levels of CLIPA3 were normalized to actin. Each dot represents 3 pooled salivary glands. (E) In vitro SGE-mediated infectivity enhancement assay using buffer alone (PBS), 1.0 SGE from control siRNA-treated mosquitoes (Control), or 1.0 SGE from CLIPA3 siRNA-treated mosquitoes. Relative levels of DENV2 were normalized to GAPDH. Data represent averages from at least 6 independent experiments. (F) Light microscopy images of NIH 3T3 MEFs shown in panel E 6 h post-SGE treatment. Student's t tests were performed to assess statistical significance when appropriate.

    Article Snippet: Mouse-adapted dengue virus type 2 (DENV2) TH-36 strain was obtained from the ATCC (VR345).

    Techniques: Infection, Fractionation, In Vitro, Quantitative RT-PCR, Expressing, Microinjection, Control, Light Microscopy