Limits...
Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

Show MeSH

Related in: MedlinePlus

Inverse correlation of pSTAT1 inhibition with DENV infectivity in prM(+) gated cellsA549 cells were infected with serial 1:3 dilutions of DENV strain 16681 beginning with an MOI = 6. Twenty-four hours post-infection cells were stimulated for 30 minutes with IFN-β. Cell staining was done using anti-pSTAT1 Alexa 488- and anti-DENV prM Alexa 647-conjugated antibodies. Quantification of cell fluorescence was performed on a FACSCalibur. An analysis gate was placed on the DENV+ population and the percent of pSTAT1+ cells was determined. Experiments were performed in triplicate. Results shown are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359095&req=5

pntd.0003468.g002: Inverse correlation of pSTAT1 inhibition with DENV infectivity in prM(+) gated cellsA549 cells were infected with serial 1:3 dilutions of DENV strain 16681 beginning with an MOI = 6. Twenty-four hours post-infection cells were stimulated for 30 minutes with IFN-β. Cell staining was done using anti-pSTAT1 Alexa 488- and anti-DENV prM Alexa 647-conjugated antibodies. Quantification of cell fluorescence was performed on a FACSCalibur. An analysis gate was placed on the DENV+ population and the percent of pSTAT1+ cells was determined. Experiments were performed in triplicate. Results shown are representative of four independent experiments.

Mentions: The pSTAT1 inhibition assay is performed akin to an ELISA assay with a standard curve. To construct the graph in Figs. 1 and 2, the percent of cells that stained DENV positive was calculated and a gate was placed on the DENV positive population at each of the virus dilutions to analyze the percent of cells that also stained positive for pSTAT1. The data points for graphs 1 and 2 display as curved shape, therefore, instead of using the formula y = Mx + b to calculate the expected percent of inhibition from DENV strains by linear regression analysis a 4-parameter logistic curve fit was performed to obtain the expected percent inhibition. The expected DENV inhibition of pSTAT1 was calculated using a 4-parameter logistic (4PL) model developed from a standard curve of serially diluted DENV strain 16681 infected cells that were stimulated with IFN-β. Serial 1:3 dilutions of 16681 were performed starting with an MOI = 6. The expected inhibition of pSTAT1 by DENV strains at the obtained infectivity was calculated using the four-parameter logistic regression (4PL) model fit using the following equation:f(x)=c+d−c1+exp(b(log(x)−log(e)))


Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

Inverse correlation of pSTAT1 inhibition with DENV infectivity in prM(+) gated cellsA549 cells were infected with serial 1:3 dilutions of DENV strain 16681 beginning with an MOI = 6. Twenty-four hours post-infection cells were stimulated for 30 minutes with IFN-β. Cell staining was done using anti-pSTAT1 Alexa 488- and anti-DENV prM Alexa 647-conjugated antibodies. Quantification of cell fluorescence was performed on a FACSCalibur. An analysis gate was placed on the DENV+ population and the percent of pSTAT1+ cells was determined. Experiments were performed in triplicate. Results shown are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359095&req=5

pntd.0003468.g002: Inverse correlation of pSTAT1 inhibition with DENV infectivity in prM(+) gated cellsA549 cells were infected with serial 1:3 dilutions of DENV strain 16681 beginning with an MOI = 6. Twenty-four hours post-infection cells were stimulated for 30 minutes with IFN-β. Cell staining was done using anti-pSTAT1 Alexa 488- and anti-DENV prM Alexa 647-conjugated antibodies. Quantification of cell fluorescence was performed on a FACSCalibur. An analysis gate was placed on the DENV+ population and the percent of pSTAT1+ cells was determined. Experiments were performed in triplicate. Results shown are representative of four independent experiments.
Mentions: The pSTAT1 inhibition assay is performed akin to an ELISA assay with a standard curve. To construct the graph in Figs. 1 and 2, the percent of cells that stained DENV positive was calculated and a gate was placed on the DENV positive population at each of the virus dilutions to analyze the percent of cells that also stained positive for pSTAT1. The data points for graphs 1 and 2 display as curved shape, therefore, instead of using the formula y = Mx + b to calculate the expected percent of inhibition from DENV strains by linear regression analysis a 4-parameter logistic curve fit was performed to obtain the expected percent inhibition. The expected DENV inhibition of pSTAT1 was calculated using a 4-parameter logistic (4PL) model developed from a standard curve of serially diluted DENV strain 16681 infected cells that were stimulated with IFN-β. Serial 1:3 dilutions of 16681 were performed starting with an MOI = 6. The expected inhibition of pSTAT1 by DENV strains at the obtained infectivity was calculated using the four-parameter logistic regression (4PL) model fit using the following equation:f(x)=c+d−c1+exp(b(log(x)−log(e)))

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

Show MeSH
Related in: MedlinePlus