Journal: bioRxiv

Article Title: Afucosylated maternal anti-dengue IgGs are a biomarker for susceptibility to dengue disease in their infants

doi: 10.1101/565259

Figure Lengend Snippet: To define the roles of FcγRIIa and FcγRIIIa in infection by dengue immune complexes, a panel of human monocytic cell lines (U937) was generated that express all combinations of these activating FcyRs. A) Expression of FcγRIIa and/or FcγRIIIa on U937 cell lines was confirmed by FACS. B) Cross-linking of FcγRIIIa induced signaling in FcγRIIIa + cells, indicated by tyrosine phosphorylation Signaling was observed in the FcγRIIa + FcγRIIIa + and FcyRII - aFcγRIIIa + cell lines but not the FcγRIIIa-deficient FcγRIIa + FcYRIIIa (ITAM-) line, FcγRIIa + FcγRIIIa - (wild-type) or FcγRIIa - FcγRIIIa - cells. C) Broadly reactive anti-E mAb 235 was expressed as a human IgG1 (mAb 235), afucosylated Fc (afucFc) mAb235, mAb 235 Fc null variant N297A. The DENV3 specific mAb 5J7 was also expressed as a human IgG1. Binding of mAbs was assessed by ELISA on dengue 2-infected cells. D) FACS gating strategy for dengue-infected cells. Infection in single, live cells was assessed by staining of envelope protein using mAb 4G2 at 24 hours unless otherwise specified. E) DENV2 infection in wild-type U937 cells increased over time when infection occurred in the presence of mAb 235 expressed as a wild-type human IgG1. Infection did not increase when mAb 235 was expressed as an Fc-null variant, N297A or when immune complexes were made with deactivated virus (iDENV). F) The degree of enhanced infectability depended on the concentration of mAb 235.

Article Snippet: The next day cells were infected with DENV2 New Guinea C (BEI Resources; NR-84) or DENV3 C0360/94 (BEI Resources; NR-48800) MOI 1 for 1 hour, washed, and incubated for 48 h in MEM, 1% methylcellulose, 2% inactivated FBS, antibiotics, and L-glutamine.

Techniques: Infection, Generated, Expressing, Phospho-proteomics, Variant Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Virus, Concentration Assay

Journal: bioRxiv

Article Title: Afucosylated maternal anti-dengue IgGs are a biomarker for susceptibility to dengue disease in their infants

doi: 10.1101/565259

Figure Lengend Snippet: A) Immune complexes composed of human IgG1 mAb 235 and DENV2 were used to infect the panel of U937 cell lines. Infectability of the FcγRIIa - FcγRIIIa - (blue) and FcγRIIa - FcyRIII + (green) cell lines was similar, demonstrating that expression of FcγRIIIa alone did not impact infection In contrast, expression of FcγRIIa alone (FcγRIIa + FcγRIIIa - , black) increased infectability and co-expression of FcγRIIa and FcγRIIIa (FcγRIIa + FcγRIIa + , red) further enhanced infection. Infection in the presence of mAb 235 N297A, an Fc mutation that abrogates FcyR binding, is shown in the unfilled circle. B) To determine whether the presence of FcγRIIIa enhances infectability of FcγRIIa + FcγRIIIa + cells by increasing virus binding to host cells or virus entry. FcyRs were individually blocked during infection. Blocking FcγRIIIa did not impact infection. C) Infection by mAb 235 dengue inmune complexes was reduced in the FcγRIIa + FcγRIIIa (ITAM-) cells which lack FcγRIIIa signaling capacity. Infection by dengue immune complexes generated from anti-envelope mAbs 747(4) B7, 2D22 or 752-2 C8 followed the pattern of being increased in FcγRIIa + FcγRIIIa - cells over FcγRIIa-defldent cells, but was maximally enhanced by co-expression of FcγRIIa and FcγRIIIa. Enhancement in FcγRIIa + FcγRIIIa + cells required FcγRIIIa signaling in all cases D) Infection by human polyclonal dengue immune complexes was maximal in FcγRIIa + FcγRIIIa + monocytes and FcγRIIIa signaling was required for enhancement in all cases.

Article Snippet: The next day cells were infected with DENV2 New Guinea C (BEI Resources; NR-84) or DENV3 C0360/94 (BEI Resources; NR-48800) MOI 1 for 1 hour, washed, and incubated for 48 h in MEM, 1% methylcellulose, 2% inactivated FBS, antibiotics, and L-glutamine.

Techniques: Expressing, Infection, Mutagenesis, Binding Assay, Virus, Blocking Assay, Generated

Journal: BMC Microbiology

Article Title: Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes

doi: 10.1186/1471-2180-7-9

Figure Lengend Snippet: The presence of DENV-2 antigen in the tracheal system correlates with early dissemination in Chetumal mosquitoes

Article Snippet: Typically to detect DENV-2 antigen by IFA, the primary antibody was the serotype-specific mouse 3H5–1 monoclonal antibody (ATCC number HB-46).

Techniques: Infection

Journal: PLoS Neglected Tropical Diseases

Article Title: Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

doi: 10.1371/journal.pntd.0002711

Figure Lengend Snippet: The monocytes from G6PD-deficient and normal controls were infected with DENV2 at an MOI of 0.1. Cells and culture supernatants were harvested at 24, 48, 72, 96, 120 hours post-infection. Number of DENV infected cells were assayed by flow cytometry ( A ), whereas virus released by the infected cells was determined by Plaque assay ( B ). Number of infected cells as well as virus titer found to be significantly higher in infected monocytes from G6PD-deficient individuals compared to the normal controls.

Article Snippet: The C6/36 mosquito A. albopictus cell line (CRL-1660 ATCC, USA) was used to propagate DENV2 (D2MY00-22563), kindly provided by Prof Shamala of University of Malaya, in Leibovitz culture medium (L-15) (SIGMA, USA) supplemented with 1% L-glutamine (SIGMA, USA), 19% tryptose phosphate broth (Hi MEDIA, India), 1% of penicillin/streptomycin (GIBCO, Grand Island, USA) and 5% fetal bovine serum FBS (GIBCO, Grand Island, USA).

Techniques: Infection, Flow Cytometry, Virus, Plaque Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

doi: 10.1371/journal.pntd.0002711

Figure Lengend Snippet: The production of nitric oxide (NO) in monocytes of both normal controls and G6PD-deficient donors increased significantly ( p <0.001) after DENV2 infection. In a time-dependent manner, monocytes from G6PD-deficient subjects produced significantly ( p <0.001) lower NO than monocytes from normal controls.

Article Snippet: The C6/36 mosquito A. albopictus cell line (CRL-1660 ATCC, USA) was used to propagate DENV2 (D2MY00-22563), kindly provided by Prof Shamala of University of Malaya, in Leibovitz culture medium (L-15) (SIGMA, USA) supplemented with 1% L-glutamine (SIGMA, USA), 19% tryptose phosphate broth (Hi MEDIA, India), 1% of penicillin/streptomycin (GIBCO, Grand Island, USA) and 5% fetal bovine serum FBS (GIBCO, Grand Island, USA).

Techniques: Infection, Produced

Journal: PLoS Neglected Tropical Diseases

Article Title: Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

doi: 10.1371/journal.pntd.0002711

Figure Lengend Snippet: The production of superoxide anions (O 2.− ) in monocytes of both normal controls and G6PD-deficient donors increased significantly (p<0.001) after DENV2 infection. In a time-dependent manner, monocytes from G6PD-deficient subjects produced significantly (p<0.001) lower O 2.− than monocytes from normal controls.

Article Snippet: The C6/36 mosquito A. albopictus cell line (CRL-1660 ATCC, USA) was used to propagate DENV2 (D2MY00-22563), kindly provided by Prof Shamala of University of Malaya, in Leibovitz culture medium (L-15) (SIGMA, USA) supplemented with 1% L-glutamine (SIGMA, USA), 19% tryptose phosphate broth (Hi MEDIA, India), 1% of penicillin/streptomycin (GIBCO, Grand Island, USA) and 5% fetal bovine serum FBS (GIBCO, Grand Island, USA).

Techniques: Infection, Produced

Journal: PLoS Neglected Tropical Diseases

Article Title: Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

doi: 10.1371/journal.pntd.0002711

Figure Lengend Snippet: The accumulation of oxidative stress in monocytes of both normal controls and G6PD-deficient donors increased significantly (p<0.001) after DENV2 infection. In a time-dependent manner, monocytes from G6PD-deficient subjects accumulated significantly (p<0.001) higher oxidative stress compared to monocytes from normal controls.

Article Snippet: The C6/36 mosquito A. albopictus cell line (CRL-1660 ATCC, USA) was used to propagate DENV2 (D2MY00-22563), kindly provided by Prof Shamala of University of Malaya, in Leibovitz culture medium (L-15) (SIGMA, USA) supplemented with 1% L-glutamine (SIGMA, USA), 19% tryptose phosphate broth (Hi MEDIA, India), 1% of penicillin/streptomycin (GIBCO, Grand Island, USA) and 5% fetal bovine serum FBS (GIBCO, Grand Island, USA).

Techniques: Infection

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: Virus Protein Antibodies.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Virus

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: CDK8 is upregulated during DENV infection. ( A , B ) Huh7 cells were either mock-infected, infected with DENV at MOI 10, or treated with equivalent UV-inactivated DENV2 (UV-DENV) over a time course of 48 h. Total cellular RNA was collected at indicated time points after infection and analyzed by qRT-PCR for DENV RNA ( A ), and CDK8 mRNA expression ( B ), relative to the time of infection and normalized to the housekeeping gene, SDHA . Results are representative of two independent experiments. ( C , D ) CDK8 ( C ) and CDK19 ( D ) mRNA expression in mock or DENV-infected Huh7 cells after 36 h of infection at MOI 10 (n = 6 biological replicates; **** p < 0.0001 unpaired, two-tailed t test). Error bars represent mean +/− SEM). ( E ) Western blot analyses of 10 µg of total protein from nuclear extracts (Soluble Nuclear Extracts) and from the remaining, salt-extracted chromatin fraction (Chromatin-Bound Proteins) from cells collected every three hours for 24 h after infection. Antibody specificities are indicated as are the relative band densities of CDK8 versus Cyclin C and histone H3 phosphorylated at serine in position 10 (H3S10-P) versus total histone H3 (H3). Infection was confirmed by presence of nuclear DENV2 NS5. The results are representative of three separate time-course infections.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Infection, Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: Knockdowns of CDK8 and CDK19 reduce DENV2 replication. ( A ) Western blot analysis of 10 µg of total protein from nuclear extracts of Huh7 cells transduced at an MOI of 1 with lentivirus-mediated non-target control or CDK8-targeted shRNA, CDK19-targeted shRNA, or cyclin C-targeted shRNA. ( B ) Lentivirus-transduced Huh7 cells were infected with DENV2 at MOI 1 for 24 h, and total cellular RNA analyzed by qRT-PCR for DENV RNA quantification relative to in vitro transcribed DENV genome equivalent (GE) standard curve ( n = 3 biological replicates per group; ** p < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean +/ SEM). ( C ) Relative optical density read in lentivirus-transduced Huh7 cells after four days of selection, then treated with CellTiter 96 Aqueous One Solution. ( n = 4 biological replicates. * p < 0.05, **** p < 0.0001; one-way ANOVA with Dunnett’s test. Error bars represent mean +/ SEM). ( D ) Lentivirus-transduced Huh7 cells were infected with DENV2 at MOI 1 for 24 h, and total cellular RNA analyzed by qRT-PCR for DENV RNA quantification normalized to the housekeeping gene, SDHA , and relative to expression in non-target controls ( n = 3 biological replicates per group; * p < 0.05, one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean +/ SEM).

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Western Blot, Control, shRNA, Infection, Quantitative RT-PCR, In Vitro, Selection, Expressing

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: CDK8/19 Chemical inhibition reduces DENV2 replication. ( A ) Relative optical density of Huh7 cells after 72 h treatment with DMSO or Senexin A at indicated doses and addition of CellTiter 96 Aqueous One Solution. ( n = 9 biological replicates. **** p < 0.0001 one-way ANOVA with Dunnett’s test. Error bars represent mean +/ SEM). ( B ) EGR 1 mRNA levels in serum-starved and serum-stimulated Huh7 cells in the presence of DMSO or 12 μM Senexin A (n = 3 biological replicates; * p < 0.05, **** p < 0.0001 one-way ANOVA with Dunnett’s test; error bars represent mean +/− SEM). ( C ) DENV2 RNAs in total cellular RNA preparations (Intracellular RNA, GE) and in culture supernatants (Extracellular RNA, GE) were determined by qRT-PCR, and supernatant virus measured by plaque assay (Infectious Particles). Huh7 cells were infected with DENV2 (MOI = 1) for 24 or 36 hpi with DMSO or 12 or 25 μM Senexin A, respectively ( n = 3 biological replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; unpaired two-tailed t test; Error bars represent mean +/ SEM). ( D ) Western blot assays of supernatant virus pellets from cells infected with 10 MOI UV-treated DENV2 (UV), or with DENV2 without (DENV) or with 25 µM Senexin A (DENV/Sen) at 24 and 48 hpi. Antibody specificities are indicated. Relative band densities of DENV2 gE, prM, and capsid proteins in DENV2-infected, Senexin A-treated versus DENV2-infected virus preparations are indicated to the right of the indicated panels. Results are representative of three biological replicates for each time point.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Inhibition, Quantitative RT-PCR, Virus, Plaque Assay, Infection, Two Tailed Test, Western Blot

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: Senexin A reduces DENV2 induction of Hexokinase 2 expression. ( A ) Huh7 cells were either mock-infected or infected with DENV at MOI 10, and HK2 mRNA expression analyzed by qRT-PCR at indicated time points. Results are representative of two independent experiments. Expression is relative to time of infection and normalized to SDHA . ( B ) HK2 expression in mock vs. infected cells at 48 hpi ( n = 7 biological replicates; *** p < 0.001 unpaired, two-tailed t test with Welch’s correction). Error bars represent mean +/− SEM. ( C ) HK2 mRNA expression in mock-infected and infected Huh7 cells treated with DMSO or 25 μM Senexin A or 12 μM Senexin B added at start of infection (MOI 10; 36 hpi). Expression relative to mock-infected, DMSO-treated cells and normalized to SDHA ( n = 3 biological replicates; ** p < 0.01, **** p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test). Error bars represent mean +/− SEM. ( D ) Western blot analysis of cellular and viral protein abundance in 2 µg mitochondrial-enriched fractions from mock-infected cells (Mock), or cells with 10 MOI UV-treated DENV2 (UV) or DENV2 without (DENV) or with or 25 µM Senexin A (DENV/Sen). Antibody specificities are indicated as are the relative band densities of HK2 versus Cox4. Relative band densities of DENV2 NS5 and prM proteins in Senexin A-treated versus DMSO-treated, DENV2-infected cell preparations are indicated to the right of the indicated panels. Results are representative of six biological replicates.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Expressing, Infection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Quantitative Proteomics

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: Senexin A reduces DENV2 induction of lipophagic gene expression. ( A ) Huh7 cells were either mock-infected or infected with DENV at MOI 10, and LC3 mRNA expression analyzed by qRT-PCR at indicated time points. Expression relative to time of infection, normalized to SDHA and representative of two independent time course experiments. ( B ) LC3 expression in mock vs. infected cells at 48 hpi ( n = 3 biological replicates; *** p < 0.001 unpaired, two-tailed t test with Welch’s correction). Error bars represent mean +/− SEM. ( C ) LC3 mRNA expression in Huh7 cells mock-infected or infected with DENV2 at MOI 10 for 36 h with DMSO or 25 μM Senexin A or 12 μM Senexin B added at time of infection (relative to mock-infected, DMSO-treated cells and normalized to SDHA ; n = 3 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test). Error bars represent mean +/− SEM. ( D ) Western blot analysis of cellular and viral protein abundance in 2 µg cytoplasmic extracts from mock-infected cells (Mock), or cells with 10 MOI UV-treated DENV2 (UV) or DENV2 without (DENV) or with or 25 µM Senexin A (DENV/Sen). Antibody specificities are indicated as are the relative band densities of LC3-I and LC3-II versus Cox4. Relative band densities of DENV2 NS3 and prM proteins in Senexin A-treated versus DMSO-treated, DENV2-infected cell preparations are indicated to the right of the indicated panels. Results are representative of six biological replicates.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Gene Expression, Infection, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Quantitative Proteomics

Journal: Viruses

Article Title: Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

doi: 10.3390/v12060654

Figure Lengend Snippet: Senexin A inhibits mitochondrial respiration. Huh7 cells were either mock-infected or infected with DENV2 at an MOI of 10 with or without DMSO or 12.5 µM Senexin A for 24 or 48 h. ( A , B ) Normalized oxygen consumption rate (OCR) was measured over time during mitochondrial stress tests. Specific values determined by the mitochondrial stress test (Basal rate, ATP production, Maximum Respiration, Spare Capacity, Proton Leak, and Non-Mitochondrial Respiration) are presented. ( C ) Normalized extracellular acidification rate (ECAR) was measured over time during a glucose stress test at 48 hpi. Specific values determined in the glucose stress test (Glycolysis levels, Glycolytic Capacity, Glycolytic Reserve, and Non-Glycolytic Capacity) are presented. Results were normalized to viable cell numbers after metabolic measurements. Parameters for individual measurements are presented in the methods (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 one-way ANOVA with Tukey’s multiple comparisons test. n = 3 biological replicates). Error bars represent mean +/ SEM.

Article Snippet: Mock- or DENV2 infected cells were incubated in DMEM with Senexin A (MedChemExpress, Monmouth Junction, NJ, USA) or Senexin B (ProbeChem, Shanghai, China) at indicated concentrations or in media with the volumetric equivalent (final 0.01%) of DMSO solvent for the indicated period of time.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A and B ) The serum concentrations of NS1(A) and MMP-9 (B) in healthy donors and severe dengue patients infected days (2, 5, 8, and 11 days) were measured by ELISA. Points represent the value in each serum sample. ( C and D ) The serum concentrations of NS1 (C) and MMP-9 (D) in each severe dengue patients infected days (2, 5, 8, and 11 days) were measured by ELISA. Points represent the value in each serum sample. ( E ) the correlations of the concentrations of NS1 and MMP-9 in the same group of severe dengue patients infected 11 days were plotted. Linear regressions were traced according to the distributions of the points. Dates were representative of two independent experiments. ns means not significant. P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***).

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were co-transfected with HA-MMP-9 and Flag-Cap, Flag-M, Flag-Prm, Flag-E, Flag-NS1, Flag-NS2A, Flag-NS2B, Flag-NS3, Flag-NS4A, or Flag-NS4B. Cell lysates were immunoprecipitataed using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( B ) HEK293T cells were co-transfected with HA-MMP9 and Flag-NS1, Cell lysates were immunoprecipitataed using anti-HA antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( C, D ) HEK293T cells were co-transfected with empty vector or VC-155-MMP-9 and VN173-NS1/E/NS4A. At 24 h post-transfection, living cells were observed by confocal microscopy. The quantification of YFP-positive cells was determined by ImageJ software (D). ND means not detection. ( E ) Purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) was incubated with purified no-tagged MMP-9 protein (3 μg) for 24 h, Mixtures were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-MMP9, anti-NS1, anti-His antibody. Untreated protein including His-SARS-CoV-2-N (1 μg), His-DENV2-NS1 (1 μg), or no-tagged MMP-9 protein (1 μg) were analyzed by immunoblotting using anti-MMP9, anti-NS1, and anti-His antibody (as input). ( F ) Yeast strain AH109 were co-transformed with combination of binding domain (BD-p53, BD-MMP-9, and BD-Lam) and activation domain (AD-T, AD-NS1) plasmid. Transfected yeast cells were grown on SD-minus Trp/Leu double dropout plates, and colonies were replicated on to SD-minus Trp/Leu/Ade/His fourth dropout plates to check for the expression of reporter genes. ( G, H ) Schematic diagram of wild-type MMP-9 protein and truncated mutants MMP-9 protein (D1 to D9) (G). HEK293T cells were co-transfected with HA-NS1 and Flag-MMP-9 truncated mutants (D1 to D9). Cell lysates were immunoprecipitated using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input (H). Dates were representative of three independent experiments.

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Software, Purification, Incubation, Western Blot, Transformation Assay, Binding Assay, Activation Assay, Expressing, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages were transfected with the different concentrations of plasmid encoding NS1 for 24 h. Cell lysates were analyzed (top) by immunoblotting. Supernatants were analyzed (middle) by gelatin zymography assays for MMP-9 proteinase activity. Intracellular MMP-9 RNA (bottom) was determined by qRT-PCR analysis. ( B ) HEK293T cells were co-transfected with the plasmid encoding MMP-9 and different concentrations of plasmid encoding NS1 for 24 h. Cell lysates were analyzed (top) by immunoblotting. Supernatants were analyzed (middle) by gelatin zymography assays for MMP-9 proteinase activity. Intracellular MMP-9 RNA (bottom) was determined by qRT-PCR analysis. ( C ) HEK293T cells were con-transfected with different concentrations of NS1 expressing plasmid and NF-κB reporter plasmid. Luciferase assays were performed 20 h after transfection. ( D ) PMA-differentiated THP-1 macrophages were firstly transfected with plasmid encoding HA-CT or HA-NS1 for 20 h, and then treated with 200 nM SC75741 for 5 h, MMP-9 protein in cell supernatants were measured by ELISA (top) and indicated proteins in cell extract were analyzed by WB (middle). Intracellular MMP-9 RNA (bottom) was determined by qRT-PCR analysis. ( E – G ) Hela cells (E), HEK293T cells (F), and PMA-differentiated THP-1 macrophages (G) were transfected with different concentrations of plasmid encoding NS1 for 24 h. The indicated proteins in cell extract were analyzed by WB. ( H ) The supernatants of HUVEC cells were incubated with BSA (3 μg/ml), NS1 (3 μg/ml), pro-MMP-9 protein (200 ng/ml), BSA (3 μg/ml) plus pro-MMP-9 protein (200 ng/ml), NS1 (3 μg/ml) plus pro-MMP-9 protein (200 ng/ml) or MMP-9 protein (200 ng/ml) for 6 h, and then the supernatants were analyzed by gelatin zymography assays for MMP-9 proteinase activity and the indicated protein expression were analyzed by immunoblotting. Dates were representative of two to three independent experiments. ns means not significant. Values are mean ± SEM, P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***).

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Transfection, Plasmid Preparation, Western Blot, Zymography, Activity Assay, Quantitative RT-PCR, Expressing, Luciferase, Enzyme-linked Immunosorbent Assay, Incubation

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages (top) or HUVEC cells (bottom) were treated with infectious or UV- inactivated DENV2 at MOI = 5 for 48h. Intracellular DENV2 E RNA (bottom) was determined by qRT-PCR analysis. Mock: untreated cells. Control: supernatant of C6/36 cells without DENV2 infection. ( B and C ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5 (B) or at different MOI for 24 h (C). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( D and E ) HUVEC cells were infected with DENV2 for different times at MOI = 5 (D C ) and at different MOI for 24 h (E). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( F ) HUVEC cells or PMA-differentiated THP-1 macrophages were infected with DENV2 at MOI = 5 for 24 h. Viral copies were quantified by RT-PCR. ( G ) HUVEC cells or PMA-differentiated THP-1 macrophages were equally distributed to four 12-hole plates and infected with DENV2 at MOI = 5 for 24 h. MMP-9 protein in cell supernatants were measured by ELISA (top) and indicated proteins in cell extract were analyzed by WB (bottom). Dates were representative of three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Quantitative RT-PCR, Infection, Activity Assay, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5, NS1 protein in Supernatants were analyzed by ELISA (top). Cell lysates were analyzed by immunoblotting (bottom). ( B ) Confluent monolayers of HUVEC cells were grown on polycarbonate membrane system and treated with the supernatants came from DENV2 infected HUVEC cells or THP-1 cells for 24 h or pre-incubated with 600nM SB-3CT (a specific inhibitor of MMP-9 protein) or 600 nM SC75741 for 1h. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) (ohm) using EVOM2 epithelial voltohmmeter. ( C – I ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. MMP-9 RNA in the blood was determined by qRT-PCR (upper) and MMP-9 protein in the serum was measured by ELISA (lower). Points represent the value of each serum samples (C). Evans blue dye was intravenously injected into mice 7 days after DENV infected groups (n = 5), control groups (n = 4) and DENV+SB-3CT (n = 5) (C–E). The dye was allowed to circulate for 2 hours before mice were euthanasia, tissues include liver (D), spleen (E) and lung (F) were collected, and the value of Evans blue was measured at OD 610 . Histopathology analysis of tissues includes Liver (G), Spleen (H) and Lung (I) after DENV infection. ( J ) Monolayers of HUVEC cells grown on Transwell inserts were incubated for 48 h with MMP-9 protein (100 ng/ml) or NS1 protein (5 μg/ml) or NS1 (5 μg/ml) plus different concentration of MMP-9 (50 ng/ml to 100ng/ml) or pre-treated with 600 nM SB-3CT or 600 nM SC7574 for 1 h, then incubated with NS1 plus MMP-9. The TEER (ohm) was measured at indicated time points. Dates were representative of two to three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane, Incubation, Permeability, Injection, Quantitative RT-PCR, Histopathology, Concentration Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A – F ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. The indicated proteins in Lung (A), spleen (B) and Liver (C) were measured by Western-blot. The expression of β-catenin in Liver (D), spleen (E), and Lung (F) by Immunohistochemistry analysis. ( G ) HUVEC cells were respectively transfected with plasmid encoding MMP-9 (2 μg) or NS1 (2 μg) or NS1 (1 ug) plus MMP-9 (1 μg) for 24 h or firstly co-transfected with plasmid encoding NS1 (1ug) plus MMP-9 (1 μg) for 12 h, then treated with 600nM SB-3CT for 12 h. The indicated proteins in cell extract were analyzed by WB. ( H–K ) HUVEC cells were treated with NS1 protein (5 μg/ml) or MMP-9 protein (100 ng/ml) or NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) or pre-incubated with 600 nM SB-3CT for 1 h, then treated with NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) for 6 h, The distribution of endogenous β-catenin (H) or ZO-1 (J) protein were visualized under confocal microscope. The quantifications of relative β-catenin (I) or ZO-1 (K) intensities were determined by ImageJ software. The quantification of protein was used by ImageJ software (A–G). (D–F) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. All dates were representative of two to three independent experiments.

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Injection, Infection, Western Blot, Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Incubation, Microscopy, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with plasmid encoding HA-NS1 plus Flag-β-catenin. Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag, anti-HA, or anti-β-catenin antibody. Cell lysates (40 μg) were used as Inputs. ( B ) Hela cells were transfected with plasmid encoding HA-NS1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( C , D ) HEK293T cells (C) or Hela cells (D) were co-transfected with plasmid encoding HA-NS1 plus Flag-ZO-1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag or anti-HA antibody. Cell lysates (40 μg) were used as Inputs. ( E , F ) HEK293T cells were transfected with Flag-β-catenin (E) or Flag-ZO-1 (F) and incubated with purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) for 24 h, cell extracts were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, anti-ZO-1 antibody. Untreated proteins including His-SARS-CoV-2-N (1 μg) and His-DENV2-NS1 (1 μg) and HEK293T cell lysates were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, and anti-ZO-1 antibody (as input). ( G , H ) Hela cells were co-transfected with plasmid encoding HA-NS1 plus Flag-MMP-9, Cell lysates were immunoprecipitated using anti-Flag (G) or anti-β-catenin antibody (H), and analyzed using anti-Flag, anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( I, J ) Hela cells were co-transfected with plasmid encoding HA-NS1, HA-MMP-9, and Flag-ZO-1 (I) or Flag-NS1, HA-MMP-9, and Flag-ZO-1 (J). Cell lysates were immunoprecipitated using anti-Flag (top) or anti-HA antibody (bottom), and analyzed using anti-Flag, anti-HA or anti-MMP-9 antibody. Cell lysates (40 μg) was used as Input. ( K , L ) HUVEC cells were treated with NS1 protein (5 μg/ml), MMP-9 protein (100 ng/ml), and NS1 protein (5 μg/ml) plus MMP-9 protein (100 ng/ml), respectively, for 6 h. The distributions of the membrane marker (Dil) (yellow), the endogenous β-catenin (yellow) and ZO-1 (yellow) proteins and the extracellular NS1 (red) and MMP-9 (green) proteins were visualized under confocal microscope. The quantifications of co-localization fluorescence were determined by ImageJ software (L). ND means not detected. All dates were representative of three independent experiments.

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Purification, Western Blot, Membrane, Marker, Microscopy, Fluorescence, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: C57BL/6 mice and MMP-9 -/- mice were injected intravenously DENV2 NS1 protein [10 mg/kg (n = 5)], the same volume of PBS was also tail vein injected to C57BL/6 mice and MMP-9 -/ - mice (n = 5) as control group. Another group of MMP-9 -/- mice (n = 5) were injected intravenously DENV2 NS1 protein (10 mg/kg) plus recombinant mouse MMP-9 protein (70 μg /kg). ( A – C ) After 24 h post-injection, mice were intravenously injected with Evans blue dye. The dye was allowed to circulate for 2h before mice were euthanized, and tissue include Lung (A), spleen (B), and Liver (C) were collected. The value of Evans blue was measured at OD 610 . ( D – I ) After 24 h post-injection, mice were euthanized and tissue were collected. The indicated proteins in Lung (D), spleen (E), and Liver (F) were measured by Western-blot. The expression of β-catenin in Lung (G), spleen (H), and Liver (I) were analyzed by Immunohistochemistry. All dates were representative of two to three independent experiments. The quantification of protein was used by ImageJ software (D–I). (G–H) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Injection, Recombinant, Western Blot, Expressing, Immunohistochemistry, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 interacts with MMP-9 and facilitates the enzyme to alter the adhesion and tight junctions and vascular leakage in human endothelial cells and mice tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mice tissues.

Article Snippet: Commercialized DENV2-NS1 protein were purchased from Native Antigen.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

doi: 10.1073/pnas.2304139120

Figure Lengend Snippet: Fig. 1. Broad-spectrum antiviral activities of s2U against Flaviviruses and CHIKV. (A) Chemical structure of 2-thiouridine (s2U). (B and C) Dose-response inhibition of DENV2 replication by s2U in VeroE6 (B) and Huh7 (C) cells. Cell lysates were collected for viral RNA determination, and viral RNA levels were determined relative to ACTB transcripts. (D) Dose-response inhibition of DENV2 propagation by s2U. Supernatants of DENV2-infected BHK-21 cells were collected at 72 hours post-infection (hpi), and dilutions were used to inoculate BHK-21 cells. Four days after inoculation, viral titers were determined by the plaque assay. (E–I) Dose- response inhibition of ZIKV (E), YFV (F), JEV (G), WNV (H), and CHIKV (I) by s2U. Cell lysates were collected for viral RNA determination, and viral RNA levels were determined relative to ACTB transcripts. (J) Dose-response inhibition of viral protein expression in the DENV2- and CHIKV- infected cells. Cells were stained with viral-specific antibodies (green, DENV2: Envelope protein, CHIKV: E1 protein) and counterstained with Hoechst 33342 nuclear dye (blue). (Scale bars indicate 200 μm.) Data are presented as mean values of biological triplicates from one of the experiments, and error bars indicate SD. Statistically significant differences were determined using a one-way ANOVA followed by Dunnett’s multiple comparisons test to compare with non-treated cells; *P < 0.01, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Article Snippet: Antiviral activity of s2U against various RNA viruses (CPE- based assay) Virus Strain Cell EC50 (μM) * Virus Strain Cell EC50 (μM) † MTT/resazurin assay DENV2 D2/hu/ INDIA/09- 74 BHK- 21 1.5 ± 0.10 RABV HEP BHK- 21 >100 AG- P10 (Mouseadapted) BHK- 21 1.3 ± 0.12 LACV ATCC VR- 1834 MDBK >100 ZIKV MR766 BHK- 21 3.8 ± 0.96 LPHV 11SB17 KB >100 YFV 17D- 204 BHK- 21 3.2 ± 1.4 LCMV Armstrong KB >100 JEV Beijing- 1 VeroE6 7.6 ± 1.3 JUNV Candid #1 293T >100 WNV NY99 BHK- 21 8.6 ± 1.8 SFTSV ArtLN/2017 MDCK >100 VeroE6 4.0 ± 0.24 RVFV MP12 MDCK >100 CHIKV SL10571 BHK- 21 3.8 ± 0.12 TPMV VRC- 66412 VeroE6 >100 VeroE6 2.2 ± 0.23 IAV H5N1 A/Hong Kong/483/97 A549 >100 HCoV 229E MRC5 0.94 ± 0.16 IAV H7N9 A/Anhui/1/2013 MA104/ TMPRSS2 >100 HCoV OC43 MRC5 4.8 ± 0.25 Virus Strain Cell EC50 (μM) * SARS- CoV- 2 WK- 521 (Ancestral) VeroE6/ TMPRSS2 2.3 ± 0.43 SARS- CoV- 2 TY38- 873 (Omicron BA.1) VeroE6/ TMPRSS2 2.0 ± 0.16 QK002 (Alpha) VeroE6/ TMPRSS2 2.2 ± 0.56 TY40- 385 (Omicron BA.2) VeroE6/ TMPRSS2 1.8 ± 0.094 TY8- 612 (Beta) VeroE6/ TMPRSS2 2.2 ± 0.23 TY41- 703 (Omicron BA.4) VeroE6/ TMPRSS2 1.8 ± 0.013 TY7- 501 (Gamma) VeroE6/ TMPRSS2 1.8 ± 0.37 TY41- 702 (Omicron BA.5) VeroE6/ TMPRSS2 2.9 ± 0.18 TY11- 927 (Delta) VeroE6/ TMPRSS2 1.9 ± 0.22 TY41- 796 (Omicron BQ.1.1) VeroE6/ TMPRSS2 2.0 ± 0.44 MA- P10 (Mouseadapted) VeroE6/ TMPRSS2 2.5 ± 0.34 TY41- 795 (Omicron XBB.1) VeroE6/ TMPRSS2 1.9 ± 0.18 Virus Strain Cell EC50 (μM) * Fold change‡ Resazurin assay rgDENV2 Wild type BHK- 21 0.86 ± 0.12 rgDENV2 NS5- G605V BHK- 21 5.1 ± 0.69 6.1 Antiviral assays were carried out as described in SI Appendix, Table S3.

Techniques: Inhibition, Infection, Plaque Assay, Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

doi: 10.1073/pnas.2304139120

Figure Lengend Snippet: Fig. 3. Molecular target and mechanism of action of s2U. (A) Ribonucleoside competition of DENV2 inhibition by s2U. DENV2 [multiplicity of infection (MOI) = 0.01]-infected BHK-21 cells were treated with 10 μM of s2U and serial dilutions of exogenous nucleosides. A resazurin reduction assay was performed at 4 days post-infection (dpi). (B) Schematic presentation of the experimental design for drug-escape mutant selection and sequence result. BHK-21 cells were infected with DENV2 in the presence of s2U. The passage of infected cells or culture supernatant was performed every 2 to 3 d. Base substitution was detected using Sanger sequencing. (C) Effect of s2U resistance mutation on anti-DENV2 activity of s2U. BHK-21 cells were infected with rgDENV2-WT or rgDENV2-NS5-G605V (MOI = 0.1) containing a serially diluted compound. A resazurin reduction assay was performed at 4 dpi. (D) Analysis of viral RdRp stalling by s2U 5′-triphosphate (s2UTP). Denaturing polyacrylamide gel electrophoresis fraction of RNA transcripts produced through primer extension by ZIKV RdRp in the presence of the indicated nucleotides. The RNA primer/template sequence used in this assay is indicated at the top (small black circles indicate the incorporation sites of UTP). (E) Relative band intensities of fluorescently labeled RNA primers. Relative fluorescence intensities of each RNA primer (white arrowhead in Fig. 3D) were normalized by the RNA sample without UTP or s2UTP (black bar, RNA only). Anti-DENV2 activities (%; A and C) are expressed relative to the values for the DMSO-treated, infected samples and non-infected samples. Data are presented as mean values, and error bars indicate SD. Statistically significant differences between wildtype and G605V viruses (C) were determined using a two-way ANOVA followed by Bonferroni’s multiple comparisons tests; *P < 0.01, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Article Snippet: Antiviral activity of s2U against various RNA viruses (CPE- based assay) Virus Strain Cell EC50 (μM) * Virus Strain Cell EC50 (μM) † MTT/resazurin assay DENV2 D2/hu/ INDIA/09- 74 BHK- 21 1.5 ± 0.10 RABV HEP BHK- 21 >100 AG- P10 (Mouseadapted) BHK- 21 1.3 ± 0.12 LACV ATCC VR- 1834 MDBK >100 ZIKV MR766 BHK- 21 3.8 ± 0.96 LPHV 11SB17 KB >100 YFV 17D- 204 BHK- 21 3.2 ± 1.4 LCMV Armstrong KB >100 JEV Beijing- 1 VeroE6 7.6 ± 1.3 JUNV Candid #1 293T >100 WNV NY99 BHK- 21 8.6 ± 1.8 SFTSV ArtLN/2017 MDCK >100 VeroE6 4.0 ± 0.24 RVFV MP12 MDCK >100 CHIKV SL10571 BHK- 21 3.8 ± 0.12 TPMV VRC- 66412 VeroE6 >100 VeroE6 2.2 ± 0.23 IAV H5N1 A/Hong Kong/483/97 A549 >100 HCoV 229E MRC5 0.94 ± 0.16 IAV H7N9 A/Anhui/1/2013 MA104/ TMPRSS2 >100 HCoV OC43 MRC5 4.8 ± 0.25 Virus Strain Cell EC50 (μM) * SARS- CoV- 2 WK- 521 (Ancestral) VeroE6/ TMPRSS2 2.3 ± 0.43 SARS- CoV- 2 TY38- 873 (Omicron BA.1) VeroE6/ TMPRSS2 2.0 ± 0.16 QK002 (Alpha) VeroE6/ TMPRSS2 2.2 ± 0.56 TY40- 385 (Omicron BA.2) VeroE6/ TMPRSS2 1.8 ± 0.094 TY8- 612 (Beta) VeroE6/ TMPRSS2 2.2 ± 0.23 TY41- 703 (Omicron BA.4) VeroE6/ TMPRSS2 1.8 ± 0.013 TY7- 501 (Gamma) VeroE6/ TMPRSS2 1.8 ± 0.37 TY41- 702 (Omicron BA.5) VeroE6/ TMPRSS2 2.9 ± 0.18 TY11- 927 (Delta) VeroE6/ TMPRSS2 1.9 ± 0.22 TY41- 796 (Omicron BQ.1.1) VeroE6/ TMPRSS2 2.0 ± 0.44 MA- P10 (Mouseadapted) VeroE6/ TMPRSS2 2.5 ± 0.34 TY41- 795 (Omicron XBB.1) VeroE6/ TMPRSS2 1.9 ± 0.18 Virus Strain Cell EC50 (μM) * Fold change‡ Resazurin assay rgDENV2 Wild type BHK- 21 0.86 ± 0.12 rgDENV2 NS5- G605V BHK- 21 5.1 ± 0.69 6.1 Antiviral assays were carried out as described in SI Appendix, Table S3.

Techniques: Inhibition, Infection, Mutagenesis, Selection, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis, Produced, Labeling, Fluorescence

Journal: PLoS ONE

Article Title: DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

doi: 10.1371/journal.pone.0020528

Figure Lengend Snippet: BHK cells were transfected with plasmids pE1D2 (A, D), pE2D2 (B, E) and pcTPA (C, F). Cells were permeabilized, fixed and treated with DENV2 hiperimmune mouse ascitic fluid (A–C) or the monoclonal DENV2 3H5 antibody (D–F), followed by incubation with anti-mouse fluorescein-conjugated goat IgG. Magnification 1000x (A, B, D, E) and 400x (C, F).

Article Snippet: Cells were then incubated for 1 h at 37°C with DENV2 hyperimmune mouse ascitic fluid (ATCC, USA), diluted 1∶1500, or the monoclonal anti-DENV2 3H5 (ATCC), diluted 1∶500.

Techniques: Transfection, Incubation

Journal: PLoS ONE

Article Title: DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

doi: 10.1371/journal.pone.0020528

Figure Lengend Snippet: Cells were metabolically labeled with [ 35 S] methionine and culture supernatants were immunoprecipitated with DENV2 hiperimmune mouse ascitic fluid. Culture supernatants of cells transfected with pE1D2 (lane 1), pE2D2 (lane 2) or pcTPA (lane 3). Arrows indicate recombinant proteins.

Article Snippet: Cells were then incubated for 1 h at 37°C with DENV2 hyperimmune mouse ascitic fluid (ATCC, USA), diluted 1∶1500, or the monoclonal anti-DENV2 3H5 (ATCC), diluted 1∶500.

Techniques: Metabolic Labelling, Labeling, Immunoprecipitation, Transfection, Recombinant

Journal: PLoS ONE

Article Title: DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

doi: 10.1371/journal.pone.0020528

Figure Lengend Snippet: The percentage (A) and degree (B) of morbidity of Balb/c mice immunized with pE1D2, pE2D2 and controls groups (non-immunized and pcTPA-inoculated animals) were analyzed after i.c. challenge with DENV2. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge (A). Differences in morbidity rates between pE1D2- and pE2D2-vaccinated mice were statistical significant (p<0.0001), as well as between pE1D2 and control animals (p<0.0001). The semi-quantitative analysis of morbidity degrees after virus challenge (B) were performed using a subjective scale ranging from 0 to 3 (0 = none, 1 = mild paralyses in one hind leg or alteration of the spinal column with a small hump, 2 = one severe hind leg paralyses and alteration of the spinal column with a small hump or two severe hind leg paralyses, 3 = two severe hind leg paralyses and deformed spinal column or death). Asterisks indicate statistically significant differences between vaccinated animals and mice inoculated with the control vector pcTPA (* p = 0.0051; ** p = 0.0002; *** p<0.0001). Data represent compilation of two independent experiments, with groups of 10 animals in each test (n = 20).

Article Snippet: Cells were then incubated for 1 h at 37°C with DENV2 hyperimmune mouse ascitic fluid (ATCC, USA), diluted 1∶1500, or the monoclonal anti-DENV2 3H5 (ATCC), diluted 1∶500.

Techniques: Infection, Control, Virus, Plasmid Preparation

Journal: PLoS ONE

Article Title: DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

doi: 10.1371/journal.pone.0020528

Figure Lengend Snippet: The neutralizing antibody titrations (PRNT 50 ) against DENV2 was evaluated in serum samples collected from pE1D2- and pE2D2-vaccinated mice (n = 20), before and after virus challenge. Individual samples were serially diluted from 1∶5 to 1∶640 and PRNT 50 were performed in 96-well plates as described in . Asterisks indicate differences that are statistically significant between pE1D2- and pE2D2-immunized animals (*, p = 0.0029) or between vaccinated mice before and after virus challenge (***, p<0.0001).

Article Snippet: Cells were then incubated for 1 h at 37°C with DENV2 hyperimmune mouse ascitic fluid (ATCC, USA), diluted 1∶1500, or the monoclonal anti-DENV2 3H5 (ATCC), diluted 1∶500.

Techniques: Virus