25 research outputs found
Dicer-2-Dependent Activation of <i>Culex</i> Vago Occurs via the TRAF-Rel2 Signaling Pathway
<div><p>Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that <i>Culex</i> Vago (<i>Cx</i>Vago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced <i>Cx</i>Vago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in <i>Cx</i>Vago promoter region is conserved in mosquito species and is responsible for induction of <i>Cx</i>Vago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates <i>Cx</i>Vago in mosquito cells <i>in vitro</i> and <i>in vivo</i>. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of <i>Cx</i>Vago.</p></div
Vago is up-regulated by flavivirus infection in mosquito cells.
<p>(<b>A</b>) Hsu (<i>Culex quinquefasciatus</i>) and RML12 (<i>Aedes albopictus</i>) cells were infected with WNV and DENV, respectively, and total RNA was collected at 48 hpi. Real-time RT-qPCR was performed using Vago-specific primers. RpL32 (ribosomal protein L32) primers were used as an internal control. Error bars represents standard error from three separate experiments with assays performed in triplicate (Student's t-test *p<0.05, comparing mRNA level between control and virus-infected cells). (<b>B</b>) Cells treated as in (A) were lysed to harvest total protein samples. Western blotting was performed using anti-Vago and anti-beta actin antibodies.</p
<i>Cx</i>TRAF, but not <i>Cx</i>TRAF3, is required for activation of WNV-induced <i>Cx</i>Vago.
<p>Hsu cells were transfected with dsRNA against TRAF-3 (TRAF-3 dsRNA) or TRAF (TRAF dsRNA). GFP dsRNA was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. (<b>A</b>) Real-time RT-qPCR was performed on uninfected cells using TRAF-3- or TRAF-specific primers. RpL32 primers were used as internal control. (<b>B</b>) Real-time RT-qPCR was performed using <i>Cx</i>Vago primers with RpL32 as control. (<b>C</b>) Real-time RT-qPCR was performed using WNV NS1 primers with RpL32 as control. (<b>D</b>) Viral titer estimation by plaque assays was conducted on the supernatant media from these cells at 48 hpi. Error bars represents standard error from three separate experiments with assays performed in triplicate (Student's t-test *p<0.05, compared to GFP dsRNA).</p
<i>Cx</i>Vago activation occurs via Dicer2-TRAF-Rel2 mediated pathway.
<p>Hsu cells were transfected with CxRel2 plasmid (with C-terminal V5 tag) and dsRNA against TRAF (<b>A</b>) or Dicer-2 (<b>B</b>). At 24 h post-transfection, the cells were infected with WNV and cell lysates was collected 48 hpi. Western blot was performed using anti-V5 and anti-beta actin antibodies. Representative blots shown here. (<b>C</b>) Female <i>Culex</i> mosquitoes were microinjected with dsRNAs against GFP, Rel2 or TRAF. After 48 h, the mosquitoes were infected with WNV (Kunjin strain). Total RNA was collected at 48 hpi and real-time RT-qPCR was performed using <i>Cx</i>Vago, <i>Cx</i>Rel2, <i>Cx</i>TRAF and WNV NS1 primers. Error bars represents standard error from experiment with assays performed in duplicates (Student's t-test *p<0.05 compared to dsRNA GFP).</p
The Rel-binding site is responsible for WNV-induced <i>Cx</i>Vago activation.
<p>(<b>A</b>) Hsu cells were transfected with <i>Renilla</i> luciferase plasmid containing Vago promoter region (∼2 kb region upstream of gene, Vago-Luc) or ∼1 kb promoter fragments (Vago1-989-Luc and Vago990-2007-Luc). Cells were also transfected with firefly luciferase plasmid under the insect promoter (<i>OpIE2</i>) as an internal control. As a positive control, cells were transfected with Renilla luciferase under OpIE2 promoter (OpIE2-Luc). After 24 h, the cells were infected with WNV and luciferase activity was measured at 24 hpi. (<b>B</b>) Hsu cells were transfected with <i>Renilla</i> luciferase plasmid containing Vago promoter (Vago-Luc) or smaller fragments of the promoter along with plasmid containing the firefly luciferase reporter (OpIE2 promoter, internal control). After 24 h, the cells were infected with WNV and luciferase activity was measured at 24 hpi. The schematics of the promoter region used in the assay is provided in the right panel. The solid promoter regions were involved in inducing luciferase expression after WNV infection. (<b>C</b>) Hsu cells were transfected with <i>Renilla</i> luciferase plasmid containing Vago promoter (Vago-Luc) or promoter with a mutated Rel-binding site (VagoRel-Luc) along with firefly luciferase plasmid (OpIE2 promoter, internal control). As a positive control, cells were transfected with Renilla luciferase under OpIE2 promoter (OpIE2-Luc). After 24 h, the cells were infected with WNV and luciferase activity was measured at 24 hpi. The <i>Renilla</i> luciferase activity values were standardised using firefly luciferase activity values (R/F) and resulting values were plotted as bar graphs. Error bars represents standard error from three separate experiments with assays performed in triplicate and values were compared to ‘Vago-Luc’ (Student's t-test *p<0.05).</p
<i>Cx</i>Rel2 is required for activation of WNV-induced <i>Cx</i>Vago.
<p>(<b>A</b>) Hsu cells were transfected with dsRNA against Rel2 (Rel2-dsRNA) or control dsRNA (GFP-dsRNA) along with plasmid containing the Vago-Luciferase reporter (and firefly luciferase control plasmid). At 24 h post-transfection, the cells were infected with WNV and luciferase activity was measured at 24 hpi. The <i>Renilla</i> luciferase activity values were standardised using firefly luciferase activity values (R/F) and resulting values were plotted as bar graphs. * indicates p<0.05 compared to GFP-dsRNA. (<b>B–D</b>) Hsu cells were transfected with dsRNA against Rel2 (Rel2-dsRNA) or control dsRNA (GFP-dsRNA). At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. (<b>B</b>) Real-time RT-qPCR was conducted using primers for <i>Cx</i>Vago, <i>Cx</i>Rel2 and <i>Cx</i>RpL32 (control). * indicates p<0.05 compared to GFP-dsRNA. (<b>C</b>) Real-time RT-qPCR was conducted using primers for WNV-NS1. * indicates p<0.05 compared with WNV infection. (<b>D</b>) Viral titer estimation by plaque assays conducted on the supernatant media from these cells at 48 hpi. Error bars represents standard error from three separate experiments with assays performed in triplicate (Student's t-test *p<0.05 compared to GFP-dsRNA).</p
(a) The number of <i>Culicoides</i> trapped (log); (b) maximum and minimum monthly rainfall (mm); and (c) maximum and minimum monthly temperature (°C) between July 2004–June 2012 is shown for four distinct climate regions: tropical, grasslands, arid and warm-temperate.
<p>Below, seasonal parameters are estimated as a function of latitude: mean peak timing and amplitude of annual peak, relative to the mean standardized time series. An asterisk indicates Pearson's R is significantly different from zero (*p<0.05; **p<0.01).</p
<i>Culex</i> cullin (Cul4) blocks the Jak-STAT pathway.
<p><b>A</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (<i>Cx</i>Cul4). Empty vector (Control) was used as transfection control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected at 48 hpi. Real-time RT-qPCR was performed using <i>Culex</i> Vir1-specific and <i>Culex</i> Vago-specific primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells). <b>B</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (CxCul4). Empty vector (Control) was used as transfection control. At 24 h post-transfection, the cells were treated with <i>Cx</i>Vago-containing medium and total RNA was collected 48 h later. Real-time RT-qPCR was performed using <i>Culex</i> Vir1-specific primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells). <b>C</b>. Hsu cells were transfected with p6x2DRAF-Luc (STAT reporter) and pAct-Renilla (control) plasmids along with dsRNA against <i>Culex</i> Cul4 (Cul4 dsRNA) or GFP (GFP dsRNA). Cells were treated with heat-inactivated <i>E</i>. <i>coli</i>, WNV or PBS for 1 h. Luciferase activity was measured 16 h post-stimulation. Fold increase over untreated control were plotted after normalising with Renilla transfection control. Error bars represent standard error from six separate samples (Student’s t-test *p < 0.05, comparing between GFP-dsRNA and Cul4-dsRNA cells for each stimulation). <b>D</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (CxCul4). Empty vector (Control) was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and treated with MG132 (1 μM) or PYR-41 (1 μM) or treated with drugs alone. Total RNA was collected at 48 hpi and real time RT-qPCR was performed using <i>Culex</i> Vir1 and WNV NS1-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells treated with or without inhibitors). <b>E.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). No dsRNA or GFP dsRNA was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. Real-time RT-qPCR was performed using <i>Cx</i>Vir1 (Vir1) and <i>Cx</i>Vago (Vago)-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (*p < 0.05, comparing between No dsRNA, GFP dsRNA and Cul4-knock-down cells). <b>F.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA) or <i>Culex</i> STAT (STAT dsRNA) or both. GFP dsRNA was used as a silencing control (Control). At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. Real-time RT-qPCR was performed using WNV NS1-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between Control and Cul4 dsRNA or STAT dsRNA cells).</p
Time series of the number of positive seroconversions per month between July 2004 - June 2012 in four distinct climate regions: tropical, grasslands, arid and warm-temperate for Akabane virus (a), bovine ephemeral fever virus (b), and bluetongue virus (c).
<p>The periodic annual function, obtained by summing the 12-monthly, 6-monthly and 3-montly harmonics (bold line) and the raw data (faded line) is shown. Vertical grid lines represent 1<sup>st</sup> July for each year, coinciding with the start of the vector season in the southern hemisphere.</p
Seasonal parameters.
<p>Linear regression analysis: a comparison between arbovirus seasonal parameters with climate (rainfall and temperature), entomology (<i>C. brevitarsis</i>) and geographic location (latitude). An asterisk indicates R-squared is significantly different from zero (*p<0.05; **p<0.01).</p><p>Seasonal parameters.</p