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ATCC denv2 strain th 36

(A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, <t>DENV2,</t> and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Denv2 Strain Th 36, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay"

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

Journal: bioRxiv

doi: 10.64898/2026.03.17.712358

Figure Legend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Techniques Used: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Image Search Results

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

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

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