Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

Background The mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti. Methodology/Principle findings We developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV). Conclusions/Significance Our demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.

Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

ABSTRACTBackgroundThe mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti.Methodology/Principle FindingsWe developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV).Conclusions/SignificanceOur demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.AUTHOR SUMMARYThe mosquito Aedes aegypti, found globally across the tropics and subtropics, transmits viral diseases with a significant global public health impact, including chikungunya, dengue, yellow fever and Zika viruses. There are no antiviral drugs to treat these diseases and few effective vaccines. One way of reducing the global burden of mosquito-borne diseases would be to develop genetically modified mosquitoes unable to transmit viruses. One approach would be to alter the mosquitoes’ immune system to allow them to better fight viral infections. To do so, we first need to understand how viruses are detected by the mosquito immune system. We developed new methods of measuring immune responses in laboratory-cultured mosquito cells and used them to show that one specific arm of the immune system, called the ‘Imd pathway’, can detect the RNA that constitutes the genome of mosquito-borne viruses. These findings pave the way for future immune studies that could inform the development of transmission-incompetent mosquitoes. We also found that another arm of the immune system, called the ‘Toll pathway’, is not functional under any experimental conditions used in this study. This finding has implications for how different laboratories interpret data from these particular cultured cells.

Monitoring transgenic mosquitoes using wing measurements and I3S Classic

Stress responses in insects can manifest as changes in size, shape and symmetry of the wings. Developing methods to measure and track such features could act as an early warning indicator of adverse events or, if all is well, provide assurance that field or laboratory colonies were fit, healthy and developing optimally. This is especially important in the case of newly developed transgenic insects, to assess morphology and as an indicator of their fitness. As body size and symmetry is known to be a significant correlate of fitness, the potential of transgenic insects is reflected in their phenotypic expression. Microsoft Paint and Photos as well as I3S Classic were used. The wings of transgenic mosquitoes DSM 1 & 2 were measured and compared to those of the parent population Anopheles gambiae G3. The right and left wings of both sexes were assessed to determine if they were symmetrical. Measurements indicated high wing symmetry in all the groups and sexes tested, indicating that the transgenic mosquitoes should be just as functional as their parents. The transgenic mosquitoes DSM 1 & 2 were found to be significantly larger in length and width than the parent population A. gambiae G3 and could be distinguished from the parent strain using I3S Classic software with 70 to 100% accuracy. I3S Classic ranked the correct sex of the test strain predominantly in the initial ranks indicating the differences in architecture of male and female wings. I3S Classic software was also used to assess wing symmetry. In keeping with the data from taking measurements, the software indicated that the wings were highly symmetrical, both the right and left wings of the correct strain were selected in the early first and second ranks in roughly equal measure. The importance of assessing the morphological characteristics of insects and of taking measurements during the investigative procedure was discussed.