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Research Project: Intervention Strategies to Support the Global Control and Eradication of Foot-and-Mouth Disease Virus (FMDV)

Location: Foreign Animal Disease Research

2017 Annual Report


1a. 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.


1b. 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.


3. Progress Report:
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 naïve 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.


4. Accomplishments
1. 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.

2. 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.

3. 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.


Review 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.

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.

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.

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 Biología 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.