The overarching objective of this application is to investigate the influence of autophagy on the replication of arthropod-borne viruses (arboviruses) in invertebrates. Autophagy is an evolutionarily conserved process that mediates the transfer of cytoplasmic material to lysosomes for degradation. Induction of autophagy results in the formation of double-phospholipid membrane vesicles termed autophagosomes which sequester targeted organelles and proteins. Subsequently, autophagosomes fuse with lysosomes which mediate degradation of their contents. This process helps maintain cellular homeostasis and survival. In addition, autophagy functions as an innate immune defense against intracellular pathogens, such as Mycobacterium tuberculosis and Toxoplasma gondii. Interestingly, autophagy has been implicated as having both pro- and antiviral activity. Its role i host-virus interactions is therefore unclear at present. Dengue virus (DENV; Flaviviridae, Flavivirus) causes tremendous morbidity and mortality worldwide, and options for control are limited. Therefore, it is imperative to elucidate factors influencing virus-vector interactions in order to develop novel control strategies. Previous studies have demonstrated that autophagy functions in a pro-DENV capacity during infection of mammalian cells, but the role of autophagy during arbovirus infection of vector mosquitoes is unknown. Therefore, we propose to examine the role of autophagy during DENV-2 infection of Aedes aegypti. We hypothesize that autophagy operates in a pro-viral capacity during DENV infection of Ae. aegypti.
Specific aim 1 addresses this question in three invertebrate cell culture systems; Ae. albopictus C6/36, Ae. albopictus U4.4, and Drosophila S2 cells. Complementary to aim 1, specific aim 2 will investigate the role of autophagy during DENV infection of Ae. aegypti. In addition, we will elucidate if autophagy functions as a determinant of Ae. aegypti vector competence for DENV using outbred mosquito strains. The role of autophagy in the virus-vector interaction will be analyzed using pharmacological inhibitors and inducers of autophagy as well as transgenic mosquitoes. We will monitored for hallmarks of autophagy using three conventional assays, electron microscopy, confocal microscopy analysis of GFP-Atg-8 punctae, and quantification of lipidated Atg-8- II by immunoblot. Further, the effects on DENV infection, replication and transmission will be assessed using standard virological assays. I have an extensive background in virus-vector interactions and have the necessary expertise and motivation to complete the proposed studies. I have a strong track record of research in the fields of vector biology and arbovirology as evident from my publication record. This is further exemplified by my former research support: a Ruth L. Kirschstein Research Service Award (5F32AI084432-02) entitled The Role of RNA Interference in West Nile virus Genetic Diversification. I have been actively pursuing tenure-track faculty positions and have recently interviewed at four highly regarded institutions. While I have yet to receive an offer, these invitations indicate that I am competitive candidate. My long-term career goals include securing a tenure-track faculty position, establishing an independent and innovative research program in vector biology and arbovirology, maintaining a well-funded laboratory, and making meaningful contributions to the scientific community that may, someday, have real-world applications. My continued interest and passion for vector biology and arbovirology combined with my professional undertakings attest to my commitment to secure a faculty position in academia. Included in this application is a career development plan that will foster my development as a young investigator and help me achieve my long-term goals. Specifically, I plan on attending the NIH's Regional Seminars on Program Funding and Grants which will not only improve my grant writing skills, but also inform me of federal regulations and policies and make me aware of current areas of special interest. Furthermore, I will be attending the American Society of Virology and the American Society of Tropical Medicine and Hygiene conferences annually as well as attending more intimate Keystone Symposia. These meetings will allow me to more easily establish collaborations with researchers outside of Colorado State University (CSU). Career development extends beyond grant writing and research. An often overlooked aspect in the career development of young investigators is teaching. CSU offers numerous courses through the Institute for Learning and Teaching dedicated to improving the teaching skills of faculty and staff. While I have been fortunate to acquire some teaching experience during my post-doc tenure, I will take advantage of the resources available through this institute such as Teaching with Technology workshops and Instructional Design Consultation to improve my teaching effectiveness. The novelty of the proposed research and its potential impact on not only virus-vector interactions, but also arthropod physiology as well as my career development plan and strong track record make me an ideal candidate for the K-22 Career Transition Award.
This application addresses the role of autophagy in Aedes aegypti vector competence for dengue virus. Autophagy is an evolutionarily conserved pathway that is involved in maintaining cellular homeostasis and immunity. Interestingly, autophagy functions in a pro-viral capacity during dengue virus infection of mammalian cells, but its function during infection of mosquitoes is unknown. Elucidating the host factors that mediate the virus/ vector interaction is imperative to developing novel control measures which are greatly needed. Dengue virus is the most significant arthropod-borne virus affecting humans and currently, there are no vaccines or antiviral and traditional control measures have proven ineffective.
|Correa, Maria A; Matusovsky, Brian; Brackney, Doug E et al. (2018) Generation of axenic Aedes aegypti demonstrate live bacteria are not required for mosquito development. Nat Commun 9:4464|
|Brackney, Doug E (2017) Implications of autophagy on arbovirus infection of mosquitoes. Curr Opin Insect Sci 22:1-6|