Investigation of the mosquito innate immune response to flavivirus infection

Mosquitoes are constantly under threats of infections by different kinds of pathogens. Due to a lack of adaptive immune system unlike higher animals, they defend themselves by using their innate immune system. In our project, we assessed the implications of the two flaviviruses (named Lammi virus and Usutu virus) on the mosquitoes’ innate immune system as well as how these flaviviruses manipulated their innate immune system during infection. First, the mosquito cell lines were examined at five different time points to identify changes of cellular structure after USUV and LAMV infections. Then, the USUV infection was evaluated by using immunofluorescence Assay (IFA staining). In this present study, we also investigated the virus load from supernatant of single and double infections separately with the help of RT-qPCR. For immune gene studies, the transcript-levels of our selected twenty-three immune genes were analyzed by using the 2-ΔΔCq Livak method from single and double infected samples with three biological replicates. After examination of the cellular structure of infected cells, all characteristics of cytopathic effects were unclear compared with the mock infection. In the supernatant samples, the USUTU virus replication was decreased but Lammi virus load was higher in the double infection that was proven to be statistically significant at five different time points. After that, RT-qPCR data of infected cells demonstrated different expressions of certain immune genes due to the presence of USUV and LAMV in the single as well as double infections. Out of 23 genes, fold changes of nine genes’ expression were verified as statistically significant. For example, the fold changes of differentially expressed REL1A and unchar_ncRNA genes were significant in the LAMV infected U4.4 cells compared with the double infected cells (USUV and LAMV together). Concurrently, some genes were up-regulated and some were down-regulated significantly in different infections at the 24 hr and 48 hr time points compared with the mock infection. In conclusion, the mosquito cell line U4.4 was infected by single (e.g. LAMV or USUV) and double infections (USUV and LAMV) separately to see the effects of these two flaviviruses and our selected immune genes revealed fold changes of differential gene expressions in both single and double infections compared with the mock infection. Furthermore, this investigation demonstrates how mosquitoes’ immune signaling pathways were manipulated by these flavivirus infections.Due to pandemic, it was a zoom presentation.