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QUANTITATIVE ANALYSIS OF DENGUE-2 VIRUS RNA DURING THE EXTRINSIC INCUBATION PERIOD IN INDIVIDUAL AEDES AEGYPTI

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2–3 log₁₀. Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

Received April 7, 2005. Accepted for publication July 29, 2005.

Acknowledgments: The authors thank the staff and students at AIDL for their assistance and helpful suggestions, especially Dr. Francisco Díaz.

Financial support: Funding for this research was provided from National Institutes of Health, Grants No. R01AI49256 and No. U01AI45430.

^* Address correspondence to William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail: wcb4{at}lamar.colostate.edu

Authors’ addresses: Jason Richardson, Alvaro Molina-Cruz, Ma Isabel Salazar, and William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, E-mails: jasonr{at}calostate.edu and web4{at}lamar.colostate.edu.

Reprint requests: William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, Telephone: (970) 491-8530, Fax: 970-491-1815, E-mail: web4{at}lamar.colostate.edu.

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