Characterization of Negeviruses and insect virome interactions during co-infection in cell culture

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

Vertical and Horizontal Transmission of Cell Fusing Agent Virus in Aedes aegypti

Cell fusing agent virus (CFAV) is an insect-specific flavivirus (ISF) found in Aedes aegypti mosquitoes. ISFs have demonstrated the ability to modulate the infection or transmission of arboviruses such as dengue, West Nile, and Zika viruses. It is thought that vertical transmission is the main route for ISF maintenance in nature. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female pupal exuviae and was 89% sensitive, but only 42% in male pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study. However, there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISF-derived arbovirus control