Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protectiveimmunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections, developing vaccine formulations and delivery targeting the mucosal immune responses. Development of novel FMD vaccine platforms. Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. Development of improved second-generation Ad5-FMD vaccines. Discovery of modified live attenuated FMDV vaccine candidates (MLAV). Discovery of cross-protective vaccines against multiple FMDV subtypes, and develop vaccine formulations. Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. Discovery/ development of novel biotherapeutics with increased potency and extended systemic half-life. Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. Evaluation of vaccine-induced immunity and FMDV carrier state. Characterization of host immunity associated with novel vaccines against FMDV. Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) . Elucidate the host-pathogen interactions of FMDV, including identifying viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions at the primary sites of infection in ruminants and pigs with focus on factors defining tropism and early host responses, and determining characteristics and mechanisms of FMDV within-hot evolution over distinct phases of infection. Determine the molecular basis for FMDV-host interactions that impact virulence. Examination of virus factors contributing to FMDV virulence. Examination of host factors contributing to FMDV virulence. Identification of molecular mechanisms associated with the establishment of FMDV persistence. Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. Investigation of within-host FMDV genomic evolution to characterize sitespecific mutational pressure, genomic variation, and potential adaptation to the host. Determination of the immune mechanisms affecting protective immunity against FMDV. Analysis of CD4 helper T-cell response to FMDV vaccination. Analysis of CD8 cytotoxic T-cell response to FMDV vaccination. Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, characterizing risk factors driving FMDV emergence and spread. Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings. Approach (from AD-416): 1. The development of intervention strategies to control and eradicate FMDV will be achieved through research on novel FMD vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates (e.g. FMDV-LL3B3D), second⿿generation Ad5-FMD vaccines, and cross-protective vaccines against multiple subtypes. Additionally, combinations of vaccine and biotherapeutics / and or adjuvants will be investigated as a way to induce mucosal immunity necessary not only to prevent disease but also to decrease persistent infection. These vaccine/ adjuvant formulations will be tested using alternate routes such as transdermal and by direct mucosal delivery. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. In addition, the interactions of the virus with specific tissues at the primary infection sites will be studied by characterizing infected tissues at the cellular and subcellular level as well as utilizing transcriptomic analyses with micro arrays and next generation RNAseq. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. Despite the challenges of the COVID pandemic, restricted access to the laboratory, and suspension of animal experiments for most of FY21, the Foot and Mouth Disease (FMD) research team achieved many of the project milestones for the year. At the same time, dedicated significant resources to multiple activities toward the National Bio and Agro-Defense Facility (NBAF) transition. This included the establishment of a biorepository task-force to help with biorepository inventories, large scale destruction of biorepository samples not going to NBAF, the establishment of a new laboratory with diagnostic capabilities for safety and freedom testing of samples, and initiation of the large scale scanning of tens of thousands of pages of laboratory records for transition to the NBAF. Under Objective 1: ⿿Develop intervention strategies to control and eradicate Foot-and-Mouth Disease Virus (FMDV), Subobjective 1.1. Development of novel FMD vaccine platforms, there were important achievements. In collaboration with Industry, under a Cooperative Research Development Agreement (CRADA) partnership, progress continued toward the development of FMDV3B3D inactivated vaccines and small-scale production of pre-Master Seed. A number of relevant vaccine strains for several serotypes have been synthesized by the commercial partner, and virus stocks have been successfully produced at PIADC. A production facility on the U.S. mainland was built by our CRADA partner, where master stocks of the main serotypes will be produced in the coming year. Additionally, using the FMDLL3B3D platform, vaccines with mosaic capsids were produced against serotypes O and Asia and successfully tested (O only) for protection against heterologous challenge. These proof-of-concept experiments served as support toward a patent application for this technology. Work has continued on exploring modified live vaccine (MLV) candidates. Testing a multifactorial mutant MLV candidate that demonstrated attenuated characteristics in vitro displayed pathogenic characteristics similar to a parental virus in swine. Similarly, MLV candidates created using codon-deoptimization showed attenuated phenotype in mice but were still virulent in pigs. This work demonstrated that MLV for FMD is still a great challenge. Under Subobjective 1.2, ⿿Development of novel biotherapeutics, important progress was made in the use of pegylation to prolong the antiviral effect of IFN against FMDV. This work, part of a CRADA, demonstrated that pegylated recombinant porcine (po)IFNa displays strong and long-lasting antiviral activity against FMDV in vitro and in vivo, completely protecting swine against FMD for at least five days after a single dose. This opens the possibility of formulating vaccines with pegylated IFNs as an emergency control approach. In Objective 2, host-pathogen interactions, key molecular mechanisms of disease involving the leader protease (Lpro) were discovered specifically regarding novel activities of this viral protein Using protein structural analysis of FMDV Lpro, amino acids associated to enzymatic activity (deUbuquitinase/deISGylase activity) were identified. Mutation of these amino acid residues in FMDV resulted in a perceptible level of attenuation in vitro and in vivo, opening possibilities to apply in MLV development. In Subobjective 2.2, research focused on studying virus evolution during persistence and particularly simulating viral coinfections as have been observed in field studies. The dynamics of infection and the predominance of different viral serotypes or subtypes at different phases of infection opened the door to understanding the emergence of novel viruses under natural conditions. Most importantly, it was demonstrated that during dual infection of cattle with 2 distinct strains, recombination of the viral genomes occurs with high frequency during the first 30 days of infection. Viral ecology studies (Objective 3) were completed in various countries in Asia and Africa, showing various patterns of virus distribution both in subclinical and in clinical infections. These patterns demonstrated the occurrence of viral ⿿waves that went across borders both in Asia and in Africa, many times initiating in areas with wildlife reservoirs. It was demonstrated that these waves of viral spread occur much more frequently than previously recognized in association with asymptomatic infections. Studies in Uganda quantified FMD dispersal directionality in cattle allowing them to anticipate viral movement and more strategically tailor intervention strategies that ultimately support FMD control in East African. In addition to our existing project milestones, research was carried out aimed at solving stakeholder problems. One example is a study done on the transmissibility of FMDV in pigs by contaminated feed. These studies demonstrated that although transmission did occur, viral serotype was a major determinant of the success and level of transmission. This data will contribute to the modelling of FMD transmission and might inform regulatory authorities on the risk of FMD from feed imports. A second study was undertaken to create an adventitious agent-free cell line with high susceptibility to all serotypes of FMDV. This new clonal cell line (MGPK) was patented and is a promising cell line to produce FMD vaccines, especially for serotypes that are difficult to adapt to production cell lines. ACCOMPLISHMENTS 01 Developing the capacity for FMD vaccine production in the United States. Foot and mouth disease FMD continues to be a major threat to U.S. agriculture. An FMD outbreak would have devastating consequences on the $1.11 trillion food and agricultural Industry. The U.S. invests millions of dollars in strategic vaccine banks for the most prevalent FMD viruses circulating around the world. However, all vaccines need to be purchased overseas as domestic vaccine production requires live virus, which is forbidden by law in the US ARS scientists developed a platform that allows safe vaccine production in the U.S. Working closely with a commercial vaccine manufacturer, the new vaccine is now approved for research and development and eventually manufacturing in the U.S. mainland, at a new multi-million-dollar facility built by the CRADA partner for this purpose. This achievement was highlighted in recent press reports (https://www.thepigsite.com/news/2021/01/usda-backs-production-of-domestic-fmd-vaccine). 02 Foot-and-Mouth Disease virus be effectively transmitted by contaminated feed. The emergence of porcine epidemic diarrhea virus (PEDV), believed to have been accidentally imported in pig feed products from China, has increased the concern of pork industry stakeholder groups regarding the potential importation of pathogens (e,g, Foot and Mouth Disease Virus- FMDV) through contaminated feed products. At the request of stakeholders, ARS researchers demonstrated the ability of contaminated feed to infect pigs through natural ingestion even 30 days after feed contamination (duration of transit from Asia). These findings are critically important to understand the risks and mitigation methods to prevent the introduction of FMDV in imported animal feed products. Work was published in Transboundary and Emerging Diseases in 2021: https://onlinelibrary.wiley.com/doi/epdf/10.1111/tbed.14230. 03 Immediate and sustained protection against Foot and Mouth Disease (FMD). Current FMD vaccines require 5 days to induce enough immunity to prevent viral spread. The time between vaccination and protection can be critical in the emergency response to an outbreak. Interferons (IFN), produced as the first line of defense against viruses, can induce almost immediate protection against FMD but usually have a short life once injected in the animals and do not protect long enough for the onset of vaccine immunity. ARS researched demonstrated that a modified (pegylated) porcine IFN induces a strong and long-lasting antiviral activity against FMDV, completely protecting swine for at least five days after injection. This discovery opens the door for testing the pegylated INF, combined with the vaccine to achieve immediate and sustained protection to control outbreaks of FMD and potentially other viral diseases. Use of Protein Pegylation to Prolong the Antiviral Effect of INF Against FMDV. (Front. Microbiol., 05 May 2021 https://doi.org/10.3389/fmicb.2021.668890).
Impacts
(N/A)
Publications
- Munsey, A., Mwiine, F., Ochwo, S., Velazquez Salinas, L., Ahmed, Z., Maree, F., Rodriguez, L.L., Rieder, A.E., Perez, A., Vanderwaall, K., Dellicour, S. 2021. Tracking the dispersal of foot-and-mouth disease virus serotype O in East Africa using phylodynamics. Molecular Ecology. https://doi.org/10. 1111/mec.15991.
- Diaz San Segundo, F.C., Medina, G.N., Mogulothu, A., Azzinaro, P.A., Attreed, S.E., Gutkoska, J.R., Lombardi, K.R., Shields, J., Hudock, T.A., De Los Santos, T.B. 2021. Use of Protein Pegylation to prolong the antiviral effect of IFN against FMDV. Frontiers in Microbiology. https:// doi.org/10.3389/fmicb.2021.668890.
- Hardham, J., Krug, P., Pacheco, J., Thompson, J., Domanowski, P., Gay, C.G. , Rodriguez, L.L., Rieder, A.E. 2020. Novel Foot-and-Mouth disease vaccine platform: Formulations for safe and DIVA-compatible FMD vaccines with improved potency. Frontiers in Veterinary Science. https://doi.org/https:// doi.org/10.3389/fvets.2020.554305.
- Medina, G., De Los Santos, T.B., Diaz San Segundo, F.C. 2020. Use of IFN- based biotherapeutics to harness the host against foot-and-mouth disease. Frontiers in Microbiology. https://doi.org/https://doi.org/10.3389/fvets. 2020.00465.
- Ramierez-Medina, E., Vuono, E., Rai, A., Pruitt, S.E., Silva, E., Velazquez-Salinas, L., Zhu, J.J., Gladue, D.P., Borca, M.V. 2020. Evaluation in swine of a recombinant African swine fever virus lacking the MGF-360-1L gene. Viruses. https://doi.org/10.3390/v12101193.
- Kloc, A., Rai, D.K., Kenney, M.A., Schafer, E.A., Rieder, A.E., Gladue, D. 2020. Residues within the foot-and-mouth disease virus 3dpol nuclear localization signal affect polymerase fidelity. Journal of Virology. https://doi.org/10.1128/JVI.00833-20.
- Chitray, M., Opperman, P., Rotherham, L., Fehrsen, J., Frischmuth, J., Rieder, A.E., Maree, F. 2020. Diagnostic and epitope mapping potential of single-chain antibody fragments against foot-and-mouth disease serotype A, SAT1 and SAT3 viruses. Frontiers in Veterinary Science. https://doi.org/10. 3389/fvets.2020.00475.
- Diaz San Segundo, F.C., Medina, G., Spinard, E., Kloc, A., Ramirez-Medina, E., Azzinaro, P.A., Mueller, S., Rieder, A.E., De Los Santos, T.B. 2021. Use of synonymous deoptimization for the development of modified live attenuated strains of foot and mouth disease virus. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.610286.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protectiveimmunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections, developing vaccine formulations and delivery targeting the mucosal immune responses. Development of novel FMD vaccine platforms. Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. Development of improved second-generation Ad5-FMD vaccines. Discovery of modified live attenuated FMDV vaccine candidates (MLAV). Discovery of cross-protective vaccines against multiple FMDV subtypes, and develop vaccine formulations. Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. Discovery/ development of novel biotherapeutics with increased potency and extended systemic half-life. Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. Evaluation of vaccine-induced immunity and FMDV carrier state. Characterization of host immunity associated with novel vaccines against FMDV. Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) . Elucidate the host-pathogen interactions of FMDV, including identifying viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions at the primary sites of infection in ruminants and pigs with focus on factors defining tropism and early host responses, and determining characteristics and mechanisms of FMDV within-hot evolution over distinct phases of infection. Determine the molecular basis for FMDV-host interactions that impact virulence. Examination of virus factors contributing to FMDV virulence. Examination of host factors contributing to FMDV virulence. Identification of molecular mechanisms associated with the establishment of FMDV persistence. Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. Investigation of within-host FMDV genomic evolution to characterize sitespecific mutational pressure, genomic variation, and potential adaptation to the host. Determination of the immune mechanisms affecting protective immunity against FMDV. Analysis of CD4 helper T-cell response to FMDV vaccination. Analysis of CD8 cytotoxic T-cell response to FMDV vaccination. Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, characterizing risk factors driving FMDV emergence and spread. Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings. Approach (from AD-416): 1. The development of intervention strategies to control and eradicate FMDV will be achieved through research on novel FMD vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates (e.g. FMDV-LL3B3D), second⿿generation Ad5-FMD vaccines, and cross-protective vaccines against multiple subtypes. Additionally, combinations of vaccine and biotherapeutics / and or adjuvants will be investigated as a way to induce mucosal immunity necessary not only to prevent disease but also to decrease persistent infection. These vaccine/ adjuvant formulations will be tested using alternate routes such as transdermal and by direct mucosal delivery. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. In addition, the interactions of the virus with specific tissues at the primary infection sites will be studied by characterizing infected tissues at the cellular and subcellular level as well as utilizing transcriptomic analyses with micro arrays and next generation RNAseq. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. Under Objective 1 (countermeasures), progress continued toward the development of FMDV3B3D inactivated vaccines through a Cooperative Research and Development Agreement (CRADA) partnership. A significant number of serotype A, O, C, Asia and Sat2 capsid constructs have been synthesized by the commercial partner and virus stocks have been successfully produced at Plum Island Animal Disease Center (PIADC). Progress has been made to establish a production facility on the U.S. mainland where master stocks of the main serotypes will be produced in the upcoming year. This will allow further development of leaderless vaccines. Work has continued on exploring modified live vaccines. In collaboration with a commercial partner, ARS scientists have been able to expand previous work using codon deoptimization on FMDV A serotype, to other subtypes/serotypes (A24, O1 Campos and Asia1). Testing in mice and swine demonstrated that mutant viruses were attenuated and induced a neutralizing antibody response. This work highlighted the use of this technology to generate attenuated strains that could be used toward the development of live attenuated or inactivated vaccine candidates. A provisional patent was filed SN: 63/030,431. In collaboration with industry partners, ARS scientists evaluated the efficacy of modified porcine interferon molecules for their capacity of providing long lasting bioavailability and efficacy in swine. This study highlighted the benefit of using a combination of this molecule with vaccines to provide immediate and long-term protection against FMD in swine. This work was performed under Material Transfer and Research Agreement (MTRA) 8064-32000-061-46H. Under Objective 2 (virus-host interactions), substantial progress was made in understanding the establishment and maintenance of the carrier state in cattle. Specifically, towards elucidating the evolution and strain emergence of FMDV during acute and persistent infection was made through a variety of experimental approaches. Highly complex in vivo experiments generated samples that were analyzed by a combination of different techniques including next generation genetic sequencing, immune- microscopy, and conventional molecular and cellular virological analyses. These approaches provided insights on the mechanisms of virus maintenance and emergence. Co-infection of cattle with distinct viral strains demonstrated that viruses may recombine within the upper respiratory tract to generate new, genetically distinct emergent viruses. Additionally, critical new insights were achieved regarding cattle gene expression during the carrier state and these results were published in several peer-reviewed publications. Progress continued in the host-response to FMDV infection. A new bovine chemokine gene named as CXCL15 was identified and annotated. The expression of this gene was significantly downregulated in FMDV carriers compared to non-carriers. The differential gene expression between carriers and non-carriers including CXCL15 detected with whole genome expression profiling in the tissues of FMDV persistent infection has led to a major progress in understanding the molecular mechanisms that could explain why carriers cannot clear the virus and vaccine cannot cure the persistent infection. The potency of an interferon biotherapeutic has been improved by approximately 20 times based on the test results in pigs, which invention has been patented. These new discoveries provide insightful information and a useful tool for FMD control. Great progress was made toward the understanding of a novel function of the FMDV Leader protein to block the innate immune response. As part of NBAF workforce development, ARS scientists discovered a novel separation of function mutation in the FMDV leader protein that uncouples two specific enzymatic activities. Impairment of novel deISGylase activity (cleavage of ISG15 from target proteins), rendered viable FMDV strains that were significantly attenuated in vitro and in vivo. In addition, ARS scientists demonstrated a novel role of ISG15 to inhibit FMDV replication. A combination of this mutation with others could be applied towards development of effective live attenuated vaccines. Furthermore, these studies highlight the potential of using ISG15 as an antiviral or vaccine adjuvant to control FMD. This work was performed under agreement 8064- 32000-061-54S with Kansas State University for the National Bio and Agro Defense Facility (NBAF) work force development program. Two manuscripts were published (logs 370339 and 369287). Progress was made in discovering a new region in the FMDV 3D protein (polymerase enzyme) important for virus replication and polymerase fidelity. It has been previously reported that the 3D enzyme encodes a sequence motif important for import of the protein to host nucleus. We identified mutations in the viral protein 3D responsible for viral replication that affect the accuracy of the enzyme during viral replication (i.e. fidelity) resulting in decreased virus growth in host cells. The alteration of 3D fidelity described uncovers a novel strategy to derive safer and more stable attenuated FMDV vaccine candidates. In response to stakeholder requests (Animal and Plant Health Service APHIS and National Pork Board) a series of FMDV experiments were carried in pigs to contribute novel scientific data of critical value for epidemiological modeling of FMD outbreaks affecting pig holdings. Specifically, the minimum infectious dose of FMDV required to cause disease in pigs exposed to FMDV-contaminated feed was established. Additionally, the duration of infectiousness of FMDV-infected live pigs, and the extent of contagion associated with carcasses from deceased FMDV- infected pigs were established and published in a peer-reviewed journals. Under Objective 3 (epidemiology and ecology), field collaborative investigations were continued in Africa and Asia. Important output included a novel, modeled meta-analysis which provided systems to determine the duration of the carrier state under differing endemic conditions. This approach demonstrated that 12 months after an outbreak of FMD, up to 51% of carrier animals may remain infected. These studies also led to discovery and report of full-length sequences of genetically unique virus strains in Vietnam, India, Kenya, and Uganda which are now available to the global research community. Important insights were achieved regarding multi-strain co-infections and interactions between viruses in cattle and wild buffalo in Kenya. Specifically, it was found that viruses are transmitted across species, yet those events occur infrequently. Additionally, we concluded a five-year project in Uganda and documenting the distribution of FMDV-exposed cattle and examining risk factors associated with the disease. As limited resources prevent routine mass vaccination, the information generated will aid on the improvement of FMD control plans for Uganda, and ultimately, support the control and eradication of the disease in the Eastern African region. Accomplishments 01 New Foot-and-Mouth Disease (FMD) vaccine candidates. Although inactivated antigen vaccines are available, they induce only a narrow and short-lived protective immunity, whereas live-attenuated vaccines induces broader or longer-lived immunity. ARS scientists in Orient, New York, in collaboration with scientists at Codagenix Inc, Farmingdale, New York, evaluated the use of codon-deoptimization to produce FMD vaccine candidates. These vaccine candidates of multiple FMD vaccine serotypes, showed promising results in vaccinated pigs. These results highlight the potential of these novel approaches toward the development of live vaccine candidates that could be used in endemic areas towards disease eradication. A patent was filed (Docket Number 137.18) for this invention.
Impacts
(N/A)
Publications
- Medina, G., Azzinaro, P.A., Ramirez-Medina, E., Gutkoska, J.R., Fang, De Los Santos, T.B. 2020. Impairment of the deISGylation activity of foot-and- mouth disease Lpro causes viral attenuation without affecting interferon expression during viral infection. Virology.
- Eschbaumer, M., Dill, V., Carlson, J.C., Arzt, J., Stenfeldt, C., Krug, P., Hardham, J., Stegner, J.E., Rodriguez, L.L., Rieder, A.E. 2020. Foot-and- Mouth disease virus lacking the leader protein and containing two negative DIVA markers (FMDV A24 LL3B3D) is fully attenuated in pigs. Pathogens.
- Yadav, S., Stenfeldt, C., Branan, M.A., Moreno-Torres, K.I., Holmstrom, L., Delgado, A.H., Arzt, J. 2019. Parameterization of the durations of phases of foot-and-mouth disease in cattle. Frontiers in Veterinary Science.
- Mwiine, F.N., Velazquez-Salinas, L., Ahmed, Z., Ochwo, S., Munsey, A., Kenney, M.A., Lutwama, J.J., Maree, F.F., Lobel, L., Perez, A.M., Rodriguez, L.L., Vanderwaal, K., Rieder, A.E. 2019. Serological and phylogenetic characterization of foot and mouth disease viruses from Uganda during cross sectional surveillance study in cattle between 2014 and 2017. Transboundary and Emerging Diseases.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protectiveimmunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections, developing vaccine formulations and delivery targeting the mucosal immune responses. Development of novel FMD vaccine platforms. Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. Development of improved second-generation Ad5-FMD vaccines. Discovery of modified live attenuated FMDV vaccine candidates (MLAV). Discovery of cross-protective vaccines against multiple FMDV subtypes, and develop vaccine formulations. Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. Discovery/ development of novel biotherapeutics with increased potency and extended systemic half-life. Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. Evaluation of vaccine-induced immunity and FMDV carrier state. Characterization of host immunity associated with novel vaccines against FMDV. Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) . Elucidate the host-pathogen interactions of FMDV, including identifying viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions at the primary sites of infection in ruminants and pigs with focus on factors defining tropism and early host responses, and determining characteristics and mechanisms of FMDV within-hot evolution over distinct phases of infection. Determine the molecular basis for FMDV-host interactions that impact virulence. Examination of virus factors contributing to FMDV virulence. Examination of host factors contributing to FMDV virulence. Identification of molecular mechanisms associated with the establishment of FMDV persistence. Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. Investigation of within-host FMDV genomic evolution to characterize sitespecific mutational pressure, genomic variation, and potential adaptation to the host. Determination of the immune mechanisms affecting protective immunity against FMDV. Analysis of CD4 helper T-cell response to FMDV vaccination. Analysis of CD8 cytotoxic T-cell response to FMDV vaccination. Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, characterizing risk factors driving FMDV emergence and spread. Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings. Approach (from AD-416): 1. The development of intervention strategies to control and eradicate FMDV will be achieved through research on novel FMD vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates (e.g. FMDV-LL3B3D), second⿿generation Ad5-FMD vaccines, and cross-protective vaccines against multiple subtypes. Additionally, combinations of vaccine and biotherapeutics / and or adjuvants will be investigated as a way to induce mucosal immunity necessary not only to prevent disease but also to decrease persistent infection. These vaccine/ adjuvant formulations will be tested using alternate routes such as transdermal and by direct mucosal delivery. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. In addition, the interactions of the virus with specific tissues at the primary infection sites will be studied by characterizing infected tissues at the cellular and subcellular level as well as utilizing transcriptomic analyses with micro arrays and next generation RNAseq. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. This year, Foreign Animal Disease Research Unit (FADRU) scientists regained limited access to animal facilities, allowing initial testing of some of the vaccines and biotherapeutics and carrying out some pathogenesis and transmission studies with foot-and-mouth disease (FMDV) in cattle and pigs. In addition, our personnel situation improved, and we were able to hire an Immunologist who started work in June 2019 and filled three scientific support positions. Under Objective 1 (countermeasures), progress continues on our lead vaccine candidate; the FMD3B3D platform which is now select agent excluded and we have started transfer of molecular constructs to our commercial partner to derive master seeds of relevant vaccine strains and serotypes in the United States mainland. Efforts have continued toward the development of FMDV modified live vaccines. In collaboration with a commercial partner, ARS scientists discovered that multiple regions of the viral genome that tolerate codon pair bias deoptimization resulting in attenuated viruses that are currently being evaluated as vaccine candidates. A new DIVA FMD ELISA test based on Nanobody-technology was developed for the detection of foot-and-mouth disease (FMD) infected cattle. This novel technology, using camelid-derived single-nanobody fragments showed great specificity and sensitivity and provides a new low- cost detection method for FMD infection in cattle. For Objective 2 (virus-host interactions) great progress was made in understanding the establishment and maintenance of the carried state in cattle. Specifically, studies on the evolution of the FMD virus during persistence using next generation genetic sequencing provided insight on the mechanisms of virus maintenance and emergence. Host gene expression showed that a previously unknown bovine gene was differently expressed between carriers than non-carriers in the epithelia where persistence occurs. Bioinformatic analyses showed that this was novel gene similar to a mouse gene important for regulating local immune responses, providing insight on a potential mechanism for persistence. For Objective 3 (ecology), we continued studies in Africa and in Asia, with important reports on virus evolution during persistent infections in Asian buffalo in India and Pakistan and characterization of FMDV circulating in Uganda, Kenya, Cameroon, Nigeria, and Vietnam. Accomplishments 01 Transmission modeling of FMDV in natural hosts. Although FMD is widely described as the most important disease affecting international trade in animal product, many aspects of the basic biology of the causative virus remain unknown. Researchers at USDA, ARS, Orient Point, New York have been working to understand how FMD virus transmits between animals and within populations. Recent work has shown that pigs can transmit the virus before any signs of disease. Research has also showed that FMD virus may be transmitted from oral fluids of carrier cows which have no signs of disease. These findings are critically important to understand how FMD spreads within natural livestock hosts, and to prepare for the possibility of an outbreak in the United States. 02 Discovering a new region in the FMDV important for vaccine efficacy. It has been previously reported that the G-H loop is a flexible structure within the FMDV capsid, which is able to induce a dominant antibody response in the animal host. Researchers at USDA, ARS, Orient Point, New York have demonstrated that modification of this structure in the Ad5-delivered FMD vaccine is tolerable and does not compromise the induction of total anti-FMD antibodies in swine, however, despite the high amount and increased reactivity, these antibodies failed to protect animals against challenge with virulent FMDV. This data suggests that although highly variable, this region of the G-H loop contributes to the protective immunity conferred by Ad5 vector- delivered FMD vaccines in swine, and cannot be substituted without a loss of vaccine efficacy.
Impacts
(N/A)
Publications
- Visser, L.J., Medina, G.N., Rabouw, H.H., De Groot, R.J., Langereis, M.A., De los Santos, T.B., Van Kuppeveld, F.J. 2018. FMDV leader protease cleaves G3BP1 and G3BP2 and inhibits stress granule formation. Journal of Virology.
- Gelkop, S., Sobarzo, A., Brangel, P., Vincke, C., Romao, E., Fedida-Metula, S., Strom, N., Ataliba, I., Mwiin, F.N., Ochwo, S., Velazquez-Salinas, L., McKendry, R.A., Muyldermans, S., Lutwama, J.J., Rieder, A.E., Yavelsky, V. , Lobel, L. 2018. The development and validation of a novel nanobody-based competitive ELISA for the detection of foot and mouth disease 3ABC antibodies in cattle. Frontiers in Veterinary Science.
- Medina, G.N., Diaz San-Segundo, F., Stenfeldt, C., Arzt, J., De los Santos, T.B. 2018. The different tactics of foot-and-mouth disease virus to evade innate immunity. Frontiers in Microbiology.
- Chen, X., Gaglione, R., Leong, T., Bednor, L., De los Santos, T.B., Luk, E. , Airola, M., Hollingsworth, N.M. 2018. Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80. PLoS Genetics.
- Barrionuevo, F., Di Giacomo, S., Bucafusco, D., Ayude, A., Schammas, J., Miraglia, M.C., Capozzo, A., Borca, M.V., Perez-Filgueira, M. 2018. Systemic antibodies administered by passive immunization prevent generalization of the infection by foot-and-mouth disease virus in cattle after oronasal challenge. Virology. 518:143-151.
- De Los Santos, T.B., Diaz-San Segundo, F., Rodriguez, L.L. 2018. The need for improved vaccines against foot-and-mouth disease. Current Opinion in Virology. 29:16-25.
- Palinski, R., Bertram, M.R., Vu, L.T., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Stenfeldt, C., Fish, I.H., Hoang, B.H., Dung, D.H., Arzt, J. 2019. First genome sequence of Foot-and-mouth disease virus serotype O sublineage Ind2001e from Vietnam. Microbiology Resource Announcements.
- Arzt, J., Bertram, M.R., Vu, L.T., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Palinski, R., Stenfeldt, C., Fish, I.H., Hoang, B.H., Dung, D.H. 2019. First detection of Senecavirus A in Vietnam. Emerging Infectious Diseases.
- Bertram, M., Bravo De Rueda, C., Garabed, R., Dickmu Jumbo, S., Moritz, M., Pauszek, S.J., Abdoulkadiri, S., Rodriguez, L.L., Arzt, J. 2018. Molecular epidemiology of foot-and-mouth disease virus in the context of transboundary animal movement in the far north region of Cameroon. Frontiers in Veterinary Infectious Diseases.
- Arzt, J., Branan, M., Delgado, A., Yadav, S., Moreno-Torres, K., Tildesley, M., Stenfeldt, C. 2019. Quantitative impacts of incubation phase transmission of foot-and-mouth disease virus. Scientific Reports.
- Arzt, J., Belsham, G.J., Lohse, L., Botner, A., Stenfeldt, C. 2018. Transmission of foot-and-mouth disease from persistently infected carrier cattle to naive cattle via transfer of oropharyngeal fluid. mSphere. 3(5) :e00365-18.
- Biswal, J.K., Ranjan, R., Subramaniam, S., Mohapatra, J.K., Sharma, M.K., Bertram, M.R., Brito, B., Rodriguez, L.L., Pattnaik, B., Arzt, J. 2019. Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India. bioRxiv.
- Omondi, G., Alkhamis, M., Obanda, V., Gakuya, F., Sangula, A., Pauszek, S. J., Perez, A., Ngulu, S., Van Aardt, R., Arzt, J., Vanderwaal, K. 2019. Phylogeographic and cross-species transmission dynamics of SAT1 and SAT2 foot-and-mouth disease virus in Eastern Africa. Molecular Ecology.
- Fernandez-Sainz, I., Gavitt, T.D., Koster, M., Ramirez-Medina, E., Rodriguez, Y.Y., Wu, P., Silbart, L.K., De Los Santos, T.B., Szczepanek, S. M. 2019. The VP1 G-H loop hypervariable epitope contributes to protective immunity against foot and mouth disease virus in swine. Vaccine.
- Arzt, J., Fish, I., Pauszek, S.J., Johnson, S.L., Chain, P.S., Rai, D.K., Rieder, A.E., Goldberg, T.L., Rodriguez, L.L., Stenfeldt, C. 2019. The evolution of a super-swarm of foot-and-mouth disease virus in cattle. Virology.
- Stenfeldt, C., Smoliga, G.R., Hartwig, E.J., Palinski, R., Fish, I.H., Silva, E.B., Pauszek, S.J., Arzt, J. 2018. Contact challenge of cattle with foot-and-mouth disease virus validates the role of the nasopharyngeal epithelium as the site of primary and persistent infection. Scientific Reports.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protective immunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections. 1.1) Development of novel FMD vaccine platforms. 1.1.1) Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. 1.1.2) Development of improved second-generation Ad5-FMD vaccines. 1.1.3) Discovery of modified live attenuated FMDV vaccine candidates (MLAV). 1.1.4) Discovery of cross-protective vaccines against multiple FMDV subtypes. 1.2) Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. 1.2.1) Discovery/development of novel biotherapeutics with increased potency and extended systemic half-life. 1.2.2) Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. 1.3) Evaluation of vaccine-induced immunity and FMDV carrier state. 1.3.1) Characterization of host immunity associated with novel vaccines against FMDV. 1.3.2) Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) Elucidation of host-pathogen interactions of FMDV in acute and persistent infections. The information derived will be used to devise effective anti-viral intervention strategies. 2.1) Determine the molecular basis for FMDV-host interactions that impact virulence. 2.1.1) Examination of virus factors contributing to FMDV virulence. 2.1.2) Examination of host factors contributing to FMDV virulence. 2.2) Identification of molecular mechanisms associated with the establishment of FMDV persistence. 2.2.1) Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. 2.2.2) Investigation of within-host FMDV genomic evolution to characterize site-specific mutational pressure, genomic variation, and potential adaptation to the host. 2.3) Determination of the immune mechanisms affecting protective immunity against FMDV. 2.3.1) Analysis of CD4 helper T-cell response to FMDV vaccination. 2.3.2) Analysis of the CD8 cytotoxic T-cell response to FMDV vaccination. 2.3.3) Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding the ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, and characterizing risk factors driving FMDV emergence and spread. 3.1) Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. 3.2) Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. 3.3) Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings. Approach (from AD-416): 1. The development of intervention strategies to control and eradicate FMDV will be achieved through the development of novel FMD vaccine platforms: including of marker FMDV-LL3B3D vaccines against relevant outbreak strains, second�generation Ad5-FMD vaccines, the discovery of modified live-attenuated FMDV vaccine candidates, and the discovery of cross-protective vaccines against multiple subtypes. Novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection will be based on the discovery/development of novel biotherapeutics with increased potency and extended systemic half-life. An evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle will be conducted. To evaluate vaccine-induced immunity and FMDV carrier state the host immunity associated with novel vaccines against FMDV will be characterized. Novel vaccines will be evaluated for their ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. The molecular mechanisms associated with the establishment of FMDV persistence will be identified through the determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T-cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. This year was challenging for the Foreign Animal Disease Research Unit (FADRU) due to the lack of access to animal facilities and a significant shortage in personnel due to multiple personnel departures and hiring freeze. However, the remaining personnel stepped up to the multiple challenges and was able to achieve most of the milestones for the year either by establishing research collaborations overseas or with university partners. Under Objective 1 (countermeasures), significant progress was made in further development of the FMD3B3D platform with select agent exclusion and approval for transfer the vaccine to our commercial partner. Additional progress was made on a next generation Ad5 vector with more robust safety and increased antigen expression. However, these vectors are awaiting in-vivo testing. Likewise, new modified live vaccine candidates made using codon deoptimization await animal testing. A new DIVA ELISA test was also developed in partnership with APHIS (Animal Plant Health Inspection Service), DHS (Department of Homeland Security), Academia and commercial partners. Several Ad5-biotherapeutics vectors were made that are still awaiting in-vivo testing. Immune assessment of FMD vaccines and testing for prevention of persistent infection was not possible due to our immunologist vacancy and lack of animal facility. For Objective 2 (virus-host interactions) the exploration of MiRNA as a antiviral mechanism was greatly limited by the departure of an ARS scientist. However, significant progress was made in understanding the molecular mechanisms mediating viral persistence in cattle. Also, some progress was made on functional genomics especially understanding the role in viral virulence of features on the viral 5� terminus (S-fragment). Important progress was made on Objective 3 (ecology) specially with collaborations overseas in Asia and Africa, where deeper understanding of the virus emergence and transmission was achieved. Overall ARS scientists made great progress in all three objectives overcoming significant limitations in personnel and animal facilities. Accomplishments 01 Safe Foot-and-Mouth Disease vaccine. Currently, production of Foot and Mouth Disease (FMD) vaccines relies on the use of live virulent virus that can escape manufacturing facilities and cause outbreaks. Live FMD virus is not allowed on the U.S. mainland. Recently, ARS researchers in Orient Point, New York used reverse genetics to create an attenuated FMD virus that is innocuous to animals but can be safely used for vaccine manufacturing. Secretary of Agriculture has authorized the movement of this modified, non-virulent version of the FMD virus from the Plum Island Animal Disease Center to the U.S. mainland for the purposes of continued vaccine development and eventually manufacturing. Vaccine manufacturer Zoetis Inc. requested and has been granted a license for this vaccine technology. Identifying a vaccine that uses a modified virus will enable vaccine manufacturing in the USA and will allow the USDA-APHIS to more quickly source and acquire FMD vaccine for the Strategic Veterinary Stockpile and in the event of an outbreak of this devastating disease. 02 Understanding the mechanisms of the Foot-and-Mouth Disease virus (FMDV) carrier state in cattle. Control and eradication of foot-and-mouth disease (FMD) is impeded by the existence of a prolonged persistent phase of infection in ruminant species which is generally referred to as the FMDV carrier state. Researchers at ARS in Orient Point, New York have spent much of the last 10 years characterizing the mechanisms of persistence in the nasopharynx (throat) of cattle. Now we have taken that information and shown that clearance of FMDV infection was associated with activation of anti-viral T cell responses, whereas the antibody-mediate immune response was stronger in animals that maintained persistent infection. Additionally, mechanisms associated with programmed cell killing (apoptosis) were activated in animals that cleared infection whereas pathways associated with prolonged cell survival were upregulated in FMDV carriers. These findings are critically important for design of novel FMD vaccines that may prevent the occurrence of persistent FMDV infection. The study was published in Scientific Reports. 03 New rapid diagnostic Kit for Foot-and-Mouth Disease. Current foot-and- mouth disease virus (FMDV) vaccines are compatible with a strategy based on �differentiating infected from vaccinated animals� (DIVA). ARS researchers at Orient Point, New York as part of a large research consortium of federal agencies, academia and animal health industry, developed and licensed a rapid-response (three-hour) Foot-and-Mouth Disease (FMD) diagnostic kit. A manuscript describing the validation of this assay that led to the licensure of the FMD diagnostic kit was published in the Journal of Veterinary Diagnostic Investigation. 04 Increased the knowledge about Foot-and-Mouth Disease virus (FMDV) emergence and transmission. Although foot-and-mouth disease (FMD) is widely described as the most important disease affecting international trade in animal products, many aspects of the natural cycle of the causative virus remain unknown. ARS researchers in Orient Point, New York have been working for several years to develop research partnerships in endemic areas of Asia and Africa to describe the behavior of FMDV under natural conditions. Some of these studies have recently culminated with critical findings from work in India which have quantitated the speed of spread of FMDV through herds of cattle during outbreaks, and the duration that the virus persists in those herds. Similarly, ARS researchers performed a study in Cameroon which demonstrated that there are benefits to introducing vaccines to herds of cattle, even if the virus is already present. Additionally, genetic sequences were acquired from viruses discovered in Vietnam which helped to show how the virus evolves over time and space to continuously create new viruses. These findings are critically important to understand how FMDV spreads within the current disease range, and to prepare for the possibility of an outbreak in the USA. 05 Understanding the virus factors critical for Foot-and-Mouth Disease Virus (FMDV) replication. ARS researchers in Orient Point, New York have used novel approaches to derive attenuated FMDV strains using modifications at the 5�terminus of the viral genome. In proof of concepts studies identified a new role attributed to a small segment of the beginning of the FMDV genome that is responsible for virus replication and also modulating the innate immune response in animals to the virus. This knowledge of the virus-unique replication processes has potential for the development of disease control strategies such as better vaccines.
Impacts
(N/A)
Publications
- Rekant, S., Lyons, M., Pacheco Tobin, J., Arzt, J., Rodriguez, L.L. 2016. Veterinary applications of infrared thermography. American Journal of Veterinary Research. 77:98-107.
- Ahmed, Z., Pauszek, S.J., Ludi, A., Larocco, M.A., Khan, E., Afzal, M., Arshed, M.J., Farooq, U., Arzt, J., Bertram, M., Brito, B., Naeem, K., Abubakar, M., Rodriguez, L.L. 2017. Genetic diversity and comparison of diagnostic tests for characterization of foot-and-mouth disease virus strains from Pakistan 2008-2012. Transboundary and Emerging Diseases. 65(2) :534-546.
- Brito, B.P., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Vu, L.T., Vu, P. P., Stenfeldt, C., Rodriguez, L.L., King, D.P., Knowles, N.J., Bachanek- Bankowska, K., Long, N.T., Dung, D.H., Arzt, J. 2018. A traditional evolutionary history of foot-and-mouth disease viruses in Southeast Asia challenged by analyses of non-structural protein coding sequences. Scientific Reports. 8:6472.
- Bertram, M.R., Delgado, A., Pauszek, S.J., Smoliga, G.R., Brito, B.P., Stenfeldt, C., Hartwig, E.J., Dickmu-Jumbo, S., Abdoulmoumini, M., Abona Olivia, A., Salhine, R., Rodriguez, L.L., Gerabed, R., Arzt, J. 2018. Effect of vaccination on cattle herds previously exposed to foot and mouth disease in Cameroon. Preventive Veterinary Medicine. 155:1-10.
- Eschbaumer, M., Stenfeldt, C., Pacheco Tobin, J., Rekant, S.I., Arzt, J. 2016. Effect of storage conditions on subpopulations of peripheral blood T lymphocytes from naive cattle and cattle infected with foot-and-mouth disease virus. Veterinary Clinical Pathology. 45:110-115.
- Stenfeldt, C., Eschbaumer, M., Smoliga, G.R., Rodriguez, L.L., Zhu, J.J., Arzt, J. 2017. Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses. Scientific Reports. 7:17800.
- Kloc, A., Rai, D.K., Rieder, A.E. 2018. The roles of picornavirus untranslated regions in infection and innate immunity. Frontiers in Microbiology. 20(9):485.
- Stenfeldt, C., Arzt, J., Pacheco, J., Gladue, D.P., Smoliga, G.R., Silva, E., Rodriguez, L.L., Borca, M.V. 2018. A partial deletion within foot-and- mouth disease virus non-structural protein 3A causes clinical attenuation in cattle but does not prevent subclinical infection. Virology. 516:115- 126.
- Kenney, M.A., Waters, R.A., Rieder, A.E., Pega, J., Perez-Filguera, M., Golde, W.T. 2017. Enhanced sensitivity in detection of antiviral antibody responses using biotinylation of foot-and-mouth disease virus (FMDV) capsids. Journal of Immunological Methods. 450:1-9.
- Hayder, S.S., VanderWaal, K., Ranjan, R., Biswal, J.K., Subramaniam, S., Mohapatra, J.K., Sharma, G.K., Rout, M., Dash, B., Das, B., Prusty, B., Sharma, A.K., Stenfeldt, C., Perez, A., Rodriguez, L.L., Arzt, J. 2017. Foot-and-mouth disease virus transmission dynamics and persistence in a herd of vaccinated dairy cattle in India. Transboundary and Emerging Diseases. 65(2):e404-e415.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protective immunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections. 1.1) Development of novel FMD vaccine platforms. 1.1.1) Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. 1.1.2) Development of improved second-generation Ad5-FMD vaccines. 1.1.3) Discovery of modified live attenuated FMDV vaccine candidates (MLAV). 1.1.4) Discovery of cross-protective vaccines against multiple FMDV subtypes. 1.2) Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. 1.2.1) Discovery/development of novel biotherapeutics with increased potency and extended systemic half-life. 1.2.2) Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. 1.3) Evaluation of vaccine-induced immunity and FMDV carrier state. 1.3.1) Characterization of host immunity associated with novel vaccines against FMDV. 1.3.2) Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) Elucidation of host-pathogen interactions of FMDV in acute and persistent infections. The information derived will be used to devise effective anti-viral intervention strategies. 2.1) Determine the molecular basis for FMDV-host interactions that impact virulence. 2.1.1) Examination of virus factors contributing to FMDV virulence. 2.1.2) Examination of host factors contributing to FMDV virulence. 2.2) Identification of molecular mechanisms associated with the establishment of FMDV persistence. 2.2.1) Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. 2.2.2) Investigation of within-host FMDV genomic evolution to characterize site-specific mutational pressure, genomic variation, and potential adaptation to the host. 2.3) Determination of the immune mechanisms affecting protective immunity against FMDV. 2.3.1) Analysis of CD4 helper T-cell response to FMDV vaccination. 2.3.2) Analysis of the CD8 cytotoxic T-cell response to FMDV vaccination. 2.3.3) Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding the ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, and characterizing risk factors driving FMDV emergence and spread. 3.1) Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. 3.2) Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. 3.3) Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings. Approach (from AD-416): 1. The development of intervention strategies to control and eradicate FMDV will be achieved through the development of novel FMD vaccine platforms: including of marker FMDV-LL3B3D vaccines against relevant outbreak strains, second�generation Ad5-FMD vaccines, the discovery of modified live-attenuated FMDV vaccine candidates, and the discovery of cross-protective vaccines against multiple subtypes. Novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection will be based on the discovery/development of novel biotherapeutics with increased potency and extended systemic half-life. An evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle will be conducted. To evaluate vaccine-induced immunity and FMDV carrier state the host immunity associated with novel vaccines against FMDV will be characterized. Novel vaccines will be evaluated for their ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. The molecular mechanisms associated with the establishment of FMDV persistence will be identified through the determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T-cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. Overall, the Foreign Animal Disease Research Unit (FADRU) scientists working on Foot-and-Mouth Disease (FMD) had a very productive year, despite major adversities including the closing of the animal facility since August 2016, the departure of two Scientists and inability to backfill these positons and the major loss of extramural funding from the Department of Homeland Security (DHS). Despite these challenges, the FADRU delivered multiple peer-reviewed publications in top scientific journals, patents and product licenses. Many of these accomplishments represent collaborative efforts within the FADRU or extramural collaborations with industry, academia and international partners. Objective 1: In FY17 we have continued to pursue the development of intervention strategies to control and eradicate FMDV including improved vaccine platforms, diagnostics and delivery systems. Under a CRADA with a pharmaceutical industry partner, we have continued advancing the development of the leaderless vaccine (FMD-LL3B3D) platform, increasing the number of serotypes now available for master seed production. We demonstrated that this vaccine platform�s built-in markers allow differentiation of infected from vaccinated animals (DIVA capacity) using either commercially available ELISA kits or an ARS-developed and newly licensed FMDV 3B-ELISA test. In collaboration with Ben Gurion University, camel nanobodies specific to FMDV nonstructural proteins 3ABC and 3Dpol were tested in ELISA assays with the intention to develop more affordable FMD diagnostics test for FMDV endemic regions of Africa. Research continued on the use of novel FMDV capsid modifications not only to allow direct binding to specific adjuvants but also to assist in purification and formulation of vaccine- adjuvant combinations. We have successfully generated mosaic FMD vaccine candidates that exhibited a broader antigenic coverage within serotype A from virus pools 1, 2, 3 and 7. We have continued advancing a second generation Ad5-FMD vaccine platform including vector modifications to improve vaccine stability and performance. In vitro, we demonstrated that the modified Ad5-FMD was stable after repeated passages and allows for detection of FMD empty capsids. We have also tested for the first time the Ad5-FMD vaccine against serotypes of relevance in Asia and demonstrated efficacy in cattle and swine. Interestingly a combination of Ad5-FMD and Ad5-IFN fully protected cattle against challenge with virulent FMDV. Research toward modified live-attenuated FMDV vaccines (MLAV) included utilizing mutations in the S-fragment region and combining with high-fidelity polymerase and genome deoptimized mutants progressed. Mutations of specific residues of the virus polymerase and deletions of the S-fragment RNA at the 5� terminus of the FMDV genome were highly attenuated in primarily cells of swine or bovine origin and importantly, exhibited significant attenuation in vivo using a mice model. We continued studies based on the use of codon usage deoptimization and polymerase targeted mutagenesis to both reduce recombination of the viral genome and attenuate FMDV. Objective 2: Substantial progress was made in pathogenesis research despite lack of access to the PIADC animal containment facility. Milestones were achieved through advanced analyses of samples from previously completed animal work, outsourcing animal research to collaborating institutions, and development of novel research techniques. Previous progress in the area of virus-host interactions associated with acute and persistent FMD in cattle was continued and further expanded this year. Previous achievements had identified the micro-anatomic site of acute and persistent FMDV infections in the bovine nasopharynx as well as the time frame for establishment of persistent infection. In FY17 we determined that activation of a cytotoxic T-cell response is a critical for successful clearance of FMDV in order to clear persistent infections. Contrastingly, induction of a strong antibody-mediated response may inhibit viral clearance. These novel findings provide information that will facilitate development of improved FMDV countermeasure products that may ultimately prevent FMDV persistence. Progress with FMDV pathogenesis studies in pigs included completion of analyses and publication of a landmark paper which challenged the notion that FMDV was not transmitted during the incubation phase. In collaboration with APHIS Center for Epidemiology and Animal Health (CEAH), we documented and described the occurrence of FMDV transmission during the incubation phase in pigs. The data obtained is critical to improve the accuracy of FMD transmission modeling and may have profound impact on the effectiveness of control strategies in the event of an FMD outbreak. Significant efforts were devoted to uncover novel interactions between host factors and viral proteins/RNA were discovered including new knowledge of a host protein necessary in FMDV replication and now that manipulating the fidelity of the FMDV polymerase could significantly attenuate virus pathogenesis. This novel approach could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches. We have discovered two novel molecular mechanisms by which FMDV blocks the host immune response. By interacting with the protein ADNP (activity dependent neuroprotective protein), FMDV affects the architecture of cellular DNA, repressing the expression of antiviral genes. In a separate study, novel insight was provided into the role of the 5�-terminal S fragment in modulating the host innate immune response and show a direct correlation between S-fragment deletion mutations and attenuated phenotypes. In proof-of-concept studies we have studied for the first time the role of noncoding host RNAs (miRNAs) during infection of cattle with FMDV. Interestingly, unique miRNA serum signatures were detected for acutely infected and persistently infected cattle. Characterization of signature serum miRNAs that are distinct to the FMDV carrier state could facilitate identification of persistently infected animals in post- outbreak surveillance. On the same topic overexpression of a synthetic mimics of specific miRNAs significantly reduced FMDV in porcine cells and was effective across multiple serotypes demonstrating the potential of these molecules as effective antivirals. New technical achievements have broadened the scope of pathogenesis research within FADRU with the implementation of next-generation sequencing (NGS) and associated data de-complexing and analyses. We have achieved reproducible high throughput, deep sequencing of FMDV full genomes and carried out quasispecies analyses of viruses from distinct anatomic sites and stages of FMDV infection including establishment of persistent infection. Initial investigations have identified mutations that are characteristic for persistent viruses, and have suggested that the selective pressures may be different in vaccinated versus na�ve hosts. Objective 3: Substantial progress was made in ongoing epidemiological studies in several endemic countries including Vietnam, Cameroon, Uganda, and India. New pilot studies were initiated with collaborators in Kenya and Nigeria. Collectively, these studies are aimed at understanding FMD disease ecology in endemic settings. Understanding and mitigating natural virus cycles in endemic regions ultimately contributes to protection and preparedness in the U.S. homeland. Numerous advances were made in projects in Asia including publishing a unique description of the duration of the FMDV carrier state in naturally infected cattle in India and discovery of the first emergence of a novel FMDV strain called Ind-2001d, in Vietnam. Additionally, we were the only laboratory in the world to generate and publish a full genome sequence of that emergent virus. Lastly, a highly novel analysis was completed and published describing movement of FMDV strains between different species and locations in Vietnam. Substantial progress was made in ongoing projects in Africa. In Uganda we identified distinct lineages of serotypes SAT1, SAT2 and O circulating in 2014/2015 as part of our ongoing surveillance program. These viruses came from probang and serum samples from twenty-two Ugandan districts that were sent to PIADC for screening to determine sero-prevalence in Ugandan and to identity circulating virus. Screening of samples from African Cape buffalo in Kenya resulted in the isolation and characterization of 60 unique FMDV strains from serotypes SAT1 and SAT2. Furthermore, these samples allowed the unprecedented accomplishment of the use of next generation sequencing (NGS) to detect several naturally occurring simultaneous co-infections of buffalo with 2 distinct serotypes of FMDV: SAT1 and SAT2. Additionally, in Cameroon we documented the emergence of serotype SAT1 in cattle. This information is crucial to gain a better understanding of FMD and for improvement of control strategies in endemic regions of Asia and Africa. The ultimate goal is to integrate such knowledge to protect herds within the USA. To accomplish this, data and analysis from these studies are directly assimilated into modeling efforts with CEAH and the University of Minnesota wherein simulations of FMD outbreaks in the U.S. are executed. This research continues to provide valuable data to maintain preparedness against global FMDV threats. Accomplishments 01 Better vaccines to fight foot-and mouth disease. Foot and mouth disease (FMD) is a highly infectious and economically devastating disease of livestock caused by highly diverse viruses. The disease has been eradicated in the U.S. since 1929 and its reintroduction could result in $15-200 billion in losses to U.S. livestock and related industries. Current foot-and-mouth (FMD) vaccines have narrow coverage, requiring specific vaccine for each strain. Therefore, USDA-APHIS invests millions of dollars to stock multiple FMD vaccines in a vaccine bank as part of the National Veterinary Stockpile. To solve this problem, ARS researchers at Plum Island, Orient, New York, in collaboration with researchers at the Los Alamos National Laboratory, engineered novel FMD mosaic vaccine that provided broad coverage against multiple FMD virus strains when tested in cattle. This is the first FMD vaccine providing broad protection against multiple FMD strains and could significantly reduce the cost of stockpiling FMD vaccines to protect the $1.5 trillion U.S. agriculture. 02 Harnessing natural defenses to protect livestock against disease. Foot and mouth disease (FMD) is a highly infectious and economically devastating disease of livestock caused by highly diverse viruses. The disease has been eradicated in the U.S. since 1929 and its reintroduction could result in $15-200 billion in losses to U.S. livestock and related industries. Vaccines usually take 1 - 2 weeks to induce protection. ARS researchers at Plum Island, Orient, New York, recently discovered a novel FMD vaccine that can protect cattle against FMD as early as 3-days post vaccination. Furthermore, combining this vaccine with a natural defense protein called interferon they achieved protection of cattle as early as 1 day post vaccination. The same vaccine was also effective in pigs. This new vaccine and its combination with this natural defense protein are very useful in protecting U.S. livestock against devastating diseases like FMD. 03 New diagnostic test for foot-and-mouth disease made in the USA now available. Foot and mouth disease (FMD) is a highly infectious and economically devastating disease of livestock caused by highly diverse viruses. The disease has been eradicated in the U.S. since 1929 and its reintroduction could result in $15-200 billion in losses to U.S. livestock and related industries. Until now, FMD diagnostic tests had to be imported from overseas at a high cost and took a long time (>6 h) to obtain a result. ARS researchers at Plum Island, Orient, New York in a government-academia-industry partnership, developed a new FMD diagnostic test that is made in the U.S. at a lower cost, is faster and works well across all livestock species. This test is the first licensed FMD diagnostic kit manufactured on the U.S. mainland, and is now available to protect U.S. livestock against this devastating disease.
Impacts
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Publications
- Gutkoska, J., Larocco, M.A., Ramirez-Medina, E., De Los Santos, T.B., Lawrence, P.J. 2017. Host microRNA-203a is antagonistic to the progression of foot-and-mouth disease virus infection. Virology. doi: 10.1016/j.virol. 2017.01.019.
- Lawrence, P.J., Rieder, A.E. 2017. Insights into jumonji c-domain containing protein 6 (JMJD6): a multifactorial role in FMDV replication in cells. Virus Genes. 53(3):340-351.
- Arzt, J., Pacheco Tobin, J., Stenfeldt, C., Rodriguez, L.L. 2017. Pathogenesis of virulent and attenuated foot and mouth disease virus in cattle. Virology. 14(1):89. doi: 10.1186/s12985-017-0758-9.
- Stenfeldt, C., Arzt, J., Smoliga, G., Larocco, M.A., Gutkoska, J., Lawrence, P.J. 2017. Proof-of-concept study: profile of circulating micro RNAs in bovine serum harvested during acute and persistent FMDV infection. Virology Journal. 14:71. doi: 10.1186/s12985-017-0743-3.
- Holinka-Patterson, L.G., O'Donnell, V., Risatti, G., Azzinaro, P.A., Arzt, J., Stenfeldt, C., Velazquez-Salinas, L., Gladue, D.P., Borca, M.V. 2017. Early protection events in swine immunized with an experimental live attenuated classical swine fever marker vaccine, FlagT4G. PLoS One. doi: 10.1371/journal.pone.0177433.
- Diaz-San Segundo, F., Medina, G., Stenfeldt, C., Arzt, J., De Los Santos, T.B. 2016. Foot-and-mouth disease vaccines. Veterinary Microbiology. doi: 10.1016/j.vetmic.2016.12.018.
- Hayer, S., Ranjan, R., Biswal, J., Mohapatra, J.K., Sharma, G.K., Subramaniam, S., Stenfeldt, C., Perez, A., Rodriguez, L.L., Pattnaik, B., Vanderwaal, K., Arzt, J. 2017. Quantitative characteristics of the foot- and-mouth disease carrier state under natural conditions in India. Transboundary and Emerging Diseases. doi: 10.1111/tbed.12627.
- Pauszek, S.J., Eschbaumer, M., Brito, B., Ferreira De Carvalho, H.C., Vu, L.T., Phuong, N.T., Hoang, B.H., Tho, N.D., Tung, N., Thuy, N.T., Long, N. T., Dung, D.H., Rodriguez, L.L., Arzt, J. 2016. Site-specific substitution (Q172R) in the VP1 protein of subclinical FMDV isolates collected in Vietnam. Virology Reports. 6:90-96.
- Brito, B., Pauszek, S.J., Eschbaumer, M., Stenfeldt, C., De Carvalho Ferreira, H., Vu, L.T., Phuong, N.T., Hoang, B.H., Tho, N.D., Dong, P.V., Minh, P.Q., Long, N.T., Dung, D.H., Rodriguez, L.L., Arzt, J. 2017. Phylodynamics of epidemic and asymptomatic foot-and-mouth disease in Vietnam 2010-2014. Veterinary Research. 48:24.
- Pacheco, J., Stenfeldt, C., Rodriguez, L.L., Arzt, J. 2016. Infection dynamics of foot-and-mouth disease virus in cattle following intra- nasopharyngeal inoculation or contact exposure. Journal of Comparative Pathology. 155(4):314-325. doi: 10.1016/j.jcpa.2016.08.005.
- Sreenivasa, B.P., Mohapatra, J.K., Pauszek, S.J., Koster, M.J., Dhanya, V. C., Tamil Selvan, R.P., Hosamani, M., Saravanan, P., Basagoudanavar, S.H., De Los Santos, T.B., Venkataramanan, R., Rodriguez, L.L., Grubman, M.J. 2017. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle. Veterinary Microbiology. 203:196-201. doi: 10.1016/j. vetmic.2017.03.019.
- Rai, D.K., Diaz-San Segundo, F., Campagnola, G., Schafer, E.A., Kloc, A., Keith, A., De Los Santos, T.B., Peersen, O., Rieder, A.E. 2017. Attenuation of foot-and-mouth disease virus by engineered viral polymerase fidelity. Journal of Virology. doi: 10.1128/JVI.00081-17.
- Ranjan, R., Biswal, J.K., Subramaniam, S., Singh, K.P., Stenfeld, C., Rodriguez, L.L., Pattnaik, B., Arzt, J. 2016. Foot-and-mouth disease virus- associated abortion and vertical transmission following acute infection in cattle under natural conditions. PLoS One. 11(12):e0167163.
- Rhyan, J., Mccollum, M., Gidlewski, T., Shalev, M., Ward, G., Donahue, B., Arzt, J., Stenfeldt, C., Mohamed, F., Nol, P., Deng, M., Metwally, S., Mckenna, T., Salman, M. 2016. Foot-and-Mouth Disease in a small sample of experimentally infected pronghorn (Antilocapra americana). Journal of Wildlife Diseases. 52(4):862-873.
- Lawrence, P.J., Rieder, A.E. 2017. Foot-and-mouth disease virus receptors: multiple gateways to initiate infection. In: Sobrino, F. and Domingo, E. Foot-and-Mouth Disease Virus: Current Research and Emerging Trends. Centro de Biolog�a Molecular "Severo Ochoa" (CSIC-UAM), Madrid, Spain. Caister Academic Press. pp. 107-136.
- Medina, G.N., Knudsen, G.M., Greninger, A.L., Kloc, A., Diaz-San Segundo, F., Rieder, A.E., Grubman, M., Derisi, J.L., De Los Santos, T.B. 2017. Interaction between FMDV Lpro and transcription factor ADNP is required for viral replication. Virology. 505:12-22. doi: 10.1016/j.virol.2017.02. 010.
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