Quantitative, time course analyses of dengue-2 virus replication and dissemination in Aedes aegypti (Diptera Culicidae)

Abstract

Aedes aegypti varies in its dengue virus (DENV) vector competence partially due to midgut infection (MIB) and escape barriers (MEB). The mechanisms of these barriers are unknown. Quantitative analyses of DENV infection may provide new insight to vector-virus interactions that impact the MIB and MEB. DENV-2 RNA was quantified during the extrinsic incubation period (EIP) in mosquito strains of varying DENV-2 susceptibility in order to better understand the dynamics of virus infection, replication, and dissemination. A SYBR Green I based strand-specific, quantitative real-time RT-PCR assay was developed to quantify DENV-2 RNA from the midgut, heads and legs of individual Ae. aegypti. DENV-2 plus and minus strand RNA was quantified at each of 14 days post infectious blood meal (dpi) in a DENV-2 competent strain from Chetumal, Mexico. Amounts of positive and negative viral RNA strands were correlated throughout the EIP. 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. The SYBR Green assay was used to quantify total DENV-2 RNA in individual Ae. aegypti from 3 strains (Ibo 11, D2S3, and D2MEB) previously selected to vary in midgut (MIR) and disseminated infection rates (DIR). A large proportion of D2MEB samples expressed a midgut escape barrier (MEB) and infected midguts of D2MEB had less DENV-2 RNA than D2S3, Ibo 11, and Chetumal. Mosquitoes with a disseminated infection had slightly higher levels of midgut DENV-2 RNA. The study of D2MEB supports a threshold model of vector competence and the hypothesis that reducing the rate of viral replication and/or destroying transcribed viral RNA in the midgut decreases overall vector competence. The effect of mosquito midgut trypsins in DENV-2 infectivity was studied. Addition of soybean trypsin inhibitor (STI) in a DENV-2 infectious blood meal resulted in a 91-97% decrease in midgut DENV-2 RNA. STI treatment also resulted in slower DENV-2 replication in the midgut, less DENV-2 E protein expression, and decreased dissemination to the thorax and the head. A second uninfected blood meal, at 7 dpi, significantly increased DENV-2 replication in the midgut and recovered oogenesis, suggesting that the lower viral infection caused by STI was in part due to a nutritional effect. Mosquitoes fed DENV-2 digested in vitro with bovine trypsin (before STI addition) exhibited a transient increase in midgut DENV-2 at 4 dpi. Blood digestion and possibly DENV-2 proteolytic processing, mediated by midgut trypsins, influence the rate of DENV-2 infection, replication, and dissemination in Ae. aegypti.