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Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes

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  • Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland; US Army Medical Research and Materiel Command, Fort Detrick, Maryland

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.

Author Notes

* Address correspondence to Michael J. Turell, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD 21702. E-mail: michael.j.turell@us.army.mil

Financial support: This work was funded, in part, by Military Infectious Disease Research Program Project U0176_09_RD.

Authors' addresses: Lawrence N. Petz, Battlefield Pain Management Research Program, US Army Institute of Surgical Research, Fort Sam Houston, TX, E-mail: lawrence.n.petz.mil@mail.mil. Michael J. Turell and Susana Padilla, Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, E-mails: michael.j.turell@us.army.mil and susana.padilla1@us.army.mil. Lewis S. Long and Vanessa R. Melanson, Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, MD, E-mails: lewis.s.long@us.army.mil and vanessa.melanson@us.army.mil. Drew D. Reinbold-Wasson, US Army Public Health Command, Aberdeen Proving Ground, MD, E-mail: Drew.reinbold@us.army.mil. Darci R. Smith, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, E-mail: darci.smith@colostate.edu. Monica L. O'Guinn, Military Infectious Diseases Research Program, US Army Medical Research and Materiel Command, Fort Detrick, MD, E-mail: monica.oguinn@us.army.mil. John S. Lee, Division of Entomology, Walter Reed Army Institute of Research, Silver Spring, MD, E-mail: john.s.lee13@gmail.com.

Reprint requests: Michael J. Turell, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD 21702, E-mail: michael.j.turell@us.army.mil.

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