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

et al. (2014) Fitness Impact and Stability of a Transgene Conferring Resistance to Dengue-2 Virus following Introgression into a Genetically Diverse Aedes aegypti Strain. PLoS Negl Trop Dis 8(5): e2833. doi:10.1371/journal.pntd.0002833 Fitness Impact and Stability of a Transgene Conferring Resistance to Dengue-2 Virus following Introgression into a Genetically Diverse Aedes aegypti Strain Alexander W. E. Franz 0 Irma Sanchez-Vargas 0 Robyn R. Raban 0 William C. Black IV 0 Anthony A. James 0 Ken E. Olson 0 Mark Quentin Benedict, University of Perugia, Italy 0 1 Department of Veterinary Pathobiology, University of Missouri , Columbia , Missouri, United States of America, 2 Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University , Fort Collins , Colorado, United States of America, 3 Departments of Microbiology and Molecular Genetics and Molecular Biology and Biochemistry, University of California , Irvine, California , United States of America 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-ofprinciple vector population replacement. - Funding: This research was supported by funds from the Regents of the University of California from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The four serotypes of dengue viruses (DENV1-4; Flaviviridae; Flavivirus) are considered the most important mosquito-transmitted arboviruses infecting humans. Epidemiologists have estimated 100390 million people per year acquire DENV infections in tropical and subtropical regions of the world [1,2]. Dengue disease symptoms range from mild febrile illness, referred to as dengue fever (DF), to severe disease dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [3]. DENV prevalence is increasing rapidly throughout South-East Asia, and Central-and SouthAmerica due to rapid urbanization, increased trade and human traffic. DENV in these regions can be hyper-endemic [4], further increasing the risk of DHF. Furthermore, virulent strains have been introduced in the last decades from South-East Asia into Central-America and the Caribbean replacing endogenous DENV2 genotypes and causing more cases of DHF and DSS among local populations [57]. Currently, there are no vaccines or therapeutic drugs readily available to the more than two billion people at risk for DENV infection or the tens of millions manifesting some level of disease [2,8]. Thus, DENV prevention relies on vector control through indoor insecticide spraying, using insecticide treated door/window curtains and reducing the number of potential oviposition sites [911]. The principal vector of DENV is the peridomestic mosquito, Aedes aegypti (L.), which is distributed widely in many regions of the world and is a major factor contributing to the global incidence of DEN disease. Novel alternative vector control strategies are now being tested that use genetically-modified Ae. aegypti carrying a dominant-lethal gene (RIDL) to reduce mosquito populations [12 14]. A second novel concept in DEN disease control is replacement of DENV-competent mosquito populations with Expression of a DENV2 sequence-derived IR RNA in the mosquito midgut initiates an antiviral intracellular RNAi response that efficiently blocks DENV2 infection and profoundly impairs vector competence for that virus in Aedes aegypti. DENV2-specific IR RNA expression in the Carb109M strain has maintained the RNAi-based, refractory phenotype for 33 generations in laboratory culture. The two transgene integration sites were stable after multiple generations and following introgression into a geneticallydiverse (GDLS) Ae. aegypti population. Introgression of the transgene into the GDLS genetic background changed GDLS from a DENV2 susceptible phenotype to a DENV2 refractory phenotype. The DENV2 refractory homozygous line, Carb109M/GDLS.BC5.HZ, exhibits (relative to GDLS) minimal fitness loss associated with the transgene. This line could be a potential candidate for proof-of-principle field studies. DENV-refractory vectors [1517]. The work presented here describes the generation of a new Ae. aegypti transgenic strain, Carb109M/GDLS.BC5.HZ, which expresses an anti-DENV2 gene construct and is highly refractory to the virus after being introgressed into a genetically diverse laboratory strain (GDLS). Ae. aegypti females acquire a DENV-containing bloodmeal from a viremic human host. DENV initially infects midgut epithelial cells and 45 days later disseminates to hemocytes, fat body, nervous system tissues, and salivary glands. The mosquito can transmit vir (...truncated)