Identification of dengue viruses in naturally infected Aedes aegypti females captured with BioGents (BG)-Sentinel traps in Manaus, Amazonas, Brazil

Abstract

Introduction:In Manaus, the first autochthonous cases of dengue fever were registered in 1998. Since then, dengue cases were diagnosed by the isolation of viruses 1, 2, 3, and 4. Methods: One hundred eighty-seven mosquitoes were collected with BioGents (BG)-Sentinel traps in 15 urban residential areas in the Northern Zone of Manaus and processed by molecular tests. Results: Infections with dengue viruses 1, 2, 3, and 4 and a case of co-infection with dengue viruses 2 and 3 were identified. Conclusions: These findings corroborate the detection of dengue in clinical samples and reinforce the need for epidemiological surveillance by the Health authorities.Keywords: Aedes aegypti. Dengue. Virus. Molecular tests.Manaus, Amazonas (AM) was infested by Aedes aegypti in 1996 and the first autochthonous cases of dengue fever were registered in March 1998 1 . Since then, dengue fever cases were diagnosed in the following years with the isolation of dengue viruses (DENV) 1, 2, 3, and 4 in 2008 2 . In the present work, all 4 serotypes of the virus were detected in naturally infected A. aegypti by reverse transcription-polymerase chain reaction (RT-PCR), the amplicon sequence of DENV-4 was analyzed, and these findings were corroborated with the detection of DENV-4 in clinical samples of patients residing in the northern zone of Manaus, AM.A total of 187 mosquitoes were collected with BioGents (BG)-Sentinel traps 3 from January 2009 to June 2010 in 15 urban residential areas in the northern zone of Manaus, AM, Brazil. Sixty BG-Sentinel traps (4 traps in each of the 15 areas) were installed in each area every 2 weeks for a 24-h time period. The captured mosquitoes were identified and sexed. Cephalothoraxes of A. aegypti were separated for each area and each collection date in 2-mL Eppendorf cups (pool size: 1-10 mosquitoes) and maintained at -70°C until analysis.The pooled samples were pulverized and macerated on ice with 300mL Trizol reagent (Invitrogen,Brazil). Viral RNA extraction was performed according to the manufacturer's instructions and reverse transcription was performed using Superscript III (Invitrogen, Brazil) and random primers. Based on the previously described protocol 4 , semi-nested PCR was conducted to amplify a region of the viral genome that contains the capsid-premembrane (C-prM) genes, with primers designed to amplify 119-bp DENV-2, 290-bp DENV-3, and 392-bp DENV-4. The generated amplicon of DENV-4 was sequenced in both directions using the BigDye Terminator cycle sequence kit (Applied Biosystems, Foster City, CA, USA).Of the total of 187 pools of Aedes aegypti that were processed between August 2009 and July 2010, which each contained 10 females, 1 pool was positive for DENV-1, 4 pools were positive for DENV-2, 3 were positive for DENV-3, and 2 were positive for DENV-4. One pool was co-infected with DENV-2 and 3. The obtained sequence DENV-4 was submitted to a basic local alignment search tool (BLAST; http://www.ncbi.nlm.nih.gov.entrez) analysis, which uses the megablast algorithm optimized for ...

Assessing the Relationship between Vector Indices and Dengue Transmission: A Systematic Review of the Evidence

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Viral Interference and Persistence in Mosquito-Borne Flaviviruses

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