BackgroundRift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.Methodology and Principal Findings Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.Conclusions/SignificanceIn both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1–4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1–4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting <3 DPO included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. Predictors of dengue in participants presenting 3–5 DPO were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue AFI. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of AFI. These findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management.

BackgroundPreviously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.Methodology and Principal Findings Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.Conclusions/SignificanceDeletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.

The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases.

BACKGROUND: In the absence of active blood donation screening, dengue viruses (DENV) have been implicated in only a limited number of transfusion transmissions worldwide. This study attempted to identify if blood from donors testing negative by an NS1-antigen (Ag) enzyme-linked immunosorbent assay (ELISA) but confirmed positive for DENV RNA caused DENV-related disease in recipients during the epidemic years of 2010 to 2012 in Puerto Rico. STUDY DESIGN AND METHODS: Donation aliquotstesting negative by an investigational NS1-Ag ELISA were stored frozen and retested retrospectively using a research transcription-mediated amplification assay (TMA) detecting DENV RNA. All RNA-reactive donations were subject to confirmatory RNA and antibody testing. Recipient tracing was conducted for all components manufactured from TMA-reactive components. Medical chart review, recipient interview, and follow-up sampling occurred for 42 recipients transfused with TMA-reactive components. RESULTS:Six of 42 recipients developed new-onset fever in the 2 weeks posttransfusion; three (50%) received RNA confirmed-positive, NS1-Ag-negative red blood cell (RBC) units. One recipient of a high-titer unit (7 3 10 7 DENV-4 RNA copies/mL) developed severe dengue, and a second recipient had only fever recorded but had a negative sepsis work-up. New fever attributable to DENV infection in a third recipient was confounded by fever potentially attributable to posttransfusion sepsis. CONCLUSIONS:In our retrospective study, NS1-Ag detected 20% of all RNA confirmed-positive donations demonstrating limitations of NS1-Ag ELISA for blood donation screening. We identified one recipient with a clinical syndrome compatible with severe dengue who had received an NS1-Ag-negative but RNA confirmed-positive RBC unit. This investigation illustrates the difficulty in confirming transfusion transmission in dengue-endemic areas among severely ill transfusion recipients.ABBREVIATIONS: ARC 5 American Red Cross; DENV 5 dengue viruses; PR 5 Puerto Rico; TMA 5 transcriptionmediated amplification assay.