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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.

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Variable inhibition of pSTAT1 by DENV clinical isolates.(A) Clinical isolates from each DENV (1–4) serotype or (B) from all DENV-2 genotypes (American, Southeast Asian, Asian/American, and cosmopolitan) were tested for their ability to inhibit IFN-α/β signaling. A549 cells were infected with DENV strains at an MOI = 2 for 24 hours and stimulated with IFN-β for 30 minutes. Cells were processed and stained with fluorescently labeled anti-DENV and anti-pSTAT1 antibodies. The percent of pSTAT1 was calculated from cells in cells infected with DENV. Experiments were performed in triplicate. Results shown are representative of four independent experiments. Data are expressed as means ± standard deviation. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. * = P < 0.05.
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pntd.0003468.g003: Variable inhibition of pSTAT1 by DENV clinical isolates.(A) Clinical isolates from each DENV (1–4) serotype or (B) from all DENV-2 genotypes (American, Southeast Asian, Asian/American, and cosmopolitan) were tested for their ability to inhibit IFN-α/β signaling. A549 cells were infected with DENV strains at an MOI = 2 for 24 hours and stimulated with IFN-β for 30 minutes. Cells were processed and stained with fluorescently labeled anti-DENV and anti-pSTAT1 antibodies. The percent of pSTAT1 was calculated from cells in cells infected with DENV. Experiments were performed in triplicate. Results shown are representative of four independent experiments. Data are expressed as means ± standard deviation. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. * = P < 0.05.

Mentions: Using our previously described method we sought to determine the ability of isolates from the four DENV serotypes to prevent STAT1 phosphorylation after stimulation with IFN-β. To determine the relative percent inhibition of pSTAT by DENV strains compared to 16681 we calculated the difference between the expected pSTAT1 inhibition at the obtained percent of infection and the observed pSTAT1 inhibition. A phylogenetic analysis of the sequenced DENV strains from our Passive Dengue Surveillance System (PDSS) was performed and we selected those that represented ancestors of recently circulating viruses (S1A-S1D Fig. and S1 Table). A description of these viruses can be seen in Table 2 (see S2 Table for plaque sizes). Our results show that all of the DENV serotypes evaluated had the capacity to block the IFN-α/β response (Fig. 3A). However, there was some variability in the levels of inhibition of pSTAT1 by these DENV serotypes compared to 16681. DENV-1 & 2 had slightly lower levels of inhibition at-16% and-10% respectively. The lowest levels of inhibition were observed with DENV-3 at-34% and the highest levels were observed with DENV-4 at 10% (Fig. 3A). The largest (44%) difference in inhibition was observed between DENV-3 and-4.


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)

Variable inhibition of pSTAT1 by DENV clinical isolates.(A) Clinical isolates from each DENV (1–4) serotype or (B) from all DENV-2 genotypes (American, Southeast Asian, Asian/American, and cosmopolitan) were tested for their ability to inhibit IFN-α/β signaling. A549 cells were infected with DENV strains at an MOI = 2 for 24 hours and stimulated with IFN-β for 30 minutes. Cells were processed and stained with fluorescently labeled anti-DENV and anti-pSTAT1 antibodies. The percent of pSTAT1 was calculated from cells in cells infected with DENV. Experiments were performed in triplicate. Results shown are representative of four independent experiments. Data are expressed as means ± standard deviation. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. * = P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003468.g003: Variable inhibition of pSTAT1 by DENV clinical isolates.(A) Clinical isolates from each DENV (1–4) serotype or (B) from all DENV-2 genotypes (American, Southeast Asian, Asian/American, and cosmopolitan) were tested for their ability to inhibit IFN-α/β signaling. A549 cells were infected with DENV strains at an MOI = 2 for 24 hours and stimulated with IFN-β for 30 minutes. Cells were processed and stained with fluorescently labeled anti-DENV and anti-pSTAT1 antibodies. The percent of pSTAT1 was calculated from cells in cells infected with DENV. Experiments were performed in triplicate. Results shown are representative of four independent experiments. Data are expressed as means ± standard deviation. Statistical evaluation was carried out by one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons. * = P < 0.05.
Mentions: Using our previously described method we sought to determine the ability of isolates from the four DENV serotypes to prevent STAT1 phosphorylation after stimulation with IFN-β. To determine the relative percent inhibition of pSTAT by DENV strains compared to 16681 we calculated the difference between the expected pSTAT1 inhibition at the obtained percent of infection and the observed pSTAT1 inhibition. A phylogenetic analysis of the sequenced DENV strains from our Passive Dengue Surveillance System (PDSS) was performed and we selected those that represented ancestors of recently circulating viruses (S1A-S1D Fig. and S1 Table). A description of these viruses can be seen in Table 2 (see S2 Table for plaque sizes). Our results show that all of the DENV serotypes evaluated had the capacity to block the IFN-α/β response (Fig. 3A). However, there was some variability in the levels of inhibition of pSTAT1 by these DENV serotypes compared to 16681. DENV-1 & 2 had slightly lower levels of inhibition at-16% and-10% respectively. The lowest levels of inhibition were observed with DENV-3 at-34% and the highest levels were observed with DENV-4 at 10% (Fig. 3A). The largest (44%) difference in inhibition was observed between DENV-3 and-4.

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