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

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

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

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

Virus-Specific Differences in Rates of Disease during the 2010 Dengue Epidemic in Puerto Rico

<div><p>Background</p><p>Dengue is a potentially fatal acute febrile illness (AFI) caused by four mosquito-transmitted dengue viruses (DENV-1–4) that are endemic in Puerto Rico. In January 2010, the number of suspected dengue cases reported to the passive dengue surveillance system exceeded the epidemic threshold and an epidemic was declared soon after.</p> <p>Methodology/Principal Findings</p><p>To characterize the epidemic, surveillance and laboratory diagnostic data were compiled. A suspected case was a dengue-like AFI in a person reported by a health care provider with or without a specimen submitted for diagnostic testing. Laboratory-positive cases had: (i) DENV nucleic acid detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in an acute serum specimen; (ii) anti-DENV IgM antibody detected by ELISA in any serum specimen; or (iii) DENV antigen or nucleic acid detected in an autopsy-tissue specimen. In 2010, a total of 26,766 suspected dengue cases (7.2 per 1,000 residents) were identified, of which 46.6% were laboratory-positive. Of 7,426 RT-PCR-positive specimens, DENV-1 (69.0%) and DENV-4 (23.6%) were detected more frequently than DENV-2 (7.3%) and DENV-3 (<0.1%). Nearly half (47.1%) of all laboratory-positive cases were adults, 49.7% had dengue with warning signs, 11.1% had severe dengue, and 40 died. Approximately 21% of cases were primary DENV infections, and 1–4 year olds were the only age group for which primary infection was more common than secondary. Individuals infected with DENV-1 were 4.2 (95% confidence interval [CI]: 1.7–9.8) and 4.0 (95% CI: 2.4–6.5) times more likely to have primary infection than those infected with DENV-2 or -4, respectively.</p> <p>Conclusions/Significance</p><p>This epidemic was long in duration and yielded the highest incidence of reported dengue cases and deaths since surveillance began in Puerto Rico in the late 1960's. This epidemic re-emphasizes the need for more effective primary prevention interventions to reduce the morbidity and mortality of dengue.</p> </div

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

Demographic and clinical characteristics of suspected dengue cases by diagnostic test result, Puerto Rico, 2010.

<p>DHF = dengue hemorrhagic fever.</p

Rates of laboratory-positive cases by municipality, Puerto Rico, 2010.

<p>Rates were calculated by dividing case numbers by municipality-specific populations and grouping by quintile of rate of all laboratory-positive cases. Rates shown are: (<b>A</b>) All laboratory-positive cases; or laboratory-positive cases with DENV-1 (<b>B</b>), DENV-2 (<b>C</b>), or DENV-4 (<b>D</b>) detected by RT-PCR.</p

Clinical characteristics of laboratory-positive cases by infecting dengue virus (DENV)-type, Puerto Rico, 2010.

<p>Relative risk ratios (RR) were calculated with 95% confidence intervals (CI) for the indicated outcomes for case-patients infected with DENV-1, DENV-2, or DENV-4. Bolded data indicate a significant risk in the indicated outcome associated with infection with the indicated DENV-type. DHF = dengue hemorrhagic fever.</p>*<p>based on a sample of 818 RT-PCR-positive specimens that were tested for evidence of primary infection; denominators for DENV-1, -2 and -4 are 520, 73, and 225, respectively.</p