Mosquito NF-κB-mediated Innate Immunity Exerts Arbovirus-Specific Antiviral Effects at Multiple Stages of the Viral Life Cycle

One third of all emerging infectious diseases are vector-borne, with the vector’s ecology and physiology playing key roles in determining whether viruses can access new vertebrate host species and spread globally. Innate immunity is a known barrier to virus replication in mosquito vectors that influences arboviral vector tropism. We here generated novel CRISPR-Cas9-mediated knockouts of the NF-κB family transcription factor Rel2 in Aedes aegypti -derived Aag2 cells and tested the impact on the replication of a diverse range of arboviruses in the Flaviviridae and Togaviridae families and the class Bunyaviricetes . We found that NF-κB-mediated innate immunity has broad antiviral activity against the Ae. aegypti -borne orthoflaviviruses dengue virus (DENV), yellow fever virus (YFV) and Zika virus (ZIKV) in mosquito cells. In contrast, little impact of NF-κB-loss-of-function was observed for the alphavirus chikungunya virus (CHIKV) or phlebovirus Rift Valley fever virus (RVFV), indicating specificity in the antiviral effects of NF-κB-mediated immunity. By comparing orthoflaviviruses with different transmission routes (mosquito-borne, tick-borne, no known vector), we demonstrated that NF-κB-mediated immunity exerts its antiviral effects both early and late in the viral replication cycle, and that NF-κB-mediated immunity is not the only molecular barrier influencing the ability of orthoflaviviruses to replicate in Ae. aegypti cells. Overall, our work demonstrates the importance of mosquito NF-κB-mediated innate immunity in suppressing arbovirus replication, and shows that the barriers for arboviruses to adapt to new vector species are multifactorial and virus-specific. Our findings increase our understanding of the molecular barriers influencing arboviral emergence, and could inform the development of refractory mosquitoes incapable of transmitting human pathogens. Author Summary Mosquito-borne viruses are causing an increasing global disease burden due to urbanisation, globalisation, changing land use and the climate change-driven invasion of mosquitoes into new locations. Few vaccines and no medicines for mosquito-borne viral infections are licensed for human use. Historically, the most effective way to reduce human disease has been to kill mosquitoes using insecticides, which also harms beneficial insects and is leading to resistance. Newer technologies with lower ecological impacts are being developed, including genetically modified mosquitoes with a reduced ability to transmit viruses. Like humans, mosquitoes have an immune system that protects against viral infections, and strengthening this immune system through genetic modification has shown promise for reducing virus transmission. We here expand our understanding of the ways in which mosquito immunity could be harnessed by demonstrating that a gene called “Rel2” helps the mosquito fight mosquito-borne virus infection. Unlike previously studied mosquito immune responses, Rel2 has a very broad (but not universal) protective capacity against viruses. Our work also indicates that Rel2 works in concert with other arms of the mosquito immune system, highlighting a need for further research to fully realise the potential of genetically modifying mosquito immune responses for the prevention of human disease. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, MR/R010315 Wellcome Trust, 096062 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BBS/E/PI/230001A, BBS/E/PI/230001B, BBS/E/PI/230002B, BBS/E/PI/23NB0004, BBS/E/PI/23NB0003, BB/W01100X/1 European Union, https://ror.org/019w4f821, 871029 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BBS/E/I/00007031, BBS/E/I/00007033, BBS/E/I/00007037, BBS/E/I/00007038