Zach Adelman - US grants

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to utilize the robust replication of an RNA virus to study gene function in disease vector mosquitoes using both forward and reverse genetic approaches. In the first aim, a portion of an RNA viral genome encoding the viral replicase will be expressed as a transgene from the mosquito genome in a tissue-specific manner, with a native mosquito gene sequence incorporated into the virus genome. As viral replication proceeds, RNA interference should be induced to both the viral RNA and the native mosquito gene. We expect that because of the viral replicase-mediated amplification step, linear variability in nuclear transcription rates, which affects the direct transcription of inverted repeat sequences and can be caused by transgene insertion sites, nutritional status or other factors, should be eliminated or substantially reduced. This should increase both the strength and the efficiency of RNAi-based reverse genetic experiments in mosquitoes as well as simplify the interpretation of RNAi knockout phenotypes. In the second aim, we seek to develop a forward genetic screen for mosquito genes involved in the RNA interference pathway. While the most obvious RNAi genes have been characterized, there are likely many critical components which remain unknown. A library-based recombinant virus system is proposed which will support virus replication if and only if a particular recombinant virus contains a portion of a gene involved in RNAi. Public Health Relevance. The mosquitoes Aedes aegypti and Anopheles gambiae are responsible for an enormous disease burden including dengue hemorrhagic fever and malaria. Developing methods to more precisely, more efficiently and more robustly interrupt mosquito gene function is critical to understanding the biology of these vectors, as well as understanding mechanisms of pathogen transmission. Understanding the genes involved in innate anti-viral immunity will allow better understanding of vector competence and modeling of transmission potential for introduced agents, either natural or through bioterrorism. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to determine the potential for selfish genes, AKA homing endonucleases, to serve as a method of dispersing and fixing genes into natural mosquito populations that reduce the vector competence of mosquitoes to transmit pathogens, such as the causative agents of dengue fever and malaria. In the first aim of this proposal we seek to determine the ability of various homing endonucleases to generate dsDNA breaks using a two-plasmid based assay. This assay has been designed so that successful cutting by the homing endonuclease will result in the loss of a negative selectable marker. Plasmid-based assays will be conducted in both cultured mosquito cells as well as in live embryos. In the second aim we seek to determine the rate at which homing endonucleases can excise a transgene from the mosquito genome. A marker gene flanked by homing endonuclease recognition sites will be inserted into the mosquito genome, followed by the introduction of a homing endonuclease which should excise the marker. Homing endonucleases found to be functional in both plasmid-based assays and transgene excision assays will serve as the foundation for future work at adapting homing endonucleases as a method of gene drive. Public health relevance: This work is part of a larger, ongoing strategy to control human pathogens using genetics. Such a genetic control strategy is founded on the hypothesis that reduced/ablated vector competence will result in a corresponding reduction/elimination of human disease, and is based on three elements: the development of anti-pathogen effector genes, the ability to introduce effector genes into mosquito vectors, and the ability to spread these effector genes into wild populations. The first two elements have seen substantial progress in the past decade, and this proposal is designed complement this work so genetic control strategies can become a reality in effecting and improving public health outcomes due to vector-borne diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Arthropod-borne viruses (arboviruses) constitute a persistent and worldwide challenge to public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. For arboviruses in nature, it is imperative that very little fitness cost be associated with infection of the mosquito. We recently demonstrated the importance of RNA interference (RNAi) in the mechanism by which alphaviruses establish a persistent, nonpathogenic infection in the mosquito vector, showing that in the absence of RNAi-based modulation, mosquitoes do not survive arboviral infection. However, very little direct experimentation has been done on mosquito RNAi genes. We have described the generation and validation of a transgenic strain of Aedes aegypti that "senses" the status of the RNAi pathway, through which we have shown that the genes Dcr-2 and Ago-2, but not Ago-3, are critical for RNAi in Ae. aegypti. We hypothesize that genetic variability in the RNAi pathway directly affects the ability of mosquitoes to become infected by, and transmit, arboviruses. In specific aim 1, we will use our transgenic "sensor strain" to determine the involvement of mosquito genes in the regulation and execution of RNAi. In specific aim 2, we will compare primary nucleotide sequences of various mosquito genes within the genus Aedes, and determine whether any evolutionary selective pressures are acting on genes involved in RNAi. In specific aim 3, we will determine the role of genetic variability in RNAi genes on the vector competence of Aedes mosquitoes for medically important arboviruses. A detailed understanding of mosquito RNAi genes, and the role played by genetic variation in those genes on virus transmission, will facilitate better evaluations of the feasibility of RNAi-based genetic control strategies, and allow us to more accurately determine the risks of new and emerging arboviruses spreading to new areas, particularly within the U.S. PUBLIC HEALTH RELEVANCE: Arthropod-borne virus (arbovirus) diseases such as yellow fever, dengue fever, and chikungunya fever remain a significant burden on global public health. This proposal deals directly with the viral pathogens that cause those diseases. We aim to characterize novel components of the mosquitoes'innate immune system which acts as a general constraint to the accumulation of virus in the mosquito. This knowledge is critical to understanding and predicting the emergence of new arboviral epidemics, as well as to understanding how arboviruses are maintained in nature, and may ultimately form the basis of RNAi-based transgenic and field diagnostic approaches for disease control.

DESCRIPTION (provided by applicant): Vector-borne diseases such as malaria and dengue hemorrhagic fever remain large public health burdens, and novel interventions are still needed. The emerging field of genetics-based control has seen a number of recent laboratory successes with the generation of pathogen- resistant mosquito strains as well as female-killing strains. The development of genetically modified mosquitoes still largely relies upon classical transposable element transformation, although several recent reports have made use of the site-specific integrase C31. While site- specificity allows the investigator to examine multiple transgenes in the same genetic environment, an unfortunate consequence of the attP-attB C31 system is that as a result of recombination the entire bacterial plasmid becomes integrated into the mosquito genome. This is undesirable, as many of the currently developed transgenic mosquito strains are intended as specific genetic interventions to control a targeted vector-borne pathogen where such antibiotic resistance genes could be transferred to native bacterial species. We hypothesize that recombinase-mediated cassette exchange (RMCE) can be an efficient means of delivering transgenes into important disease vector species in a site-specific manner, without the negative consequences of co-integrating bacterial sequences. As such, we propose to (1) test a panel of heterospecific lox sites for their ability to resist intramolecular recombination i cis and promote RMCE in the embryos of the dengue vector Aedes aegypti and the malaria vector Anopheles gambiae; (2) generate transgenic docking strains based on the best candidate heterospecific lox sites and determine the rate of RMCE for each using the phage P1 cre recombinase. Such an alternative system which preserves the ability to perform site-specific recombination, but avoids the integration of bacterial sequences and remains as easy to use as TE-based helper plasmids would likely be much more widely adopted, and would help to drive the field of novel genetics- based control strategies for vector-borne diseases.