Stability and loss of a virus resistance phenotype over time in transgenic mosquitoes harbouring an antiviral effector gene

Abstract

Transgenic Aedes aegypti were engineered to express a virus-derived, inverted repeat (IR) RNA in the mosquito midgut to trigger RNA interference (RNAi) and generate resistance to dengue virus type 2 (DENV2) in the vector. Here we characterize genotypic and phenotypic stabilities of one line, Carb77, between generations G9 and G17. The anti-DENV2 transgene was integrated at a single site within a non-coding region of the mosquito genome. The virus resistance phenotype was strong until G13 and suppressed replication of different DENV2 genotypes. From G14 –G17 the resistance phenotype to DENV2 became weaker and eventually was lost. Although the sequence of the transgene was not mutated, expression of the IR effector RNA was not detected and the Carb77 G17 mosquitoes lost their ability to silence the DENV2 genome.

Genetic control ofAedesmosquitoes

“…Transgene-based expression of a hairpin RNA corresponding to part of the DEN2 virus in either the midgut 79 or salivary glands 80 has been shown to provide a strong block to virus transmission. However, for the midgut-expressing line, expression of the anti-DEN2 hairpin and the associated refractory phenotype were lost after about 13 generations, 81 suggesting that expression may impose a significant fitness cost, and also perhaps that the unusual inverted repeat structure involved may be subject to some form of epigenetic silencing.…”

Biological Control Strategies for Mosquito Vectors of Arboviruses

“…aegypti transformed by the mariner transposable element [125]. However, the resistance phenotype was only transiently maintained for several generations in the laboratory, precluding the possibility of conducting field trials to demonstrate its efficacy in disease control [168]. A similar approach was later used to generate additional lines of genetically modified Ae.…”

Disruption of dengue virus transmission by mosquitoes

“…Carb77 females exhibited a midgut infection barrier to the virus, which could be circumvented by intrathoracic injection of the virus [25]. After 17 generations in laboratory culture, these Carb77 mosquitoes lost their resistance phenotype even though the transgene itself was not mutated [49], possibly due to hetero-chromatin rearrangement that silenced the IR effector gene. In a subsequent experiment a new transgenic line, Carb109, was generated, which harbored the identical transgene as Carb77 [26**].…”

“…The identical DENV2 targeting IR effector was also transgenically overexpressed in fat body tissue under control of the bloodmeal inducible vitellogenin 1 promoter and in salivary glands from the 30K promoter [27**,49]. Silencing DENV2 in fat body did not affect the mosquito’s overall vector competence for the virus, indicating that this tissue can be circumvented by DENV2 during its route to the salivary glands.…”