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United States Department of Agriculture

Agricultural Research Service

2009 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
Continued studies focusing on understanding the interactions between Foot-and-Mouth Disease Virus (FMDV) and its natural host cattle using functional genomics and immunological approaches. We carried out detailed characterization of the tissues and cells infected by FMDV early after infection (before generalization) and determined that epithelial cells in the pharynx play a critical role in as early replication and lung replication sites are important during generalization. The aerosol inoculation model developed in FY 2008 was used to characterize the pathogenesis of FMDV wild type and mutants generated by random and targeted mutagenesis of the FMDV cDNA infectious clones. Mutant viruses containing insertions between the two functional AUG (inter AUG) had altered leader protein (Lpro) were attenuated in cattle. A novel conserved domain (SAP) in Lpro associated with nuclear retention of this viral protease and its ability to induce NF- kappa B degradation was characterized. Mutation of the SAP domain resulted in FMDV attenuation. In preliminary studies, inoculation of swine with this mutant resulted in high neutralizing antibody titer, and protection against challenge suggesting that it could be used to derive a candidate live attenuated FMDV vaccine. Mutations in two other FMDV regions; the 3’untranslated region and protein 3B were tolerated but did not affect virulence in vivo. Continued studies of interactions of host proteins with the 5’untranslated region, showed that RNA helicase H and Sam68 form ribonucleoprotein complexes with viral RNA and 3C protease and are critical for viral growth. Cell receptor usage was determined for a SAT1 vaccine strain which possesses excellent growth properties in BHK-21 cells. This knowledge can be utilized to improve growth of poorly performing SAT vaccine strains. Primary cell cultures derived from bovine pharynx (a primary site of virus replication and persistence) were persistently infected with FMDV. Demonstrated that adaptation between virus and host cell during the persistence process, results in a stable persistent state. We demonstrated treatment with interferon gamma was capable of eliminating the FMDV infection from the persistent cells. Immune evasion by FMDV during infection of cattle and swine is a central part of disease pathogenesis. We have demonstrated that the innate responses of both dendritic cells (DCs) and natural killer cells (NK cells) are inhibited during acute infection of swine. These cells are a major source of anti-viral cytokines including type 1 and type 2 interferons. In cattle, the opposite happens, NK cells are activated during infection. These results are consistent with the observation that pigs shed much more virus than cattle during acute infection.

1. Discovered novel pathogenesis mechanisms for FMDV that may lead to safe and effective attenuated vaccines. A conserved domain (SAP) of the leader protease (Lpro) which is associated to nuclear retention and NF-kappa B degradation was identified. Mutation of the SAP domain resulted in FMDV attenuation not only in tissue culture but also in swine. When used as a vaccine, FMDV Leader SAP mutant induced a robust immune response and protected swine from FMDV challenge. This discovery can be used for the rational design of novel live attenuated FMD vaccines.

2. Demonstrated that natural killer (NK) cells are dysfunctional during acute infection in swine. One of the primary sources of interferon gamma (IFNg) in swine PBMC is NK cells. We showed that NK cells from FMDV infected pigs had little capacity to kill target cells and greatly reduced INFg secretion. We have initiated studies to understand the intracellular mechanisms that lead to reduced function of NK cells during infection.

3. Determined that the region between the two functional AUG of FMDV contain a virulence determinant for cattle. Viruses containing transposon-inserts derived from a mutagenized cDNA infectious clone of FMDV grew at a slower rate and had a smaller plaque size phenotype than the parental virus (A24-WT). When tested by aerosol inoculation in cattle mutant viruses containing insertions in inter-AUG did not cause clinical disease or viremia. However, viruses that partially or totally removed the tn insertion during animal infection reverted to virulence. Therefore, this study identified inter-AUG as an FMDV viral virulence determinant in cattle infected by aerosol route. These viral mutants are not only useful in studying FMDV pathogenesis in cattle but could also lead to live vaccine strategies.

4. Established a stable FMDV persistent culture in cells derived from the bovine pharynx. In order to study the mechanisms of induction and maintenance of FMDV persistent infections in cattle we established persistence in primary cell cultures derived from bovine pharynx, a primary site of virus replication. Persistently infected cells continue to harbor FMDV without showing appreciable cytopathic changes even after 23 passages. The persistent virus released from the culture, exhibited phenotypic changes including small-plaque morphology, and higher level of replication in pharynx cells compared to the parental virus. Characterization of persistently infected pharynx cells and viruses growing in them demonstrated an adaptation between virus and host cells during the persistence process, that resulted in a stable persistent state. Cytokine responses in persistent and acutely infected cells showed differences in a number of cytokines and chemokines involved in interferon gamma (IFNg) responses. Based on this we tested the effect of IFNg treatment on persistent cells and demonstrated that this treatment was capable of eliminating FMDV infection from the persistent cells. This in vitro persistent model will allow a better understanding of the molecular mechanisms of induction and maintenance of FMDV in carrier animals.

5. IFN gamma inhibition. One of the primary sources of interferon gamma in swine PBMC is the NK cell. We demonstrated that these cells are dysfunctional during acute infection with FMDV showing little capacity to kill target cells and greatly reduced secretion. We have initiated studies to understand the intracellular mechanisms that lead to reduced function of NK cells during infection.

6. Inhibition of the CTL response to FMDV in swine. Using a strain of pigs, the Yucatan X/X, we have developed the technology to determine whether FMDV infection or vaccination can induce cytotoxic T lymphocytes (CTL) specific for FMDV infected cells. This is a critical aspect of FMDV immune responses that is still unknown in any susceptible species. We have now demonstrated that induction of CTLs can be achieved using the Adeno5 vectored empty capsid vaccine. In collaboration with University of Copenhagen, we are generating new reagents, MHC-FMDV tetramers, to analyze the CTL response in swine.

6.Technology Transfer

Number of Invention Disclosures Submitted1

Review Publications
Golde, W.T., Nfon, C.K., Toka, F.N. 2008. Immune Evasion During Foot-and-Mouth Disease Virus (FMDV) Infection of Swine. Review Article. 225:85-95.

Piccone, M.E., Pauszek, S.J., Pacheco Tobin, J., Rieder, A.E., Kramer, E., Rodriguez, L.L. 2009. Molecular Characterization of a Foot-and-Mouth Disease Virus (FMDV) Containing a 57-Nucleotide Insertion in the 3' Untranslated Region (3'UTR). Archives of Virology. 154:671-676.

Pacheco Tobin, J., Arzt, J., Rodriguez, L.L. 2008. Early Events in the Pathogenesis of Foot-and-Mouth Disease in Cattle After Controlled Aerosol Exposure. The Veterinary Journal. 183(1):46-53.

Rainwater-Lovett, K., Pacheco Tobin, J., Packer, C., Rodriguez, L.L. 2009. Detection of Foot-and-Mouth Disease Virus Infected Cattle Using Infrared Thermography. The Veterinary Journal. 180:317-324.

De Los Santos, T.B., Diaz-San Segundo, F., Zhu, J.J., Koster, M.J., Dias, C., Grubman, M.J. 2008. A Conserved Domain in the Leader Proteinase of Foot-and-Mouth Disease Virus is Required for Proper Sub-Cellular Localization and Function. Journal of Virology. 83(4):1800-1810.

Last Modified: 8/25/2016
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