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United States Department of Agriculture

Agricultural Research Service

2008 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 2008, we continued studies focusing on understanding the interactions between Foot-and-Mouth Disease Virus (FMDV) and its natural hosts using a functional genomics approach. Pathogenesis studies included both in-vitro and in-vivo approaches utilizing an aerosol inoculation model we developed this year that mimics natural infection. Predictive biology was utilized to identify novel functional domains in FMDV leader protein. These domains were then mutagenized and demonstrated to mediate pathogenesis by retaining the viral leader protein in the nucleus where it interferes with the host innate response. In-vitro studies showed two novel virus-host interactions; interaction of the viral 5’NTR with two host proteins (RNA helicase and Sam68) that were shown necessary for efficient viral replication. We also used random insertion mutagenesis of the FMDV genome as an exploratory tool to find viral genomic regions critical for viral virulence. Viral genomic regions were identified that accepted significant insertions or deletions in untranslated and non-structural protein regions. Furthermore, a novel determinant of virulence was identified when mutagenized viruses containing insertions in near the translation initiation codons were shown to be attenuated in cattle after aerosol exposure. The FMDV tissue localization during the pre-viremic phase of infection is being studied using a trimodal approach for virus detection including virus isolation, real-time RT-PCR and immunohistochemistry. Knowledge derived from these studies will serve to identify the primary site of viral replication utilizing wildtype and mutant viruses with restricted distribution. We also developed an FMDV in-vitro persistence system in relevant primary bovine cells and characterized the virus-host cell interaction. In FY 2009, these studies will be continued to determine the molecular basis of infection, transmission and persisitence of FMDV in natural hosts. The progress of this project relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

1. Discovery of a novel domain in the Foot-and-Mouth Disease Virus (FMDV) leader protein effecting host innate response

We have previously demonstrated that a protein (Lpro) of FMDV is involved in antagonizing the natural defenses by shutting off production of proteins involved in fighting the viral infection such as interferon (IFN). Our recent studies have shown that Lpro is associated with degradation of a subunit of the transcription factor NF-kB, which further reduces host ability to fight infection. Understanding the mechanism by which FMDV antagonizes the host innate immune response may allow developing new strategies to control virus spread including the generation of possible new attenuated vaccines and tools to study viral pathogenesis.

Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

2. Development of a Foot-and-Mouth Disease (FMD) aerosol infection model in cattle

Early events of Foot-and-Mouth Disease Virus (FMDV) infection in cattle remain unclear. A novel aerosol inoculation method was developed to simulate natural, airborne transmission of FMDV and thereby allow the identification of early replication sites. Aerosol inoculation of FMDV consistently resulted in virus detection by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and viral isolation in soft palate, pharynx, and lungs with viral antigens also detected by immunohistochemistry in all of these tissues. Aerosol exposure resulted in typical FMD clinical signs. This aerosol method is highly reproducible regarding inoculum dose and volume, and allowed the detailed study of early events in FMDV-infected cattle. Our extensive post-mortem sampling and sensitive viral detection systems including virus isolation, real-time PCR and immunohistochemistry allows a more precise determination of FMDV localization than previously reported. This methodology is currently being used to characterize the tissue distribution of wild type and mutant FMDV. Deeper understanding of the early processes in viral infection will allow the development of more effective countermeasures. Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

3. Discovered two cellular proteins influencing Foot-and-Mouth Disease Virus (FMDV) growth

There is little understanding of the mechanisms that FMDV uses to grow inside cells. Foot-and-Mouth Disease (FMD) relies heavily on the interaction of host cell and viral proteins with viral RNA structures for many aspects of the virus life cycle. The interruption or modification of such interactions can have a major effect in pathogenesis and might result in attenuated viruses. Using bioinformatics, biochemical and genetic approaches, we have identified two cellular RNA-binding proteins required for FMDV replication. These host-cell proteins (RNA helicase A (RHA) and Sam68) were shown to bind to the viral RNA and relocalize from the nucleus to the cytoplasm as a result of FMDV infection. Moreover inhibition of RHA-gene expression by siRNA resulted in significant reduction of viral titers. This discovery will help in development of effective anti-FMDV approaches. Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

4. Inhibition of the innate immune response and immune evasion during Foot-and-Mouth Disease Virus (FMDV) infection

Infection with Foot-and-Mouth Disease (FMD) results in a decrease of the host defenses by various mechanisms that are not clear. Our research showed that activation of natural killer (NK) cell response capable of killing FMDV-infected fibroblasts is greatly inhibited in NK cells isolated from FMDV infected swine. Activated natural killer cells (NK Cells) obtained from healthy swine have low cytotoxicity for porcine fibroblast cell lines but are efficient at killing the same cells after they are infected with FMDV. We also showed that FMDV infection in pigs decreases the ability of dendritic cells to produce interferon alpha in response to virus. This discovery will focus efforts to develop antiviral therapeutics for FMDV outbreaks. Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

5. Developed Infrared thermography for detection of Foot-and-Mouth Disease Virus (FMDV) infected cattle

The development of pen-side screening tests to quickly identify potentially infected animals for confirmatory diagnostic testing has the potential to save not only the lives of animals but also time and effort of farmers, veterinarians, laboratory technicians, and government officials. We assessed infrared thermography (IRT) as a means of detecting Foot-and-Mouth Disease Virus (FMDV)-infected cattle before the development of clinical signs. Preliminary IRT imaging demonstrated that foot temperatures increased in FMDV-infected animals. A pre-clinical cut-off value was established at 34.4ºC (sensitivity=61.1%, specificity=87.7%) with the aim of detecting FMDV-infected animals in herds. With this test, we are able to detect FMDV-infected animals 24 to 48 hours prior to the development of foot vesicles. These results show IRT as a promising screening technology for detection of FMDV-infected animals with potential application during FMDV outbreaks. Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

6. Genomic characterization of bovine rhinovirus 2; the closest relative of Foot-and-Mouth Disease Virus (FMDV)

Foot-and-mouth disease virus (FMDV) is one of the most contagious agents known to man and causes disease of devastating economic consequence in livestock. Therefore a high level of biocontainment is required to carry out research with this agent. In fact in the USA the only laboratory allowed to work with FMDV is Plum Island Animal Disease Center. In an effort to look for potential surrogate viruses that allow working at lower biosafety levels we have determined the first full genomic sequence (except for a small section on non-translated sequence) and organization of bovine-rhinovirus type 2 (BRV-2). This virus was shown to be the closest relative to FMDV described to date. However, unlike FMDV this virus causes only mild respiratory disease in cattle and therefore it can be studied without the requirement of stringent biocontainment. This is an important discovery since BRV-2 has the potential to be used as a model for dissecting the molecular biology and pathogenesis of FMDV.

Relates to component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Diseases of the National Program in Animal Health, within the NP 103 Action Plan.

5.Significant Activities that Support Special Target Populations
Nothing to report.

6.Technology Transfer

Number of the New MTAs (providing only)2
Number of Non-Peer Reviewed Presentations and Proceedings11

Review Publications
Fowler, V.L., Paton, D.J., Rieder, A.E., Barnett, P.V. 2008. Chimeric Foot-and-Mouth Disease Viruses: Evaluation of Their Efficacy as Potential Marker Vaccines in Cattle. Vaccine. 26:1982-1989.

Vagnozzi, A., Stein, D.A., Iversen, P.L., Rieder, A.E. 2007. Inhibition of Foot-and-Mouth Disease Virus in Cell Culture with Antisense Morpholino Oligomers. Journal of Virology. 81:11669-11680.

Rieder, A.E. 2007. Picornaviridae. Book Chapter. 2007. P523-545.

Oem, J., Yeh, M., Mckenna, T., Hayes, J.R., Rieder, A.E., Giuffre, A.C., Robida, J.M., Lee, K., Cho, I., Fang, X., Joo, Y., Park, J. 2008. Pathogenic Characteristics of the Korean 2002 Isolate of Foot-and-Mouth Disease Virus Serotype O in Pigs and Cattle. Journal of Comparative Pathology. 138(4):204-214.

Last Modified: 8/27/2016
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