title: Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti.
abstract: 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 log(10). 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.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, Colorado, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Richardson
authors.forename: Alvaro
authors.initials: A
authors.lastname: Molina-Cruz
authors.forename: Ma Isabel
authors.initials: MI
authors.lastname: Salazar
authors.forename: William
authors.initials: W
authors.lastname: Black
mesh_terms: D000330:Aedes / Q000821:virology*; D000818:Animals; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D012367:RNA, Viral