Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes.

Background. Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. Method. Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. Results. A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. Conclusion. Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.