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.

Quantitative Analysis of Replication and Tropisms of Dengue Virus Type 2 in Aedes albopictus

“…aegypti reported by others. 22,28 Subsequently, Western blots indicated that DENV-2 protein expression was parallel to virus RNA replication and increased with time, but it had no decline. However, the results reported before indicated that DENV-2 titer peaked at an early time and then, declined in Ae.…”

“…[25][26][27] q-RTPCR as a rapid and sensitive assay has been used for quantitative analysis of the interaction between dengue virus and mosquitoes. [20][21][22][28][29][30] The quantitative replication of DENV-2 RNA (+) in Ae. albopictus was studied by q-RTPCR in this study.…”

“…The quantitative replication of DENV-2 in mosquito midgut was already studied by using q-RTPCR. [20][21][22] In this study, besides midgut, the quantitative replications of DENV-2 in other mosquitoes organs, including brain, fat body, salivary gland, malpighian tubes, and ovary, were also determined by q-RTPCR.…”

“…aegypti midguts and legs. [20][21][22] But, there have no reports about the quantitative analysis of DENV replication in other mosquito organs and in Ae. albopictus organs.…”

Evaluation of the Dengue NS1 Ag Strip®for Detection of Dengue Virus Antigen inAedes aegypti(Diptera: Culicidae)

“…A DENV 2-specific one-step quantitative RT-PCR assay targeting a 177-bp region of the NS5 gene (Richardson et al 2006) was performed in a LightCycler 2.0 system using a LightCycler RNA Master SYBR Green Kit I (Roche Diagnostics, GMbH). Real-time quantitative RT-PCR assay conditions follow that of Lai et al 2007.…”