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Transmission experiments are a critical component of vector competence studies. In this study, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to enumerate the amount of West Nile virus (WNV) secreted in mosquito saliva following oral infection. Culex pipiens quinquefasciatus were allowed to feed on WNV-infected blood, and saliva was collected on days 14 and 21 post-infection (pi). The amount of virus at these two time points varied significantly, with mean equivalent plaque-forming units (pfu) of approximately 30,500 on day 14 pi and 5,800 on day 21 pi. Individual mosquitoes secreted up to 2 x 105 pfu of virus. Titer of whole mosquitoes and immunofluorescence assay of salivary glands from mosquitoes collected at these two time points were also used for supplemental comparison. This report describes the first use of a real-time RT-PCR to quantify the amount of WNV in mosquito saliva.
Received January 20, 2004. Accepted for publication March 1, 2004.
Acknowledgments: We thank Dr. R. B. Tesh for providing the WNV used in this study and for his review of the manuscript, and B. S. Davis for technical advice on WNV primers and PCR optimization.
Financial support: This research was supported by National Institutes of Health grant AI-47246 and Centers for Disease Control and Prevention (CDC) grant U50/CCU620539. Bradley S. Schneider was supported by CDC training grant TOI/CCT622892.
Authors’ addresses: Dana L. Vanlandingham, Bradley S. Schneider, Kimberly Klinger, David Beasley, Jing Huang, Patricia Hamilton, and Stephen Higgs, E-mail: sthiggs{at}utmb.edu. Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. Joseph Fair, Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112.
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