Jason L. Rasgon, PhD - US grants

vertical transmission; Rickettsiales disease; animal population study; epizootiology; Culicidae; histocompatibility; epidemiology; cytoplasm; genetic strain; animal genetic material tag; mathematical model; statistics /biometry; field study; polymerase chain reaction;

DESCRIPTION (provided by applicant): Knowledge of how the genetics of vector populations condition the spread of arboviruses is critical for understanding dynamics of disease incidence, development of risk assessment strategies for novel virus introductions and development of virus transmission biomarkers that can be used to efficiently target control efforts. Since its introduction in 1999, West Nile Virus (WNV) has spread completely across the contiguous United States and has been responsible for over 23,000 confirmed human cases with almost 900 deaths. The most efficient laboratory vector has been demonstrated to be Culex tarsalis, which has been identified as a very important vector in the western United States. Prior studies and our preliminary data have demonstrated significant genetic structure among Cx. tarsalis populations and geographic variation in the ability of Cx. tarsalis to transmit WNV orally and vertically. We hypothesize that genetic variation related to vector competence is partly responsible for temporal and spatial variation seen in WNV transmission in Cx. tarsalis. We will use the Cx. tarsalis/WNV relationship as a model system to investigate how the genetics of mosquito populations govern the successful invasion and maintenance of an introduced arbovirus in natural populations. We have developed multiple genetic marker systems for use in Cx. tarsalis. Our specific aims are to: 1) Conduct population genetic analysis of Cx. tarsalis populations using mitochondrial sequence data and our newly-developed microsatellite markers, 2) Identify quantitative trait loci (QTL) responsible for WNV vector competence in Cx. tarsalis using an Advanced Intercross Design and 3) Identify QTL explaining WNV vector competence in natural Cx. tarsalis populations. Our results will guide future efforts to identify and validate specific candidate genes responsible for WNV susceptibility in Cx. tarsalis, determine the role of specific genes in maintaining WNV transmission in natural Cx. tarsalis populations and understand how the genetics of natural vector populations condition the successful introduction and maintenance of introduced exotic pathogens. Our work will ultimately lead to the identification of mosquito genetic biomarkers for WNV transmission that can be used for risk assessment and effective targeting of vector control efforts. This work also has important relevance for bioterrorism issues because it will provide insight into how intrinsic vector genetic factors in mosquito populations affect the epidemiology of a released Category B agent.

[unreadable] DESCRIPTION (provided by applicant): Failure of traditional methods to control malaria has stimulated efforts to create transgenic mosquitoes for malaria control. Transgenes must be actively driven into natural Anopheles populations to high frequency for transgenic strategies to be successful. Wolbachia are maternally inherited endosymbionts associated with cytoplasmic incompatibility (CI) i.e., reduced egg hatch when an infected male mates with an uninfected female. Matings of infected females are fertile regardless of the infection status of the male. CI confers a reproductive advantage to infected females and allows Wolbachia to spread rapidly through insect populations to high frequency. Transgenic traits tightly linked to Wolbachia are expected to be driven into the population by genetic hitchhiking, replacing the natural population with one that is refractory to parasite transmission. While Wolbachia infections are common in mosquitoes, they have never been observed in any species of Anopheles. Thus, Wolbachia-based strategies for malaria control require the artificial transfer of infection into Anopheles species responsible for parasite transmission. Artificial Wolbachia transfections have succeeded in several medically-important Aedes species but have not yet succeeded in Anopheline mosquitoes. Our in vitro studies show that the Anopheles gambiae genetic background is competent to harbor at least 3 diverse Wolbachia infections, and thus, previous unsuccessful Anopheles transfection attempts are likely due to technique rather than an intrinsic genetic block to infection. We will use recently-developed embryonic and adult injection techniques to establish Wolbachia infections in Anopheles gambiae and examine the interaction between Wolbachia and Anopheles at the phenotypic, genetic and population levels. By the end of the proposed research, we will 1) artificially transfect Anopheles gambiae with Wolbachia, 2) determine the effect of Wolbachia infection on expression of candidate Anopheles genes previously identified by cell line experiments and 3) evaluate the potential for Wolbachia to drive maternally-inherited transgenes into Anopheles gambiae cage populations. These results will lay the foundation for the successful deployment of genetically-modified mosquitoes for malaria control. Transgenic strategies for malaria control require the active spread of introduced transgenes to high frequency in natural Anopheles populations. Wolbachia endosymbionts can theoretically act as a mechanism to spread genes into populations. We propose to evaluate transfer Wolbachia infection into Anopheles gambiae and evaluate the symbiont as a candidate transgene drive system for malaria control. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): One of the greatest obstacles facing basic and applied molecular Anopheles gambiae research is the prohibitively low efficiency of transgenesis for this species. In the twenty years since first report of a stable germline transformation, only a handful of successful transgenic An. gambiae lines have been published. The dearth of tools for targeted gene knockouts and gene replacements in Anopheles gambiae is currently recognized as a critical research need. In other systems, Zinc-Finger Nucleases (ZFNs) are very efficient tools for targeted gene-disruption by inducing a double strand break (DSB). When no homologous donor template is provided, ZFN-induced DSBs result high efficiency gene-disruption through non-homologous end-joining (NHEJ)-mediated frame-shifts. When a DSB occurs, if a homologous template (such as a plasmid) is available the DSB can be repaired by the homologous recombination (HR) pathway, inserting new genetic material into the genome. It has been shown in other systems that when a single-stranded parvovirus genome is used as the homologous template with a DSB, site-specific gene replacement occurs at a frequency of 10-1 to 10-3 events per cell - up to 10 million times greater efficiency than spontaneous HR (no DSB) with a traditional plasmid template. Our laboratory possesses the only known parvovirus capable of infecting An. gambiae (the Anopheles gambiae densonucleosis virus;AgDNV), which we believe will serve as a high-efficiency vector for the delivery of agents capable of precise manipulation of the Anopheles gambiae genome. In this proposal, we will investigate this question using an in vitro system. Our overall hypothesis is that AgDNV can be used to transduce specific ZFNs alone or in conjunction with homologous gene replacement cassettes for high- efficiency gene knockout or gene replacement. This hypothesis will be examined by the following 3 specific Aims: 1) establish a system for targeted gene-disruption by AgDNV-based transduction of site-specific ZFNs in Anopheles gambiae cells, 2) use recombinant AgDNV donor vectors for gene replacement by homologous recombination in Anopheles gambiae cells and 3) examine the synergistic effect of ZFN-mediated site-specific DSBs on AgDNV-mediated gene-replacement. PUBLIC HEALTH RELEVANCE: One of the greatest obstacles facing basic and applied molecular research on the major malaria vector Anopheles gambiae is the prohibitively low efficiency of transgenesis for this species. The Anopheles research community will benefit tremendously from tools that enable targeted gene knock-outs and targeted integration of transgenes. In this proposal, we will develop a system for ultra- high efficiency site-specific gene knock-outs and knock-ins for the Anopheles gambiae genome.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Genetic manipulation is a powerful technique for addressing research questions in arthropods of medical importance. Current approaches rely upon delivering DNA or endonucleases to preblastoderm embryos via embryonic microinjection. However, embryonic microinjection is technically challenging, is limited to a small number of arthropod taxa, and is inefficient even in optimized species. As such, there is a critical need to develop methods for arthropod genetic manipulation that are simple, accessible for many researchers and generally compatible for a large variety of arthropod species. During oogenesis, insects transfer yolk protein precursors to developing oocytes by receptor-mediated endocytosis (RME). When Drosophila melanogaster yolk protein 1 (DmYP1) is injected into pre-vitellogenic adult female Anopheles mosquitoes, it is transduced into the germline by RME. We show that DmYP1 can be used to transduce cargo such as protein or DNA with 100% efficiency to the developing Anopheles germline. We term this technique Receptor-Mediated Ovary Transduction of Cargo, or ReMOT Control, which we hypothesize can be used to transduce cargo into the Anopheles germline for stable and heritable editing of the mosquito chromosomal genetic sequence. This hypothesis will be investigated by two specific Aims. The first aim is to use ReMOT Control to specifically delete genes in the germline of Anopheles stephensi. DmYP1 will be fused to transcription activator-like effector nucleases (TALENs) targeting GFP. After injection into GFP-transgenic mosquitoes, deletion lines will be identified by loss of fluorescence and sequencing. The second aim is to use ReMOT Control to create transgenic Anopheles stephensi by transduction of transposable elements into the mosquito germline. DmYP1 will be used to transduce GFP-containing transposons into transgenic transposase-expressing mosquitoes or co-injected with DmYP1-transposase fusion enzymes. Transgenic lines will be identified by GFP gain-of- function; insertion sites will be identified by PCR, sequencing and southern blot. Once optimized, ReMOT Control will dramatically change the landscape of molecular entomology research, allowing easy, flexible genetic manipulation of a wide variety of vector arthropods and non-model species.

? DESCRIPTION (provided by applicant): Efforts to control vector-borne pathogens have been hindered by evolution of insecticide resistance and failing drug therapies. To improve the sustainability and efficacy of control efforts, alternative vector control strategies are being considered. Infection with the maternally inherited endosymbiont Wolbachia pipientis has been demonstrated to inhibit viruses and parasites in vector arthropods. Wolbachia-infected mosquitoes are currently being released into nature to control human disease. However, a worrying trend is emerging whereby Wolbachia infections enhance rather than suppress pathogens in insect vectors. We have now demonstrated Wolbachia-mediated extent of Wolbachia-induced pathogen enhancement, and the mechanism(s) leading to this phenotype enhancement of several arboviral human pathogens in the mosquito vector Culex tarsalis; a sobering reminder that the pathogen inhibitory effects resulting from Wolbachia infection in some insects cannot and should not be generalized across vector-pathogen systems. Understanding the general are critical for estimating how likely Wolbachia-based control strategies are to fail or make things worse, for identifying potential points where Wolbachia-based control is likely to break down in the field, and for planning risk mitigation strategies in he case of unforeseen harmful outcomes. In this research, we will investigate the hypothesis that Wolbachia-induced modulation of the mosquito hologenome can lead to increased arbovirus infection/transmission in some vector-pathogen systems of human importance. This hypothesis will be examined in the following three Specific Aims: (1) Determine the generality of Wolbachia- induced arbovirus enhancement in multiple mosquito-pathogen systems; (2) Determine the role of Wolbachia- microbiome interactions in mediating WNV enhancement in Cx. tarsalis; (3) Determine the role of Wolbachia- induced modulation of mosquito gene transcription in mediating WNV enhancement in Cx. tarsalis.

Zika virus is a mosquito-borne flavivirus first isolated in the Zika forest of what is now Uganda. For many decades, Zika virus was of no major epidemiological concern, causing occasional small outbreaks in Africa and Southeast Asia with only a handful of human cases recorded. This changed in 2007, when the first outbreak outside of Africa or Asia occurred on the island of Yap in Micronesia with approximately 100-200 confirmed or suspected cases. Zika virus is no longer a mild infection limited to Africa and Asia ? it has now been introduced to the western hemisphere, with autochthonous Zika transmission documented in Brazil since May 2015, in other countries in central and south America, and over 250 imported cases in the United States (as of March 2016). Due to newly observed associations with major birth defects, the World Health Organization has declared Zika a global emergency and is estimating approximately 3-4 million cases by the end of 2016. Similar to dengue and Chikungunya viruses, the mosquito Aedes aegypti is thought to be the primary vector for Zika virus. Aedes albopictus has also been demonstrated to be a highly competent vector in laboratory studies. However, Zika virus has been detected in over 25 species of mosquitoes from 5 genera. Although detecting virus in a mosquito is not proof of transmission, these studies emphasize our lack of knowledge about the transmission biology of this emergent pathogen. Some identified vector species (Aedes albopictus and Aedes aegypti) are present in the United States and these vectors alone open the possibility of outbreaks and even local transmission in parts of the USA. If other native or established mosquito species are competent to transmit Zika, the virus could potentially move into the USA beyond areas currently colonized by aegypti and albopictus, similar to what was observed with the mosquito Culex tarsalis and the invasion of West Nile virus into the USA during the early 2000's. In this proposal, we will (1) investigate the potential for common, widespread mosquito species present in the United States to transmit Zika virus and (2) investigate geographic variation among Aedes albopictus populations to transmit Zika virus. Proactive knowledge about the potential role that North American mosquito fauna may play in the introduced epidemiology of Zika virus is absolutely critical for the development of efficient Zika virus control strategies and risk management policies in the USA.  

Project Summary Human malaria, responsible for inordinate mortality, morbidity and economic loss worldwide, is caused by protozoan parasites in the genus Plasmodium that are obligatorily transmitted by Anopheles mosquitoes. Failure of traditional control methodologies has stimulated efforts to develop novel strategies to control the mosquito vectors of malaria, particularly An. gambiae. While transgenic manipulation of Anopheles species has been accomplished, routine manipulation of An. gambiae has proven challenging, and the technology to do so is not broadly available among non-specialized laboratories. The development of novel, easy to use tools for routine forward genetics in An. gambiae is critical for both applied strategies for malaria control and basic research into the genetics and host/parasite interactions of this important mosquito vector species. Densonucleosis viruses, or ?densoviruses? (DNVs), are single-stranded DNA viruses in the family Parvoviridae with very small genomes (4-6 kb) that are flanked by terminal hairpin structures at the 5-prime and 3-prime ends. The entire viral genome can be placed into an infectious plasmid from which functional virus will be produced upon transfection into an appropriate cell line. In our laboratory, we have identified only known densovirus (AgDNV) capable of infection and dissemination in Anopheles gambiae. AgDNV replicates preferentially in adult mosquito tissues to very high titer, but is completely non-pathogenic. We have developed and validated novel techniques to use AgDNV to express secreted effectors or microRNAs that can modulate or alter patterns of Anopheles gene expression. Our overall hypothesis is that AgDNV can be used overexpress or knock down expression of specific genes of interest in Anopheles gambiae, leading to phenotypes of basic and applied importance. This overall hypothesis will be addressed in the following specific aims: 1) Develop an AgDNV-based gene transduction system for routine forward genetics in An. gambiae, focusing on modulation of Plasmodium falciparum infection/transmission; 2) Develop an AgDNV-based system for routine reverse genetics in Anopheles gambiae, focusing on modulation of P. falciparum infection/transmission and mosquito fitness; 3) Characterize and quantify AgDNV infection of the male mosquito reproductive system, and determine the potential for using auto-dissemination to introduce AgDNV into mosquito cage populations. This research will result in the development of a novel toolset for addressing basic questions in Anopheles and Plasmodium biology, as well as the development of potential control agents for human malaria.

Non-technical paragraph: Genetic manipulation is a powerful technique for addressing research questions in arthropods. Current approaches rely upon delivering gene-editing material to arthropod eggs by embryonic microinjection (EM). However, EM is very challenging, is limited to a small number of arthropod species, and is inefficient even in optimized species. There is a critical need to develop methods for arthropod genetic manipulation that are simple, accessible for many researchers and generally compatible for a large variety of arthropod species. We have developed a technology called Receptor-Mediated Ovary Transduction of Cargo, or ReMOT Control, to specifically deliver gene-editing cargo to the developing arthropod germline by easy injection into female arthropods during egg development. ReMOT Control can bring the power of genetic modification technology to any model or non-model species without the need for injecting embryos, allowing any lab to use these powerful tools for their research questions. Using workshops, social media, symposia, and by making reagents publically available, we will ensure that the ReMOT Control technology is available to any interested researchers. We will also use numerous outreach venues to educate the public about the benefits of these amazing tools. We will develop outreach activities and leverage existing outreach venues that engage K-12 students and members of the public to educate them about how these techniques can benefit their everyday lives.

Technical paragraph: The ReMOT Control technology is based on the identification of small peptide ligands with very specific tropism for the developing arthropod germline. These ligands are used to transduce the CRISPR/Cas9 ribonucleoprotein complex to developing eggs after injection into vitellogenic females. In this proposal, we will develop both specific and generalizable ReMOT Control technologies that work across diverse arthropod taxa, opening the true power of this technique to all researchers interested in genetic engineering techniques. ReMOT Control will break down barriers to genetic modification, allowing researchers in diverse animal systems to move beyond correlation to accurately and precisely study gene function. Our overarching conceptual goal for this proposal is nothing less than the complete democratization of gene editing capability for all researchers working in any arthropod system, be it model or non-model. This goal will be realized by the following three Specific Aims: 1) Identify species-specific and universal ligands for applying ReMOT Control technology to diverse oviparous and viviparous arthropod species; 2) Use identified ligands to transduce CRISPR/Cas9 to the host germline for targeted gene deletion and gene knock-in in diverse arthropod species; 3) Disseminate ReMOT Control information, technology and methodology to broad communities of scientists involved in research on animal behavior, animal physiology, insect-plant interactions, sustainable agriculture, and public health. By the end of the research project this transformative technology will bring genetic modification technology within the reach of everyday scientists regardless of their research system.

Knowledge of the factors influencing pathogen spread in vector populations is critical for understanding dynamics of disease incidence, development of risk assessment strategies for novel pathogen introductions and development of transmission biomarkers that can be used to efficiently target control efforts. Although novel pathogens such as Zika virus receive inordinate media attention, the Category B Priority Pathogen West Nile Virus (WNV) is the most widespread locally transmitted mosquito-borne pathogen in the USA. Since its introduction in 1999, WNV spread completely across the United States with over 50,000 confirmed human cases and over 2,000 deaths. The most efficient laboratory vector is Culex tarsalis, which has been identified as a very important vector in the western United States. Our recent work showed strong correlation between the genetic structure of Cx. tarsalis and the invasion pattern of WNV across the western United States; one of the first studies to directly use mosquito population genetics to implicate their role in pathogen invasion. Traditionally, variation in vector competence has been attributed to genetic differences between mosquito strains or individuals. However, no organism exists in isolation; organisms are a community consisting of the host and its associated microorganisms (bacteria, fungi and viruses) that, collectively, make up the holobiome. Little is known about the mosquito holobiome factors influencing pathogen invasion in the field. In this proposal, we will delineate the hologenomic (genetics of the mosquito and its associated microorganism) factors underlying WNV phenotypic variation across field populations of Cx. tarsalis. Our overall hypothesis is that variation in the mosquito hologenome determines variation in WNV infection, transmission and/or dissemination in the field. This hypothesis will be investigated in the following Specific Aims: (1) Delineate fine-scale landscape genetics of Cx. tarsalis in the United States; (2) Determine the relationship between the Cx. tarsalis microbiome and WNV infection, dissemination and transmission in field populations of Cx. tarsalis; and (3) Use a replicated pooled genome-wide association study (PoolGWAS) to identify genomic loci associated with WNV infection, dissemination and transmission in field populations of Cx. tarsalis. Identification of hologenomic loci affecting pathogen vector competence and studies of correlated genetic variation between populations are critical for understanding patterns of pathogen transmission and disease outbreaks. This work has particular importance for bioterrorism issues because it will provide conceptual insight into how intrinsic vector hologenomic factors in natural populations affect the epidemiology of a released Category B agent.