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United States Department of Agriculture

Agricultural Research Service

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

Location: Foreign Animal Disease Research

2013 Annual Report

1a.Objectives (from AD-416):
Foot-and-mouth disease virus (FMDV) causes a highly contagious, viral disease of domestic and wild cloven-hoofed animals effecting cattle, swine, sheep, goats and deer, which is characterized by fever, lameness and vesicular lesions and results in a high morbidity but low mortality in adult animals. FMD is the number one foreign animal disease threat to the United States livestock industry. Disease outbreaks result in the inhibition of trade in susceptible animals and their products. This research project will provide an understanding of virus-host interactions and disease pathogenesis that will aid in the development of alternative vaccine candidates and biotherapeutics. The objectives of this research project are: 1. Develop an understanding the mechanisms of FMDV host-pathogen interactions at the molecular and in vivo levels will aid in the development of improved disease control measures.

2. Develop new and improve current novel vaccines and biotherapeutic countermeasures that will enable rapid and effective prevention of FMDV infection in natural hosts and thereby contribute to global eradication of FMD.

3. Determine the epidemiology of FMDV in enzootic regions by biological characterization of variant FMDV strains and identifying the epidemiological importance of persistence on transmission and maintenance.

1b.Approach (from AD-416):
1. To gain an understanding of the mechanisms of FMDV host-pathogen interaction, we will determine the molecular mechanisms of FMDV pathogenesis and viral-host interactions responsible for virulence, transmission and host-range specificity. Parental wild-type and attenuated viruses will be compared both in cell culture and in susceptible animals to identify virulence determinants; comparative pathogenesis studies will be conducted with multiple serotype and attenuated viruses to determine FMDV tissue distribution in susceptible hosts. The mechanisms mediating long term immunity induced by FMDV infection will be studied as well as the mechanisms of FMDV persistence and development of the carrier state.

2. The development and continued improvement of biotherapeutic countermeasures will be achieved through the discovery of biotherapeutic platforms to improve early protection against infection. Improved vaccine platforms will be identified by examining a series of vaccine approaches including inactivated vaccines derived from genetically engineered attenuated viruses with DIVA markers, live-attenuated vaccines, and adenovirus vectored FMDV. Studies will focus on the identification of the mechanisms to broaden the immune response of vaccines and to increase the duration of immunity conferred.

3. The characterization of FMDV epidemiological factors will be achieved through the collection of field samples and corresponding epidemiological information supplied by collaborating international partners from countries in which FMDV is currently circulating. Transmission, carrier state and epidemiological information will be compiled to gain a better understanding of FMD transmission and viral ecology in enzootic regions.

3.Progress Report:
Significant progress was made on the three major objectives: 1- pathogenesis; 2- countermeasures 3- epidemiology. In pathogenesis we identified three novel cellular host factors interacting with FMDV proteins or RNA that are essential for FMDV growth. We found that FMDV modulates expression of cellular vimentin as part of its life cycle. We reported a direct association between a deletion in FMDV protein 3A and complete attenuation in cattle, but not swine. Using functional genomics and bioinformatics analyses we reported the molecular mechanisms associated with FMDV tissue tropism in cattle. In countermeasures, we focused on three vaccine approaches: inactivated antigen, Ad5-FMD and live-attenuated vaccines. Significant advancement was made on early development of the FMD-Leaderless 3B3D platform for inactivated FMD vaccine production. We conducted genetic stability, safety studies in cattle and swine, and showed that the platform is safe for vaccine production. Two companion DIVA diagnostic tests based on antigenic markers in 3B and in 3D proteins have been developed, with the 3B test now transferred to industry. Research continued to increase the potency of the Ad5-FMD vaccine platform including: vector improvements to increase antigen expression, adjuvant testing and route of delivery studies. Research continues toward identifying a LAV vaccine candidate, focusing on the stabilization of attenuating mutations in the leader region of the SAP mutant and adding antigenic markers in other genomic regions of this virus to achieve further attenuation and increase genetic stability. A strong component of our countermeasure development is the identification of more effective, longer-lasting biotherapeutics. Work continued on IFN-lambda, the only interferon thus far identified to be fully protective against FMDV challenge in cattle. Studies on porcine IFN lambda have demonstrated that swine can be protected against FMD in a dose dependent manner. All type I interferons from cattle were tested in-vitro for potency. The most potent IFN-alpha was cloned in Ad5 vector and induced potent antiviral activity in cattle. This knowledge will be used to design vectors for in-vivo testing. Other molecules and viral vectors were tested and shown to increase potency of Ad5-IFNalpha constructs in swine. In FMD epidemiology, collaborators from Pakistan, Vietnam and Cameroon have provided access to over 100 field isolates that were analyzed by genomic sequencing and vaccine matching. In Pakistan we identified that serotypes A and Asia1 circulating were not well covered by existing vaccine, prompting the Pakistani authorities to change current vaccine formulation to achieve better coverage. This increased our knowledge of circulating strains in endemic regions. We identified numerous carrier steers and buffalo in Vietnam. These animals have been placed in transmission cells with susceptible negative sentinel animals, with no evidence of transmission observed thus far. Relevant tissues were harvested from persistently infected buffalo in Vietnam and sent to PIADC for screening to determine the role of the carrier Asian buffalo in FMD transmission.

1. Advanced development of a new platform for FMDV vaccine production. Foot-and-Mouth Disease (FMD) is an extremely contagious viral disease of cloven-hoofed ungulates including domestic livestock and a variety of wild animals. Manufacturing of inactivated antigen FMD vaccines requires the large-scale growth of virulent virus, which is then chemically inactivated. This process requires manufacturing under expensive biosafety level 3 conditions and poses the risk of virus escape from the manufacturing facilities. ARS researchers at Greenport, New York discovered and filed a patent for a rationally designed vaccine production platform based on FMD virus lacking a critical protein that determines virulence. In addition they were able to introduce two antigenic markers in two non-structural proteins (NSP) 3B and 3D, that allow development of companion diagnostic tests to differentiate infected from vaccinated animals (DIVA). Finally the platform contains genetic features that allow it to be used for the multiple serotyes and subtypes of FMDV. The new virus, termed FMDV-LL3B3D, which is currently under development with a CRADA partner, was shown to be totally harmless to cattle and swine after inoculation with the live virus and inactivated antigen vaccines manufactured with the platform were fully protective in cattle and swine. This vaccine production platform sets a new paradign for safety in FMD vaccine production, allowing manufacturing at BSL2 or lower facilities, reducing costs of production and allowing to DIVA diagnostic tests development for at least two of the most immunogenic viral proteins.

2. Developed a new test for differentiating infected from vaccinated animals (DIVA). During the control of Foot-and-Mouth Disease (FMD) outbreaks it is critical to be able to distinguish vaccinated animals from infected animals in order be able to preserve the former and remove the later. Some such tests are commercially available but are expensive and manufactured outside the US. In order to gain domestic production of these tests, an interagency team including ARS researchers at Greenport, New York developed a competitive ELISA that uses a 3ABC recombinant protein and a monoclonal antibody. Unlike existing tests, the new test can be used not only for the conventional FMD vaccines, but also is compatible with novel FMDV marker vaccines, such as the Ad-FMDV or FMD-LL3B3D vaccine platforms. A commercial partner is now manufacturing this kit which will be further evaluated and made available for the National Veterinary Stockpile. This is the first FMD DIVA test manufactured in the US.

3. Identification of genetic determinants of tissue tropism in cattle for Foot-and-Mouth Disease Virus (FMDV). Foot-and-Mouth Disease is a devastating disease of cloven hoof animals. After entry into the bovine host via the respiratory tract, the virus replicates at specific sites in the back of the throat then invades lung and goes into the blood stream to the lesion sites. They can exist in various amount in different tissue. Despite many decades of active research there is little understanding for the very specific tropism for specific tissues at those sites. Based on the recently published whole bovine genome, ARS researchers at Greenport, New York have designed a microarray containing molecular probes for all the cattle genes and used it to determine what genes are over-represented or under-represented in tissues preferred for FMD replication and persistence. The results showed that three groups of genes including (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear virus infected cells via death receptor signaling, play roles in determining FMDV tissue tropism. Additionally increased extracellular matrix turnover, induced by FMDV infection, play a key role in the pathogenesis of vesicular lesions. This work represents a roadmap to increasing fundamental understanding of FMDV pathogenesis in cattle and is already guiding development of vaccines and biotherapeutic tools targeting critical steps in virus infection.

Review Publications
Lawrence, P., Uddowla, S., Pacheco Tobin, J., Kotecha, A., Fry, E., Rieder, A.E. 2012. Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis. Virology. 136(1):150-161.

Alejo, D.M., Moraes, M.P., Liao, X., Dias, C.C., Tulman, E.R., Diaz San Segundo, F.C., Rood, D., Grubman, M.J., Silbart, L.K. 2013. An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine. Vaccine. 313(18):2302-2309.

Montiel, N.A., Smoliga, G.R., Arzt, J. 2012. Time-dependent biodistribution and transgene expression of a recombinant human adenovirus serotype 5-luciferase vector as a surrogate agent for rAd5-FMDV vaccines in cattle. Veterinary Immunology and Immunopathology. 151(1-2):37-48. DOI: 110.1016/j.vetimm.2012.10.003.

Loughran, G., Libbey, J.E., Uddowla, S., Scallan, M.F., Ryan, M.D., Fujinami, R.S., Rieder, A.E., Atkins, J.F. 2013. Theiler’s murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism. Journal of General Virology. 94(2):348-353.

Pega, J., Bucafusco, D., Di Giacomo, S., Malacari, D., Capozzo, A., Arzt, J., Perez-Beascoechea, C., Maradei, E., Rodriguez, L.L., Borca, M.V., Perez-Filgueira, M. 2012. Early adaptive immune responses in the respiratory tract of foot and mouth disease-infected cattle. Journal of Virology. 87(5):2489-2495.

Pacheco Tobin, J., Tucker, M.T., Hartwig, E.J., Bishop, E.A., Arzt, J., Rodriguez, L.L. 2012. Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences. The Veterinary Journal. 193(2):456-463.

Lawrence, P., Larocco, M.A., Schafer, E.A., Baxt, B., Rieder, A.E. 2013. Examination of soluble integrin resistant mutants of foot-and-mouth disease virus (FMDV). Virology Journal. 10(1):2.

Dias, C.C., Moraes, M.P., Weiss, M., Diaz San Segundo, F.C., Perez-Martin, E., Salazar, A.M., De Los Santos, T.B., Grubman, M.J. 2012. Novel antiviral therapeutics to control foot-and-mouth disease. Journal of Interferon and Cytokine Research. 32(10):462-473.

Grubman, M.J., Diaz San Segundo, F.C., Dias, C.C., Moraes, M.P., Perez-Martin, E., De Los Santos, T.B. 2012. Use of replication-defective adenoviruses to develop vaccines and biotherapeutics against foot-and-mouth disease. Future Virology. 7(8):767-778.

Nene, V., Svitek, N., Golde, W.T., Barlow, J., Buus, S., Nielsen, M. 2012. Designing bovine T-cell vaccines via reverse immunology. Ticks and Tick Borne Diseases. 3(2012):188-192.

Pedersen, L.E., Harndahl, M., Nielsen, M., Patch, J.R., Jungersen, G., Buus, S., Golde, W.T. 2012. Identification of peptides fromm foot-and-mouth disease virus structural proteins bound by class I swine leucocyte antigen (SLA) alleles, SLA-1*0401 and SLA-2*0401. Animal Genetics. 44(3):251-258. DOI: 10.1111/j.1365-2052.2012.02400.x

Gladue, D.P., O'Donnell, V., Baker-Branstetter, R., Pacheco Tobin, J., Holinka-Patterson, L.G., Fernandez Sainz, I.J., Lu, Z., Brocchi, E., Baxt, B., Piconne, M.E., Rodriguez, L.L., Borca, M.V. 2012. Foot and mouth disease virus non structural protein 2C interacts with Beclin1 modulating virus replication. Journal of Virology. 86(22):12080-12090. doi:10.1128/JVI.01610-12.

Rai, D., Rieder, A.E. 2012. Homology modelling and analysis of structure predictions of the bovine rhinitis B virus RNA-dependent RNA polymerase (RdRp). International Journal of Molecular Sciences. 2012(3):8998-9013.

Uddowla, S., Hollister, J., Pacheco Tobin, J., Rodriguez, L.L., Rieder, A.E. 2012. A safe foot-and-mouth disease vaccine platform with two negative markers for differentiating infected from vaccinated animals (DIVA). Journal of Virology. Nov,86(21):11675-11685.

Brito, B.P., Perez, A.M., Jamal, S.M., Belsham, G.J., Pauszek, S.J., Ahmed, Z., Rodriguez, L.L. 2012. Foot-and-mouth disease virus serotype O phylodynamics: genetic variability associated with epidemiological factors in Pakistan. Transboundary and Emerging Diseases. DOI:10.1111j.1865-1682.2012.01366.x.

Larocco, M.A., Krug, P.W., Kramer, E., Ahmed, Z., Pacheco Tobin, J., Duque, H., Baxt, B., Rodriguez, L.L. 2013. A continuous bovine kidney cell line constitutively expressing bovine alpha v beta 6 integrin has increased susceptibility to foot-and-mouth disease virus. Journal of Clinical Microbiology. 51(6):1714-1720. DOI:10.1128/JCM.03370-12.

Ehizibolo, D.O., Perez, A.M., Carrillo, C., Pauszek, S.J., Al Khamis, M., Ajogi, I., Umoh, J.U., Kazeem, H.M., Ehizibolo, P.O., Fabian, A., Berninger, M., Moran, K., Rodriguez, L.L., Metwally, S.A. 2013. Epidemiological analysis, serological prevalence, and genotypic analysis of foot-and-mouth disease in Nigeria 2008-2009. Transboundary and Emerging Diseases. DOI:10.1111/tbed.12054.

Rai, D.K., Schafer, E.A., Singh, K., Mcintosh, M., Sarafianos, S., Rieder, A.E. 2013. Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of Foot-and-Mouth Disease Virus in host cells. Antiviral Research. 98(3):380-385 DOI:10.1016/j.antiviral.2013.03.022.

Zhu, J.J., Arzt, J., Puckette, M.C., Smoliga, G.R., Pacheco Tobin, J., Rodriguez, L.L. 2013. Mechanisms of foot-and-mouth disease virus tropism inferred from differential tissue gene expression. PLoS One. 8(5):e64119. DOI:10.1371/journal.pone.0064119.

Di Giacomo, S., Brito, B.P., Perez, A.M., Bucafusco, D., Pega, J., Rodriguez, L.L., Borca, M.V., Perez-Filgueira, M. 2013. Heterogeneity in the antibody response to foot-and-mouth disease primo-vaccinated calves. Transboundary and Emerging Diseases. DOI:10.1111/tbed.12130.

Patch, J.R., Kenney, M.A., Pacheco Tobin, J., Grubman, M.J., Golde, W.T. 2013. Characterization of cytotoxic T lymphocyte function following foot-and-mouth disease virus infection and vaccination. Viral Immunology. 26(4):239-249 DOI:10.1089/vim.2013.0011.

Maree, F.F., Blignaut, B., De Beer, T.A., Rieder, A.E. 2013. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues in virus stability. PLoS One. 8(5):e61612 DOI:10.1371/journal.pone.0061612.

Diaz San Segundo, F.C., Dias, C.C., Moraes, M.P., Weiss, M., Perez-Martin, E., Owens, G., Custer, M., Kamrud, K., De Los Santos, T.B., Grubman, M.J. 2013. Venezuelan Equine Encephalitis Virus replicon particles can induce rapid protection against Foot-and-Mouth Disease Virus. Journal of Virology. 87(10):5447-5460. DOI: 10.1128/JVI.03462-12.

Gladue, D.P., O'Donnell, V., Baker-Branstetter, R., Holinka-Patterson, L.G., Pacheco Tobin, J., Fernandez Sainz, I.J., Lu, Z., Brocchi, E., Piccone, M.E., Fletcher, P., Ambroggio, X., Rodriguez, L.L., Borca, M.V. 2013. Foot-and-mouth disease virus modulates cellular vimentin for virus survival. Journal of Virology. 87(12):6794-6803.

Last Modified: 4/17/2014
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