Progress 07/01/17 to 06/28/22

Outputs
PROGRESS REPORT Objectives (from AD-416): Objective 1. Develop the workforce needed to staff NBAF and fulfill the Foreign Animal Disease Research Unit⿿s mission to detect and control foreign animal diseases. Resources will be provided for academic-related expenses and the research projects that will enable the trainees to successfully achieve the academic requirements for obtaining degrees in one of the seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. Objective 2. Implement research projects under the direction and guidance of ARS scientists at the Foreign Animal Disease Research Unit (FADRU), PIADC, Orient Point, New York, and others in collaboration with FADRU. Approach (from AD-416): Division A of the Consolidated Appropriations Act, 2017 (P.L. 115-31) contains an increase of $900,000 (NTL) for research on NBAF Workforce Development at the Center for Grain and Animal Health Research, in Manhattan, Kansas. The increased funds are to be used to establish a new ARS project, which will be held in the Office of the Center Director for the Center for Grain and Animal Health Research. There is a shortage of qualified scientists, including the availability of doctors of veterinary medicine (DVM) with a Ph.D degree, to conduct animal health research at the NBAF when the facilities become available in 2022. This will be addressed by specifically training scientists in the following seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. The objective and desired outcome is a mechanism to ensure a viable and qualified scientific workforce is available to implement a program to recruit and train scientists with expertise in biodefense research, with a focus on foreign and emerging animal diseases, including dangerous zoonotic pathogens. The mechanism for training scientists in biodefense research will be established in collaboration with the guidance of the American Association of Veterinary Medical Colleges. ARS does not presently have high containment facilities (BSL-3E, BSL-3Ag, and BSL-4) to train biodefense research scientists in Manhattan, Kansas. (However, through collaboration with Kansas state University, BSL-3Ag laboratories are available.)Therefore, the research projects needed to obtain a doctoral degree in one of the seven core scientific disciplines listed in the previous section will be conducted at the Plum Island Animal Disease Center (PIADC), Orient Point, New York, and/or the research facilities of collaborators contributing to the implementation of the ARS biodefense research programs. Regarding Objective 1, progress was made in developing the workforce needed to staff the ARS research location in Manhattan, Kansas and to fulfill the mission to detect and control foreign animal diseases. A virologist has been trained at Auburn University, and has been working on the impact on antigenicity in vaccinated cattle, Bovine Viral Diarrhea virus (BVDV)-naïve steers. Comparative virus neutralization assays were performed in Madin-Darby bovine kidney (MDBK) cells to determine the anamnestic response to vaccination. A computer engineer has been hired and has developed a bioinformatic workflow on a local server for viral population genetic analysis. Regarding Objective 2, three new cooperative projects were established. The first will provide field and laboratory training to selected veterinary and post-graduate students in detection, surveillance, and characterization of emerging diseases in livestock and feral swine. The second will provide training to selected veterinary and post-graduate students in tick-borne disease research, specifically a bacterial pathogen causing Heartwater disease. The final project supports a transboundary animal disease course which recruits, provides initial training to, and exposure of potential new technical staff and graduate students. All these projects provide opportunities to attack, evaluate, and create enthusiasm in the research completed in Manhattan, Kansas, and other ARS Biosafety Level 3 research facilities. A post-doctoral researcher was hired to work in developing a feeding behavior library for Culicoides. ACCOMPLISHMENTS 01 Methods to better characterize feeding behavior for Culicoides. Detecting changes in feeding behavior of Culicoides is providing insight into virus transmission and pathogenicity and contribute to identify actions needed to break the transmission cycle with collaboration with ARS in Manhattan, Kansas. 02 Workforce development for National Bio- and Agro-Defense Facility. Two Virologist and a Bioinformaticians have been trained and are eligible to apply for positions at ARS in Manhattan, Kansas.

Impacts
(N/A)

Publications

Progress 10/01/20 to 09/30/21

Outputs
PROGRESS REPORT Objectives (from AD-416): Objective 1. Develop the workforce needed to staff NBAF and fulfill the Foreign Animal Disease Research Unit⿿s mission to detect and control foreign animal diseases. Resources will be provided for academic-related expenses and the research projects that will enable the trainees to successfully achieve the academic requirements for obtaining degrees in one of the seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. Objective 2. Implement research projects under the direction and guidance of ARS scientists at the Foreign Animal Disease Research Unit (FADRU), PIADC, Orient Point, New York, and others in collaboration with FADRU. Approach (from AD-416): Division A of the Consolidated Appropriations Act, 2017 (P.L. 115-31) contains an increase of $900,000 (NTL) for research on NBAF Workforce Development at the Center for Grain and Animal Health Research, in Manhattan, Kansas. The increased funds are to be used to establish a new ARS project, which will be held in the Office of the Center Director for the Center for Grain and Animal Health Research. There is a shortage of qualified scientists, including the availability of doctors of veterinary medicine (DVM) with a Ph.D degree, to conduct animal health research at the NBAF when the facilities become available in 2022. This will be addressed by specifically training scientists in the following seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. The objective and desired outcome is a mechanism to ensure a viable and qualified scientific workforce is available to implement a program to recruit and train scientists with expertise in biodefense research, with a focus on foreign and emerging animal diseases, including dangerous zoonotic pathogens. The mechanism for training scientists in biodefense research will be established in collaboration with the guidance of the American Association of Veterinary Medical Colleges. ARS does not presently have high containment facilities (BSL-3E, BSL-3Ag, and BSL-4) to train biodefense research scientists in Manhattan, Kansas. (However, through collaboration with Kansas state University, BSL-3Ag laboratories are available.)Therefore, the research projects needed to obtain a doctoral degree in one of the seven core scientific disciplines listed in the previous section will be conducted at the Plum Island Animal Disease Center (PIADC), Orient Point, New York, and/or the research facilities of collaborators contributing to the implementation of the ARS biodefense research programs. In regard to Objective 1, progress has been made in developing the workforce needed to staff the ARS location in Manhattan, Kansas, and fulfill the mission to detect and control foreign animal diseases. A computer engineer has been hired, and has developed a bioinformatic workflow on a local server for viral population genetic analysis. The engineer has obtained access and is adapting this workflow to be available to scientists through ARS Scientific Computer Initiative (SCINet). This will include a graphical user interface for easy end-user analysis. A post-doctoral associate has been hired and received training on the specialized equipment needed for the research. Additional accessories have been obtained to adapt the equipment to use with biting insects. Preliminary experiments have been initiated. The Doctor of Veterinary Medicine, PhD Candidate recently completed and passed her large animal internal medicine residency and board certification exam. She is now full-time in the PhD program at Auburn University and expects to complete her PhD in the next two years. One postdoctoral trainee was hired and trained for two years in recombinant African swine fever virus (ASFV) technology and sequencing of ASFV field isolates. One postdoctoral trainee was identified and received two years of specialized experience in bioinformatics and transcriptomic analyses of Foot-and mouth disease virus infection in cattle. In regard to Objective 2, three new cooperative projects were established. The first will provide field and laboratory training in detection, surveillance, and characterization of emerging diseases in livestock and feral swine. The second will provide training in tick-borne disease research, specifically a bacterial pathogen causing Heartwater disease. The final project supports a transboundary animal disease course which recruits, provides initial training and exposure of potential new technical staff and graduate students. All these projects provide opportunities to attack, evaluate, and create enthusiasm in the research completed in Manhattan, Kansas, and other ARS Biosafety Level 3 research facilities. A collaboration was established between College of Veterinary Medicine- Mississippi State University and ARS-USDA which enabled the data transfer and establishment of analytic pipelines for future collaborative endeavors. A collaboration was established with The Ministry of Agriculture Animal Industry and Fisheries, at Entebbe, Uganda. African swine fever virus (ASFV) samples were collected for sequencing of field isolates of ASFV. Record of Any Impact of Maximized Teleworking Requirement: Despite the SARS-CoV2 pandemic, this cooperative agreement made good progress in their specific objectives for FY2021. However, limited access to the laboratory during quarantines and lack of supplies due to supply- chain disruptions, caused some negative impact.

Impacts
(N/A)

Publications

Progress 10/01/19 to 09/30/20

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop the workforce needed to staff NBAF and fulfill the Foreign Animal Disease Research Unit⿿s mission to detect and control foreign animal diseases. Resources will be provided for academic-related expenses and the research projects that will enable the trainees to successfully achieve the academic requirements for obtaining degrees in one of the seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. Objective 2. Implement research projects under the direction and guidance of ARS scientists at the Foreign Animal Disease Research Unit (FADRU), PIADC, Orient Point, New York, and others in collaboration with FADRU. Approach (from AD-416): Division A of the Consolidated Appropriations Act, 2017 (P.L. 115-31) contains an increase of $900,000 (NTL) for research on NBAF Workforce Development at the Center for Grain and Animal Health Research, in Manhattan, Kansas. The increased funds are to be used to establish a new ARS project, which will be held in the Office of the Center Director for the Center for Grain and Animal Health Research. There is a shortage of qualified scientists, including the availability of doctors of veterinary medicine (DVM) with a Ph.D degree, to conduct animal health research at the NBAF when the facilities become available in 2022. This will be addressed by specifically training scientists in the following seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. The objective and desired outcome is a mechanism to ensure a viable and qualified scientific workforce is available to implement a program to recruit and train scientists with expertise in biodefense research, with a focus on foreign and emerging animal diseases, including dangerous zoonotic pathogens. The mechanism for training scientists in biodefense research will be established in collaboration with the guidance of the American Association of Veterinary Medical Colleges. ARS does not presently have high containment facilities (BSL-3E, BSL-3Ag, and BSL-4) to train biodefense research scientists in Manhattan, Kansas. (However, through collaboration with Kansas state University, BSL-3Ag laboratories are available.)Therefore, the research projects needed to obtain a doctoral degree in one of the seven core scientific disciplines listed in the previous section will be conducted at the Plum Island Animal Disease Center (PIADC), Orient Point, New York, and/or the research facilities of collaborators contributing to the implementation of the ARS biodefense research programs. In collaboration with ARS researchers at Manhattan, Kansas, and Kansas State University, the following new projects were initiated in fiscal year 2020: Related to Objective 1, a workshop was originally planned in person, however with COVID-19, the meeting was moved to a virtual platform and took place August 11th-12th 2020. Attendees participated in the following activities: breakout groups and polls, attendees heard from scientists about their work and their careers; learned about the National Bio and Agro-Defense Facility⿿s vision and culture from the director; and about Human Resources for Animal and Plant Health Inspection Service and ARS. Objective 2 is to develop a computational pipeline for analysis of genetic population diversity. This pipeline will be applicable across disciplines but will initially be focused on viral population genetic diversity selected by host source using datasets already planned to be developed in ARS research projects through the following goals: develop comparative next generations sequence datasets for Japanese Encephalitis Virus isolated from invertebrate and vertebrate cell lines and organisms; establish a computational pipeline to provide quantitative analysis of the genetic diversity between virus populations from various sources; and integrate the virus populations genetics into epidemiology models to provide insight on how viruses may evolve when introduced into new environments. Using transcriptomics and proteomics to understand arbovirus effects on midge sensory function with goals of assessing the effects of orbivirus infection on sensory perception in female Culicoides sonorensis and potential applications receiving training. The research associate, whose background should be in molecular biology and bioinformatics within any discipline, will receive training in virology, biting insect biology, insect micromanipulation techniques and insect behavioral assays. The techniques used and questions generated in this study can be applied to any vector-virus system, including vesicular stomatitis virus research and potentially bovine ephemeral fever virus, as well as research on Rift Valley Fever Virus and Japanese Encephalitis Virus interactions with mosquito vectors. Additionally, while the proposed project is focused on vector-virus interactions, these genetic and proteomic approaches can be used in a variety of other studies including vector-host, vector- environment and host-pathogen interactions. Therefore, this expertise has application across all disciplines of vector-borne disease research. Electrical Penetration Graph is a new technology for blood-feeding insects. This proposed research will establish this technology and will be utilized to determine the effects of different pathogens on the feeding behavior of Culex mosquitoes and Culicoides. Detecting changes in feeding behavior will provide insight into virus transmission and pathogenicity and will identify actions needed to break the transmission cycle. There are two main goals of this research. The first is understanding effects of virus infection in Culex feeding behavior, which include establishing the feeding behavior library for a Culex mosquito species, determining effects of Rift Valley Fever virus infection on feeding behavior of Culex mosquitoes, and determining effects of Japanese Encephalitis Virus infection on the feeding behavior of Culex mosquitoes. The second goal is to establish the feeding behavior of Culicoides, which includes a method of creating electrical circuit containing Culicoides and developing a feeding behavior library for Culicoides. Three new agreements were established with Kansas State University to develop expertise in arthropod-borne diseases. Accomplishments 01 Workforce development virtual symposium. A virtual symposium to bring together workforce development students across ARS and Animal and Plant Health Inspection Service was held August 11-12, 2020. The symposium brought together more than 50 participants, with 8 speakers, multiple breakout sessions, and a career panel of 6 specialists across the industry. Attendees received an overview of the mission, vision, culture and operation, of the facility. Attendees discussed best practices for mentorship and shared some of the scientists⿿ research projects.

Impacts
(N/A)

Publications

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop the workforce needed to staff NBAF and fulfill the Foreign Animal Disease Research Unit⿿s mission to detect and control foreign animal diseases. Resources will be provided for academic-related expenses and the research projects that will enable the trainees to successfully achieve the academic requirements for obtaining degrees in one of the seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. Objective 2. Implement research projects under the direction and guidance of ARS scientists at the Foreign Animal Disease Research Unit (FADRU), PIADC, Orient Point, New York, and others in collaboration with FADRU. Approach (from AD-416): Division A of the Consolidated Appropriations Act, 2017 (P.L. 115-31) contains an increase of $900,000 (NTL) for research on NBAF Workforce Development at the Center for Grain and Animal Health Research, in Manhattan, Kansas. The increased funds are to be used to establish a new ARS project, which will be held in the Office of the Center Director for the Center for Grain and Animal Health Research. There is a shortage of qualified scientists, including the availability of doctors of veterinary medicine (DVM) with a Ph.D degree, to conduct animal health research at the NBAF when the facilities become available in 2022. This will be addressed by specifically training scientists in the following seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. The objective and desired outcome is a mechanism to ensure a viable and qualified scientific workforce is available to implement a program to recruit and train scientists with expertise in biodefense research, with a focus on foreign and emerging animal diseases, including dangerous zoonotic pathogens. The mechanism for training scientists in biodefense research will be established in collaboration with the guidance of the American Association of Veterinary Medical Colleges. ARS does not presently have high containment facilities (BSL-3E, BSL-3Ag, and BSL-4) to train biodefense research scientists in Manhattan, Kansas. (However, through collaboration with Kansas state University, BSL-3Ag laboratories are available.)Therefore, the research projects needed to obtain a doctoral degree in one of the seven core scientific disciplines listed in the previous section will be conducted at the Plum Island Animal Disease Center (PIADC), Orient Point, New York, and/or the research facilities of collaborators contributing to the implementation of the ARS biodefense research programs. Dr. McVey, in collaboration with the National Programs Office, Plains Area Office, Atlantic Area Office, and PIADRU/ABADRU scientists will develop the execution plan for this project that will be in affect by September, 2017. It is expected that this will require agreements with multiple university partners. The agreements will define the operational methods and outcome expectations for training scientists capable of working on select agent animal diseases that are threats to U.S. animal agriculture. The following projects (initiated in FY17 and FY18) continue through Mississippi State University and Kansas State University as reported in FY18: A. Bioinformatics-based approaches to Identifying host mechanisms of Foot-and-Mouth Disease carrier state divergence B. Inferred antigenic emergence associated with Quasispecies Dynamics and Subconsensus Variants of FMDV C. Development and evaluation of a next generation ASFV live attenuated vaccine D. Mechanisms of host-specific virulence and vector-enhanced immunity to orbiviruses E. Research supporting workforce development for the National Bio-Agro Defense Facility: Virus-host interactions of Vesicular Stomatitis Virus F. Research supporting workforce development for the National Bio-Agro Defense Facility: Development of CSF subunit vaccines G. Research Supporting Workforce Development for the National Bio-Agro Defense Facility: Development of novel Foot-and-Mouth Disease virus candidates The following new projects were initiated in FY19: 1. Heterologous Flavivirus Infections of Swine and Cattle Specific Objectives: A. Continue relevant training through graduate courses and clinical case work that will allow scientist to complete the requirements for board- certification by the American College of Veterinary Internal Medicine and the Auburn University Graduate School for a doctoral degree in Biomedical Sciences B. Investigate the implications of heterologous BVDV infection on the viral genome following congenital infections in pigs, infectivity of BVDV for cattle, and antigenic changes for immune recognition in vaccinated cattle. a. Investigate whether viral genetic change allows clearance of BVDV in some congenitally infected piglets. b. Determine whether persistently or chronically infected (PI or CI) heterologous hosts can cause spill-back infections to cattle c. Evaluate antigenic changes of BVDV propagated in heterologous hosts during immune recognition in vaccinated cattle d. Determine the prevalence of BVDV and BVDV antibody in white-tailed deer and feral swine in Alabama e. Increasing the efficacy of the Ad5-FMD vaccine using a multi-gene delivery system 2. Increasing the efficacy of the Ad5-FMD vaccine using a multi-gene delivery system Objective: A replication-defective human adenovirus type 5 (Ad5) vectored vaccine that delivers Foot-and-Mouth Disease Virus (FMDV) capsid and capsid processing (3C protease) genes has been shown to provide full protection against FMDV challenge in swine and cattle. However, efforts are needed to improve the efficacy, and the genetic stability during large scale production with the ultimate goal of providing early and long-lasting protection against disease in a cost-effective manner. One approach to improve the efficacy of this vaccine is to increase the amount, stability and immunogenicity of the delivered antigen. The 3C protease strictly necessary for antigen processing in the Ad5-FMD vaccine has been shown to be toxic presumably due to nonspecific targeting of cellular proteins during vector production. Mutations in the FMDV 3C coding region will be incorporated to improve vector yield during vaccine production, and at the same time increase express higher amounts of antigen after vaccination. It is expected that vectors containing this mutation will provide a stronger immune response. ARS, PIADC has recently designed new Ad5 vectors that presumably display increased genetic stability by modifying genomic sequences in the vector that are also present in the cell line used for vaccine propagation. These vectors will be used to express the FMD type O cassette including the identified 3C mutation. Viruses will be characterized in vitro for expression of empty capsids and genetic stability. Candidate vectors will be selected for in vivo potency/ efficacy studies in the FMDV natural host, swine and/ or cattle. SPECIFIC OBJECTIVES 1. Evaluation of an Ad5-FMD type O vaccine containing 3C identified L127P mutation. 2. Evaluation of an Ad5-FMD vaccine that delivers simultaneously FMDV antigens and a biotherapeutic/ adjuvant. 3. Epidemiology Workforce Development Goal: This research project is aimed at developing capacity on applied epidemiology to contribute to the workforce of the National Bio- and Agro- Defense Facility (NBAF) in Manhattan, Kansas. Specific desired areas of expertise include study design, data analysis, interpretation of results, and recommendation of management practices and policy, for the ARS with a focus on FMD and /or ASF. The strategic integration of ARS research programs with epidemiology research will be paramount for the development of effective biosecurity and disease control programs for those diseases. Objectives: 1. To support workforce development for the FADRU/ARS in the transition from PIADC to NBAF by training two PhD students/post docs that could eventually be eligible for a position with the ARS on applied epidemiology of ASF and FMD, respectively, using data of interest for the ARS-collected in South/South East Asia, Africa, and any other endemic region of interest, or experimental data generated at the ARS FADRU laboratory. 2. To produce scientific evidence of the training in the form of presentation to scientific meetings and publications. Accomplishments 01 ARS workforce development. In FY19, ARS created Workforce Development training agreements with the Auburn University, the University of Connecticut and the University of Minnesota. All three Universities are new NBAF training partners with ARS. These agreements will support 4 new (FY19) trainees in immunology/vaccinology, epidemiology and disease pathogenesis. There are seven trainees currently in place at Mississippi State University and Kansas State University (agreements established in FY17 and FY18). ARS scientists are collaboratively involved in all of these research training projects. ARS will hold a research symposium for the workforce development trainees, and their USDA and University mentors in Manhattan, Kansas, on August 27, 2019.

Impacts
(N/A)

Publications

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop the workforce needed to staff NBAF and fulfill the Foreign Animal Disease Research Unit�s mission to detect and control foreign animal diseases. Resources will be provided for academic-related expenses and the research projects that will enable the trainees to successfully achieve the academic requirements for obtaining degrees in one of the seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. Objective 2. Implement research projects under the direction and guidance of ARS scientists at the Foreign Animal Disease Research Unit (FADRU), PIADC, Orient Point, New York, and others in collaboration with FADRU. Approach (from AD-416): Division A of the Consolidated Appropriations Act, 2017 (P.L. 115-31) contains an increase of $900,000 (NTL) for research on NBAF Workforce Development at the Center for Grain and Animal Health Research, in Manhattan, Kansas. The increased funds are to be used to establish a new ARS project, which will be held in the Office of the Center Director for the Center for Grain and Animal Health Research. There is a shortage of qualified scientists, including the availability of doctors of veterinary medicine (DVM) with a Ph.D degree, to conduct animal health research at the NBAF when the facilities become available in 2022. This will be addressed by specifically training scientists in the following seven core scientific disciplines: pathology, virology, immunology, entomology, epidemiology, microbiology, and computational biology. The objective and desired outcome is a mechanism to ensure a viable and qualified scientific workforce is available to implement a program to recruit and train scientists with expertise in biodefense research, with a focus on foreign and emerging animal diseases, including dangerous zoonotic pathogens. The mechanism for training scientists in biodefense research will be established in collaboration with the guidance of the American Association of Veterinary Medical Colleges. ARS does not presently have high containment facilities (BSL-3E, BSL-3Ag, and BSL-4) to train biodefense research scientists in Manhattan, Kansas. (However, through collaboration with Kansas state University, BSL-3Ag laboratories are available.)Therefore, the research projects needed to obtain a doctoral degree in one of the seven core scientific disciplines listed in the previous section will be conducted at the Plum Island Animal Disease Center (PIADC), Orient Point, New York, and/or the research facilities of collaborators contributing to the implementation of the ARS biodefense research programs. Dr. McVey, in collaboration with the National Programs Office, Plains Area Office, Atlantic Area Office, and PIADRU/ABADRU scientists will develop the execution plan for this project that will be in affect by September, 2017. It is expected that this will require agreements with multiple university partners. The agreements will define the operational methods and outcome expectations for training scientists capable of working on select agent animal diseases that are threats to U.S. animal agriculture. The project team is evaluating multiple potential projects at multiple universities for inclusion in this project. The research projects listed below have been initiated at Mississippi State University to achieve these objectives: 1. Mechanisms of host-specific virulence and vector-enhanced immunity to orbiviruses. Need for Research: Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are animal orbivirus pathogens that cause noncontagious, insect-transmitted, hemorrhagic disease in domestic and wild ruminants world-wide (1-3). Severe economic losses from outbreaks result from effects on animal production and non-tariff trade restrictions on the sale and movement of animals and animal products. Losses to U.S. livestock industries attributed to BTV and EHDV have been conservatively estimated at $144 million annually (4). These viruses are of growing concern to livestock producers in North America due to 1) the emergence of new viral serotypes, 2) increased reports of spillover and clinical disease in cattle, and 3) increased spread and adaptation to new geographic areas. Although closely related and sharing distinctive common properties such as segmented double stranded RNA (dsRNA) genomes and characteristic non- structural proteins, BTV and EHDV cause disease in different specific hosts, primarily in sheep and white-tailed deer (WTD), respectively (5, 6) . Recent progress in reverse genetics, viral sequencing, and in vitro and in vivo models have improved our understanding of orbiviral pathogenesis. However, the molecular mechanisms that determine orbiviral host specificity, the viral-insect interactions affecting virulence and clinical outcome, and the viral and insect factors which modulate protective innate and adaptive immune responses against orbiviruses remain elusive. Previous studies demonstrated that BTV capsid proteins VP2 and VP5 are involved in cell attachment and viral penetration into mammalian host cells, and neutralizing antibodies to VP2 and VP5 confer protective immunity (7, 8). Yet the molecular targets that determine host specificity are unknown. Toward this research need, we propose to identify molecular determinants of orbiviral capsid proteins that confer host susceptibility. (Obj. 1.) Unlike natural, midge-transmitted BTV infections which can result in 30- 50% mortality, routine experimental infection of sheep by inoculation only rarely produces clinical disease beyond a transient fever and mild clinical signs depending on the infectious dose, inoculation route, and the BTV serotype used. It has been hypothesized that this difference is due to intradermal vs. subcutaneous routes of virus delivery, as well as interactions of the vector�s saliva with either the virus, the animal�s immune responses, or with both. Such �vector-enhanced� transmission and pathogenesis has been reported for several arboviruses (9-11). Ongoing vector-host research with Culicoides midges and mice suggests the saliva deposited into the animal�s dermal layer during blood feeding elicits a very strong innate immune response which likely affects orbiviral infection. Additionally, Culicoides saliva contains proteins such as D7, protease inhibitors, and maltase which can induce hypersensitivity responses and may also affect orbiviral infection (12, 13). Understanding this vector-elicited response, the molecular determinants which confer host susceptibility, and the potential insect factors that may modulate orbiviral virulence and transmissibility, are critical to informing more efficacious vaccine strategies and developing potential methods for interrupting vector-borne transmission. Toward this research need, we propose to determine the effect of insect salivary proteins on orbiviral immunity in a natural host. (Obj. 2.) 2. Development and evaluation of a next generation ASFV live attenuated vaccine. Project objectives and Deliverables. Objective 1) Develop and assess next generation candidate ASFV vaccines using bioinformatics to determine additional genetic determinants of virulence in ASFV. (75% of Postdoctoral effort). Deliverable: a new vaccine candidate for ASF, incorporating an additional deletion to ASFV-G-delta9GLdeltaUK. Objective 2) Assess the host response to candidate ASFV vaccine by utilizing NGS technology to determine the differences in RNA expression profile of host target cells (15% of Postdoctoral effort). Deliverable: RNAseq analysis of host response in swine macrophages to new PIADC ASFV vaccine. Objective 3) Assess current circulating ASFV strains in Uganda by obtaining and sequencing by NGS field isolates from Uganda. (10% of Postdoctoral effort). Deliverable: Sequence of circulating ASFV strains in Uganda. 3. Inferred antigenic emergence associated with Quasispecies Dynamics and Subconsensus Variants of FMDV Development of effective, next-generation vaccines against exotic viruses that threaten U.S. livestock depends upon understanding of the mechanisms through which viruses evolve to evade host immunity. Specifically, viruses may elude vaccine-induced immunity by extremely rapid genomic mutation leading to antigenic variation. Recent work within the ARS Foreign Animal Disease Research Unit (FADRU) in Orient Point, New York, has characterized genomic substitutions in foot-and-mouth disease virus (FMDV) during distinct phases of infection in cattle. However, additional analyses are required to elucidate how these genomic changes affect FMDV antigenic structure and function. Scientists at Mississippi State University (MSU) have demonstrated expertise in computational approaches to correlate alterations of viral structural proteins with underlying genomic variability. The intention of this project will be to utilize existing next generation sequencing data from completed FMDV pathogenesis experiments to conduct novel analyses aimed at elucidating viral quasispecies dynamics and antigenic correlations. More specifically, FMDV evolution including genomic substitution and antigenic variation will be characterized throughout distinct stages of infection including the acute, transitional, and carrier phases. The execution of the project will include recruitment and training of a postdoctoral fellow who will be trained with the intention of future staffing of the National Bio- and Agro-Defense Facility (NBAF) in Manhattan Kansas. 4. Bioinformatics-based approaches to Identifying host mechanisms of Foot- and-Mouth Disease carrier state divergence. The development of the next-generations of vaccines against viruses that threaten U.S. agriculture, such as foot-and-mouth disease virus (FMDV), will depend upon understanding and enhancing the mechanisms of effective anti-viral immunity. Specifically, elucidating the host-mechanisms which determine clearance of infection under natural conditions may indicate critical pathways which may be exploited by novel countermeasures. Recent work within the ARS Foreign Animal Disease Research Unit (FADRU) in Orient Point, New York, has used microarray-based transcriptomic analyses to characterize host mechanisms of immunity to FMDV in cattle during distinct phases of infection. However, additional analyses are required to more thoroughly elucidate the relevant host pathways. Scientists at Mississippi State University (MSU) have demonstrated expertise in developing computational resources that improve the functional information of bovine genes such as addition of Gene Ontology, prediction of genes involved in host-pathogen interactions, etc. The intention of this project will be to utilize existing microarray data from completed FMDV pathogenesis experiments to conduct novel analyses aimed at elucidating endogenous and vaccine-induced mechanisms of anti-FMDV immunity in cattle. More specifically, we will utilize a number of open source bioinformatics tools including some developed at Mississippi State University for enhanced understanding of the functions and pathways in bovine immune mechanisms that contribute to carrier state versus clearance of FMDV. The execution of the project will include recruitment and training of a graduate student who will be trained with the intention of future staffing of the National Bio- and Agro-Defense Facility (NBAF) in Manhattan Kansas. Accomplishments 01 Mechanisms of host-specific virulence and vector-enhanced immunity to orbiviruses. Project planning has been completed between Mississippi State University (MSU) and Foreign Animal Disease Reserach Unit (FADRU) scientists. Recruitment has been initiated for the project trainee. 02 Development and evaluation of a next generation ASFV live attenuated vaccine. Project planning has been completed between Mississippi State University (MSU) and Foreign Animal Disease Reserach Unit (FADRU) scientists. A post-doctoral student has been hired and work has been initiated. 03 Inferred antigenic emergence associated with Quasispecies Dynamics and Subconsensus Variants. Project planning has been completed between Mississippi State University (MSU) and Foreign Animal Disease Reserach Unit (FADRU) scientists. Recruitment has been initiated for the project trainee. 04 Bioinformatics-based approaches to Identifying host mechanisms of Foot- and-Mouth Disease carrier. Project planning has been completed between Mississippi State University (MSU) and Foreign Animal Disease Reserach Unit (FADRU) scientists. Recruitment has been initiated for the project trainee.

Impacts
(N/A)

Publications