Negeviruses are a newly described taxon of insect-specific viruses (ISVs) that show potential to be used as a virus-based pathogen control strategy through superinfection exclusion. Due to its recent discovery, little is known about the biology of these ISVs or how they interact with the insect’s virome. It was recently demonstrated that both wild-type and genetically modified Negeviruses can inhibit the replication of Alphaviruses in mosquito cell culture. For Negeviruses to be used in wild mosquito populations they will have to compete with other viruses that infect mosquitoes, typically other wild-type Negeviruses and ISVs. Thus, I performed co-infection assays in different cell types to observe homologous and heterologous exclusion during virus infection. The cell lines used were Aag2 cells derived from Aedes aegypti, and C7/10 cells derived from Aedes albopictus. C7/10 cells have a dysfunctional RNA interference response, while Aag2 cells have a functional RNA interference pathway and pre-existing chronic infections with two ISVs: CFAV and PCLV. Homologous exclusion will be tested through additional infectious clones of Negeviruses that have fluorescent reporter genes inserted into their genome. Analyzing the growth trends of different Negeviruses in these cell lines allow us to determine which Negevirus isolates can establish infection in the presence of existing or co-infections with other viruses. Results demonstrated the successful cloning of PIUV ORF3 mScarlet, but with concerns regarding insert stability. The variability in outcomes during co-infection experiments was attributed to cell type differences, mainly RNAi competency, and pre-existing infections. Fitness variations were observed among the viruses, with NEGV isolates and LORV demonstrating higher fitness. These findings contribute to our understanding of Negevirus biology and highlight the importance of further research to overcome the challenges encountered in cloning and characterization of the behavior of these viruses
