Fitness Impact and Stability of a Transgene Conferring Resistance to Dengue-2 Virus following Introgression into a Genetically Diverse Aedes aegypti Strain

Abstract

In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement.

Disruption of dengue virus transmission by mosquitoes

“…An anti-DENV effector gene that efficiently triggers the RNAi pathway against the virus consists of an inverted-repeat (IR) DNA encoding virus-derived sequences of 300–600 bp length in sense and antisense orientation [25,26**,27**]. A small, functional intron placed between the sense and antisense sequences is spliced out during transcription, thereby supporting stable dsRNA formation and establishing the trigger for RNAi.…”

“…One of them is population reduction (elimination) based on Release of Insects with Dominant Lethality (RIDL) [18–20,21**–22], the other concept includes population replacement strategies, which will be the focus of this article. Population replacement implies that an arbovirus-competent mosquito population is replaced with laboratory generated, incompetent mosquitoes harboring specifically engineered antiviral effector genes [23–25,26**,27**]. Until now, the majority of research on disrupting mosquito-borne viral disease transmission has been concentrated on DENV and Ae.…”

“…Besides overcoming several tissue barriers in the mosquito, DENV has to cope with several innate immune defenses, Toll, JAK-STAT, and RNA interference (RNAi), the last of which is the most potent antiviral immune pathway capable of eliminating the virus [32*,33*,34*,35,36*]. Because the midgut is the initial tissue of DENV infection in the mosquito, it also constitutes the ideal site for over-expressing/activating an antiviral effector [25,26**]. This way DENV can be attacked at an early, vulnerable step of its replication cycle before the virus has established any infection foci.…”

Mosquito immune responses to arbovirus infections

“…In a more recent study, we developed new transgenic lines using the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 infection for >33 generations [79]. Genetic analyses and physical chromosome mapping identified two closely linked transgene integration sites in Carb109M mosquitoes associated with chromosome 3.…”

“…Introgression of the transgene into GDLS, with a different genetic background from Carb109M, changed the GDLS population from a highly DENV2-permissive phenotype to a DENV2-refractory phenotype. Significantly, the DENV2-refractory homozygous line, Carb109M/GDLS.BC5.HZ, exhibited (relative to GDLS) minimal fitness loss associated with the transgene [79]. …”