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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
1: Develop a defined model to identify early (pre-viremic) events in the pathogenesis of FMDV in its natural hosts. 2: Develop tools to evaluate host-pathogen interactions at the genomics level. 3: Identify critical FMDV virulence determinants associated with mechanisms of invasion, infection, replication, persistence and transmission. 4: Understand the mechanisms of FMDV persistence and the viral carrier state in animals. 5: Determine mechanisms of FMDV interference with host responses.

1b.Approach (from AD-416)
1: A defined model will be accomplished by developing and validating an inoculation model that mimics natural FMDV infections in target hosts.

2: Development of tools to evaluate the host-pathogen interaction will be accomplished through the design and development of a whole genome bovine microarray.

3: Identification of virulence determinants will be accomplished by: a.) Developing molecular tools to assess viral virulence factors. b.) Identify molecular events during FMDV-receptor interactions. c.) Identify components of the FMDV replication complex and the role of viral NSPs (non-structural proteins) in virus replication. d.) Determine the function of the FMDV 5' and 3' untranslated regions.

4: An understanding of FMDV persistence in the carrier state will be accomplished by: a.) Establishment and characterization of persistently infected cells. b.) Characterization of virus released from persistently infected cells. c.) Determine the role of autophagy in persistent infection.

5: Determination of FMDV interfernce with host responses will be completed by: a.) Examining the interference with the innate response in FMDV-infected cells. b.) Examining the mechanisms of FMDV immune evasion.

3.Progress Report
In FY 2010, we continued studies focusing on understanding the interactions between Foot-and-Mouth Disease Virus (FMDV) and cattle, one of its natural hosts using an aerosol infection method we developed in 2008. Using a combination of classic pathology, functional genomics and immunological approaches we were able to identify the cells responsible for virus replication at the primary sites of infection in the pharynx and lung. These are epithelial cells expressing cytokeratin but negative for MHC-II antigens. In the lung these infected cells were found in close association with blood vessels suggesting that the lung is likely the source of viremia early during infection. We generated an attenuated viral strain containing insertions between the two functional AUG (inter AUG) and showed that this virus did not infect beyond the primary replication site in the pharynx and was not capable of generalizing and causing clinical disease. This virus will be useful in further determining the host determinants of viral disease particularly early after infection. We continued to characterize the host proteins interacting with the 5’ untranslated region (UTR) of FMDV. We demonstrated for the first time that RNA helicase A (RHA) plays an essential role in the replication of FMDV and potentially other picornaviruses through ribonucleoprotein complex formation at the 5' end of the genome and by interactions with proteins 2C, 3A, and PABP. We also showed that reduction in the expression of RHA, using RHA-specific small interfering RNA constructs, effectively inhibited FMDV replication. We have continued to work on primary cell cultures derived from bovine pharynx (a primary site of virus replication and persistence) that are persistently infected with FMDV. We showed that these cells are refractory to superinfection with FMDV but not with vesicular stomatitis virus, indicating a novel mechanism of resistance to viral infection in persistent cells. Using microarray technology the gene expression profile of bovine cells infected with wild type (WT) or leaderless FMDV was determined. Thirty nine out of approximately 22,000 bovine genes were selectively up-regulated by 2 fold or more in leaderless versus WT FMDV infected cells. A novel biotherapeutics candidate was identified during this study that is being pursued as an anti-viral strategy. Immune evasion by FMDV during infection of cattle and swine is a central part of disease pathogenesis. Work has continued on the effect of FMDV infection on NK cells showing a decrease in function by these cells that correlates with the interruption of proinflammatry cytokine production by dendritic cells in infected animals. We have initiated studies on adaptive and CTL mediated immune responses in cattle and will continue this research in 2011.

1. Characterized early events of Foot-and-Mouth Disease Virus pathogenesis in cattle. Understanding the basic mechanisms of viral pathogenesis, including the viral and host determinants of virulence is pivotal to developing effective control and eradication tools. ARS researchers at PIADC, Greenport, NY have combined veterinary pathology with molecular biology and bioinformatics approaches in order to better understand the early (previremic) FMDV – host interaction after aerosol infection in cattle. FMDV was most consistently localized to nasopharyngeal tissues, thereby indicating this region as the most important site of primary viral replication. The earliest site of microscopic localization of FMDV antigens was cytokeratin-positive pharyngeal epithelial cells. Onset of viremia coincided with marked increase of viral loads in pulmonary tissues and with substantial decrease of viral detection in nasopharyngeal tissues. These data indicate that subsequent to aerogenous exposure to FMDV, the temporally defined critical pathogenesis events involve (1) primary replication in epithelial cells of the pharyngeal mucosa-associated lymphoid tissue crypts and (2) subsequent widespread replication in pneumocytes in the lungs, which coincides with (3) the establishment of sustained viremia. A mutant FMDV with impaired initiation of translation replicated in the pharynx but did not invade the lung, had no detectable viremia and did not cause clinical signs confirming that lung invasion is necessary for viral generalization and disease. The application of functional genomics to the understanding of the FMD virus – host interaction in a relevant animal inoculation model uncovered novel pathogenesis mechanisms and deepened our understanding of this relevant animal disease.

2. Showed inhibition of innate immune responses to early infection with Foot-and-Mouth Disease Virus (FMDV). Analysis of peripheral blood cells from swine by ARS researchers at PIADC, Greenport, NY showed a cell population that is the primary source of early anti-viral response. These cells, termed "the natural killer" (NK) cells, produce the antiviral protein interferon gamma (IFN gamma). Analysis of these NK cells isolated from animlas infected with FMDV show inhibition of the IFN gamma production, compromising the anti-viral response in infected pigs. This correlates with the interruption of similar responses by other cells, specifically dendritic cells, shown previously in infected animals. These data show that infection with FMV renders important, early responding cell populations less active, allowing the virus to spread throughout the animal and between animals.

3. Identified a key factor for Foot-and-Mouth Disease Virus replication. Work by ARS researchers at PIADC, Greenport, NY in this period has demonstrated for the first time the involvement of the RNA helicase A (RHS), a nuclear cell protein, in foot-and-mouth disease virus replication. This protein, which in normally located in the nucleus was shown to relocate to the cell cytoplasm to enhanced virus replication. The molecular mechanisms of RHA function was shown to relate to its ability to interact with both, virus proteins and with the viral genome. This information uncovered a novel host factor and aid in our understanding of the mechanism of virus-host interaction during infection.

4. Determined host-cell gene expression in response to FMDV viral infection. ARS researchers at PIADC, Greenport, NY using microarray technology the gene expression profile of bovine cells infected with wild type (WT) FMDV or an attenuated virus lacking the leader protein (leaderless FMDV) was determined. Thirty nine out of approximately 22,000 bovine genes were selectively up-regulated by 2 fold or more in leaderless versus WT FMDV infected cells. Many of these genes were involved in regulation of the innate immune responses. A novel host response gene was identified in response to FMDV infection and is currently being explored as a biotherapeutics candidate to prevent FMDV infection.

Review Publications
Piccone, M.E., Pacheco Tobin, J., Pauszek, S.J., Borca, M.V., Rieder, A.E., Kramer Jr, E.V., Zhu, J.J., Rodriguez, L.L. 2010. The region between the two polyprotein initiation codons of foot-and-mouth disease virus is critical for virulence in cattle. Virology. 396:152-159.

Arzt, J., Gregg, D.A., Clavijo, A., Rodriguez, L.L. 2009. Optimization of immunohistochemical and fluorescent antibody techniques for localization of foot-and-mouth disease virus in animal tissues. Journal of Veterinary Diagnostic Investigation. 21(6):779-792.

Nfon, C.K., Toka, F.N., Kenney, M.A., Pacheco Tobin, J., Golde, W.T. 2010. Loss of plasmacytoid dendritic cell function coincides with lymphopenia and viremia during foot-and-mouth disease infection. Viral Immunology. 23(1):29-41.

Arzt, J., White, W.R., Thomsen, B.V., Brown, C.C. 2010. Agricultural diseases on the move early in the third millennium. Veterinary Pathology. 47(1):15-27.

Rodriguez, L.L., Grubman, M.J. 2009. Foot-and-mouth disease virus vaccines. Vaccine. 27(4):D90-4.

Zhu, J.J., Weiss, M., Grubman, M.J., De Los Santos, T.B. 2010. Differential gene expression in bovine cells infected with wild type and leaderless foot-and-mouth disease virus. Virology. 404(1):32-40.

Toka, F.N., Nfon, C., Dawson, H.D., Golde, W.T. 2010. Natural killer cell dysfunction during acute infection with foot-and-mouth diseaase virus. Clinical and Vaccine Immunology. 16(12):1738-1749.

Last Modified: 8/28/2016
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