Inverse correlation of pSTAT1 inhibition with DENV infectivity in prM(+) gated cells

<p>A549 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.</p

Comparison of DENV replication in A549 cells detected by RT-PCR.

<p>A549 cells were infected with representative DENV strains at an MOI = 0.1 for 1 hour. After washing, cells were incubated for the time period indicated (12, 24, 48, or 72 h) and cell supernatants were harvested for quantification of viral genomes by real-time RT-PCR as described in Materials and Methods. Experiments were performed in triplicate. Results shown are representative of two independent experiments.</p

STAT1 is phosphorylated in non-human primate cell lines infected with sylvatic DENV and stimulated with IFN-β.

<p>Dengue sylvatic strains Daka510, DakAr 75505, and DKD811 were used to infect (<b>A</b>) LLCMK2 and (<b>B</b>) Vero cells at an MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and co-stained with anti-pSTAT1 Alexa 647- and anti-dengue prM Alexa 488-conjugated antibodies. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using FlowJo software. Results shown are representative of two independent experiments.</p

Percent inhibition of pSTAT1 in DENV infected A549 cells at different MOIs.

<p>Percent inhibition of pSTAT1 in DENV infected A549 cells at different MOIs.</p

Inhibition of IFN-β signaling in DENV-2 16681 in human and non-human primate cell lines.

<p>DENV-2 16681 was used to infect human (A549 & Huh7) and NHP (LLCMK2 & Vero) cell lines at a MOI of 5 for 24 hours. Cells were then stimulated for 30 min. with IFN-β (500 U/ml) and processed for (<b>A</b>) flow cytometry of pSTAT1 or (<b>B</b>) Western blots of pSTAT1, STAT1, STAT2, NS4B and GAPDH. Results shown are representative of two independent experiments.</p

Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus

<div><p>Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an <i>in-vitro</i> diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.</p></div

Laboratory Results for the Fatal Laboratory Positive Dengue Cases, 2007, Puerto Rico.

<p>DPO = day post onset of fever; DENV = dengue virus; RT-PCR = reverse transcriptase polymerase chain reaction; MAC-ELISA = IgM antibody capture enzyme linked immunosorbent assay; ELISA = enzyme linked immunosorbent assay; WNV = West Nile virus; PRNT = plaque reduction neutralization test; IgM Lepto = IgM ELISA ImmunoDOT assay for <i>Leptospira;</i> IHC = immunohistochemical microscopy; NEG = negative test result; POS = positive test result; N/A = not applicable for patient; QNS = quantity not sufficient to perform test indicated.</p>†<p>These samples where whole blood collected at time of death.</p

Clinical Features of the Fatal Laboratory-positive Dengue Cases at Time of Death or End of Hospital Stay, 2007, Puerto Rico.

<p>This table describes the clinical features of laboratory positive dengue case-patients at time of death or end of hospital stay.</p>†<p>DHF = dengue hemorrhagic fever; PLT = platelet; HCT = hematocrit; GI = gastrointestinal; ICH = intracranial hemorrhage; LOS = length of stay.</p>‡<p>DHF criteria as defined by World Health Organization in Dengue Hemorrhagic Fever: Diagnosis, Treatment, Prevention and Control. Second edition. Geneva: World Health Organization.</p

Limit of detection.

<p>The limit of detection of the Assay was evaluated by detection and quantification of (8) ten-fold dilutions of laboratory-adapted DENV-1–4 strains, 5 replicas per dilution. The Assay was performed in singleplex <b>(A)</b> and multiplex formats <b>(B)</b>. Viral RNA concentration was quantified using a standard curve measured in genome copy equivalents per milliliter of serum or plasma (GCE/mL). A linear regression was estimated for dilutions of virus stocks in plasma (dashed lines) or serum (solid line). Regression coefficient (R²) is shown for each serotype. Error bars indicate standard deviation of measurements from 5 replicas in GCE/mL.</p

CDC DENV-1–4 Real Time RT-PCR Assay specificity against clinically relevant concentrations of other pathogens.

<p>CDC DENV-1–4 Real Time RT-PCR Assay specificity against clinically relevant concentrations of other pathogens.</p