Virus titers of DV2 in sera of DV2-infected wild-type and TLR6 KO mice.

<p>Virus titers of DV2 in sera of DV2-infected wild-type and TLR6 KO mice.</p

Expression of TLR2 and TLR6 of DV2-infected PBMC.

<p>(A) Growth curve of DV2 in PBMC was determined using plaque assays (0 hr, 6 hrs, 1 to 5 days). Values plotted are mean ± SEM of three independent experiments performed in triplicates obtained using separate PBMC from three donors. Mock-infected and DV2-infected PBMC on day 3 post-infection were stained using rabbit anti-TLR6 antibody, goat anti-rabbit DyLight 633 (APC) antibody and mouse anti-CD14 antibody conjugated with FITC. The cell debris was excluded by gating as shown in (B) and the stained cells were analyzed using flow cytometry (C). The percentages of cell subset population were indicated on the representative results of three independent experiments obtained using separate PBMC from three donors (i, ii & iii). Data for day 1 and day 2 post-infection were shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005053#ppat.1005053.s001" target="_blank">S1 Fig</a>. The percentages of TLR6+CD14+ cells of mock-infected and DV2-infected PBMC were plotted on a graph (E). Mock-infected and DV2-infected PBMC on day 3 post-infection were also stained using mouse anti-TLR2 antibody, goat anti-mouse DyLight 633 (APC) antibody and mouse anti-CD14 antibody conjugated with FITC. The cell debris was excluded by gating as shown in (B) and the stained cells were analyzed using flow cytometry (D). The percentages of cell subset population were indicated on the representative results of three independent experiments obtained using separate PBMC from three donors (i, ii & iii). Data for day 1 and day 2 post-infection were shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005053#ppat.1005053.s002" target="_blank">S2 Fig</a>. The percentages of TLR2+CD14+ cells of mock-infected and DV2-infected PBMC were plotted on a graph (F). The PBMC from three donors (i, ii & iii) were harvested on day 3 post-infection and stained using rabbit anti-TLR6 antibody, goat anti-rabbit DyLight 633 (APC) antibody and mouse anti-TLR2 antibody conjugated with FITC (G). 30, 000 cells were analyzed and gating was set to remove cell debris. Region 1 (R1) contains cells which are negative for TLR2 but positive for TLR6. Region 2 (R2) contains cells which are positive for both TLR2 and TLR6. Region 3 (R3) contains cells which are negative for both TLR2 and TLR6. Region 4 (R4) contains cells which are positive for TLR2 but negative for TLR6. The percentages of TLR2+TLR6+ cells of mock-infected and DV2-infected PBMC were plotted on a graph (H).</p

DV2-infected mice up-regulates IL-6, TNF-α and produces DV NS1 protein.

<p>ELISA was performed to quantify the amount of IL-6 (A) and TNF-α (B) in the sera of mice infected with 2.7 x 10⁸ PFU of DV2 at 1–2 day-old via intraperitoneal injection. There were 24 mice in each time point (12 DV2-infected and 12 mock-infected, 12 wild-type mice and 12 TLR6^-/- mice). Using Kruskal-Wallis test, the amount of IL-6 and TNF-α detected in the sera of DV2-infected wild-type and TLR6^-/- mice were significantly different among the respondents. Wild-type (C) and TLR6^<i>-/-</i> (D) mice at 1–2 day-old were infected with 2.7 x 10⁸ PFU of DV2 strain 16681. DV NS1 protein in the sera of the mice was assayed using Bio-rad Platelia Dengue NS1 Antigen detection kit.</p

DV NS1 protein up-regulates TLR2 and TLR6 of PBMC.

<p>Mock-infected, His-tag-treated, DV2-infected, DV NS1 protein-treated, LPS-treated PBMC were lysed and equal total protein was loaded into SDS-PAGE gel. PBMC of two donors were used. The presence of TLR6 protein in the cell lysate was detected using rabbit anti-TLR6 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (A). Actin was used as loading control. The band intensity was measured using GelQuantNET software program. The intensities of the TLR6 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (B). The presence of TLR2 protein in the cell lysate was detected using rabbit anti-TLR2 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (C). Actin was used as loading control. The intensities of the TLR2 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (D). The relative intensities of the bands were indicated on the charts. PBMC were untreated (E & J), mock-infected (F & K), DV2-infected (G & L), His-tag-treated (H & M) and DV NS1 protein-treated (I & N). The PBMC were harvested at day 3 post-treatment. TLR6 was stained red using primary rabbit anti-TLR6 antibody and secondary goat anti-rabbit DyLight 633 antibody (E to I). TLR2 is stained red using primary mouse anti-TLR2 antibody and secondary goat anti-mouse DyLight 633 (J to N). CD14 was stained green using anti-CD14 antibody conjugated with FITC (E to N). Cell nuclei are stained blue with DAPI. Representative images from triplicate experiments obtained using separate PBMC from three donors are shown.</p

Activation of TLR2 and TLR6 of DV2-infected PBMC.

<p>ELISA was performed to quantify the amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by mock-infected and DV2-infected PBMC. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s <i>t-</i>tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of DV2-infected PBMC to that of the mock-infected PBMC. ELISA was also performed to quantify the amount of IL-6 (C) and TNF-α (D) secreted into the supernatant by mock-infected, DV2-infected, LPS-treated and MALP-2 treated PBMC on day 2 post-treatment. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s <i>t-</i>tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of TLR2 and TLR6 blocked PBMC to that of the isotype control PBMC.</p

TLR6 knockout enhanced the survivability of DV2-infected mice.

<p>(A) Wild-type (184 mice) and TLR6^<i>-/-</i> (203) mice were infected with 2.7 x 10⁸ PFU of DV2 strain 16681 at 1–2 day-old via intraperitoneal injection. Mock-infected wild-type (140 mice) and TLR6^<i>-/-</i> (146) mice were injected with 50 μl of C6/36 cell culture supernatant. The survival of the mice was monitored from day 1 to day 21 on a daily basis. Kaplan-Meier survival curves were shown. Log-rank test was performed to determine if there was significant difference in survivability between DV2-infected wild-type and TLR6^<i>-/-</i> mice. The blood and tissues of mice were harvested on day 1, 2, 3, 4, 5 and 21 post-infection via cardiac puncture. The virus in the sera (B), livers (C), limbs (D) and brains (E) of mice were quantified by plaque assays. Virus titers of 0 PFU/ml were set to 0 Log10 PFU/ml to be reflected on the log scale graph. No actual virus titer of 0 Log10 PFU/ml was obtained from any of the samples. Each sample group is in triplicates. Each set of samples was in triplicates.</p

DV NS1 protein activates TLR2 and TLR6 of PBMC.

<p>The amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by PBMC treated with dengue viral proteins (capsid, envelope, premembrane, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, each 50 μg/ml), His-tag-treated PBMC, untreated PBMC, UV-inactivated DV2-treated PBMC (10 virus particles per cell), LPS-treated PBMC (5 μg/ml, 10 μg/ml, 50 μg/ml) on day 2 post-treatment were quantified using ELISA. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s <i>t-</i>tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of viral protein-treated PBMC to that of the His-tag-treated PBMC, the mean amount of IL-6/TNF-α detected in culture supernatants of LPS-treated PBMC to that of the untreated PBMC, the mean amount of IL-6/TNF-α detected in the culture supernatants of His-tag-treated PBMC to that of the untreated PBMC and the mean amount of IL-6/TNF-α detected in the culture supernatants of UV-inactivated DV2-treated PBMC to that of the untreated PBMC. The amount of IL-6 (C) and TNF-α (D) secreted into the supernatant by DV NS1 protein-treated PBMC (1 μg/ml) on day 1 to day 3 post-treatment were quantified using ELISA. Data represent mean ± SEM of three independent experiments. Student’s <i>t-</i>tests were used to compare mean amount of IL-6/TNF-α detected in culture supernatants of DV NS1 protein-treated PBMC to that of the His-tag-treated PBMC. The amount of IL-6 (E) and TNF-α (F) secreted into the supernatant by DV NS1 protein-treated PBMC (1 μg/ml) on day 1 post-treatment were quantified using ELISA. TLR2 and TLR6 of the PBMC were blocked prior to the treatment. Data represent mean ± SEM of three independent experiments obtained using separate PBMC from three donors. Student’s <i>t-</i>tests were used to compare mean amount of IL-6 detected in culture supernatants of DV NS1 protein-treated PBMC isotype control to that of DV NS1 protein-treated TLR2/6 blocked PBMC. HEK 293 cells transfected with SEAP reporter plasmid (G), HEK 293 cells transfected with both SEAP reporter plasmid and pDUO-hTLR6/TLR2 plasmid (H) were treated with His-tag (1 μg/ml), DVNS1 recombinant protein (1 μg/ml), LPS (5 μg/ml), MALP-2 (50 ng/ml), mock-infected or DV2-infected (M.O.I of 10). The supernatants of the HEK 293 cell cultures were harvested on day 1 to day 3 post-treatment. The SEAP in the supernatants were quantified using SEAP reporter assay kit. Data represent mean ± SEM of three independent experiments. Student’s <i>t-</i>tests were used to compare mean amount of SEAP detected in culture supernatants of DV NS1 protein-treated HEK 293 cells to that of His-tag-treated HEK 293 cells, mock-infected HEK 293 cells to that of DV2-infected HEK 293 cells, LPS-treated HEK 293 cells to that of untreated and MALP-2 HEK 293 cells to that of untreated HEK293 cells.</p

DV NS1 protein activates TLR6 of mice.

<p>ELISA was performed to quantify the amount of IL-6 (A) and TNF-α (B) secreted into the supernatant by His-tag (1 μg/ml), DV NS1 recombinant protein (1 μg/ml), MALP-2 (50 ng/ml), Ultrapure LPS (2.5 x 10⁴ EU/ml), mock-infected or DV2-infected (M.O.I of 10) wild-type or TLR6^<i>-/-</i> murine peritoneal macrophages on day 2 post-treatment. (C) Wild-type (23 mice) and TLR6^<i>-/-</i> (9) mice were injected with 20 μg of DV NS1 protein at 1–2 day-old via intraperitoneal injection. Control wild-type (18 mice) and TLR6^<i>-/-</i> (9) mice were injected with 20 μg of His-tag. The survival of the mice was monitored from day 1 to day 7 on a daily basis. Kaplan-Meier survival curves were shown. Log-rank test was performed to determine if there was significant difference in survivability between DV NS1-treated wild-type and TLR6^<i>-/-</i> mice. ELISA was performed to quantify the amount of IL-6 (D) and TNF-α (E) in the livers of wild-type mice injected with 20 μg of DV NS1 protein or His-tag at 1–2 day-old via intraperitoneal injection. Each set of samples was in triplicates.</p

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

<div><h3>Background</h3><p>Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.</p> <h3>Methods</h3><p>Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The <em>in vivo</em> efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.</p> <h3>Results</h3><p>Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.</p> <h3>Conclusion</h3><p>Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.</p> </div

CHIKV infection in mice pre-treated with shRNA E1.

<p>In contrast to the wildtype and scrambled E1 (sE1)-treated groups, pre-treatment of mice with single doses of plasmid-shRNA E1 via intraperitoneal (i.p.) route was shown to confer strong protective effect against CHIKV disease (<i>n = 5</i> per treatment group). Survival of these shRNA E1-treated mice suggested a dose-dependent inhibition against CHIKV pathology throughout 15 days p.i. CHIKV infection was carried out using 10⁶ PFU. WT refers to the non-treated group; For the mock-inf, sterile PBS was inoculated in replacement of infectious CHIKV; shRNA sE1 refers to the shRNA scrambled E1 plasmid.</p