1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
FAD continue to be a highly significant animal health concern. Today, the threat posed by these diseases is unprecedented due to the increasing globalization of trade, movement of people and agricultural products and changes in farm practices including movement to more intensive agriculture. Recent FAD outbreaks in Europe and Asia (1997-2001) have been catastrophic resulting in devastating economic consequences for the countries involved. A similar scenario would be expected should these diseases be introduced into the United States. New disease control strategies are needed to reduce this threat.

New disease control strategies are needed to reduce the threat posed by high consequence foreign animal diseases (FAD) . Functional pathogen genomics ' offers an unprecedented opportunity for addressing this critical issue. High throughput pathogen genome sequencing together with detailed genome analysis will be used to provide improved tools for pathogen detection, disease diagnosis and epidemiological disease investigations, improved vaccines and therapeutics, and in all likelihood, completely novel disease control strategies with increased efficacy and utility. Specifically the goal of this research is to:.
1)Develop methods for rapid strain identification and epidemiological investigations for key FAD agents;.
2)Develop rapid field diagnostic tests for key FAD agents;.
3)Develop improved vaccines for key FAD; and.
4)Develop new methods on strategies for efficient and effective disease control.

2.List the milestones (indicators of progress) from your Project Plan.
1) Develop rapid diagnostic tests based on microbial genomics, nucleic acid sequences and immuno-assays for key Foreign Animal Disease Agents (FAD); 2) Develop improved vaccines and vaccine delivery vectors for key FAD; 3) Develop improved methods for pathogen detection on microbial genomics, nucleic acid sequences, and immuno-assays.

3.Milestones:
Future research work in FY-05: Comparative genomic analysis will be used to identify optimal genomic targets for strain identification and epidemiological investigations of key FAD high consequence agents. Genomic targets will be used to develop rapid PCR-based field detection assays. Evaluation of Lumpy Skin Disease assays will be completed. Comparative genomics and genetic techniques will be used to identify and characterize pathogen virulence, host range and immune evasions mechanisms, targets that may be exploited to improve vaccine design and/or delivery or to develop novel intervention/therapeutic approaches.

FY-06: Comparative Genomics and assay development/evaluation for Rinderpest virus will be completed. CBPP genomic targets will be identified and initial evaluation of CPBB diagnostic tests is anticipated

FY-07: Complete evaluation of CBPP diagnostic test and initiate work on other identified priority FAD pathogens.

4.What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishments during FY 2004: Comparative analysis of 103 FMDV genomes representing all seven serotypes was completed. Data reveal novel highly conserved genomic regions indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Genomic regions/signatures with potential epidemiological and forensic value were identified. Phylogenetic incongruencies observed between different viral genomic regions suggested FMDV recombination is more prevalent in nature than previously thought. A novel virus lineage containing nonstructural proteins distinct from both SAT and euroasiatic lineages was identified. Work significantly impacts understanding of FMDV genome variability, evolution and ecology.

B. Other Significant Accomplishment(s) if any: FMDV genetic variability (rates of mutation) during passage in the animal host was assessed. FMDV was serially passed 13 times by contact transmission in pigs. Resulting viruses at each passage level were sequenced and analyzed to identify genetic changes. In vivo, substitutions per nucleotide per infectious cycle were found to be approximately 100-fold lower than those previously reported for virus passed in vitro with non-synonymous substitutions significantly reduced. Notably, no changes were observed for the structural protein coding region of the genome during in vivo passage. Most significantly, results indicate that extrapolation of variability data from in vitro experiments are not accurate and that FMDV mutation rates have been overestimated. These results will permit improved phylogenetic and forensic analysis of FMDV as well as providing new insights into strain origin, routes of transmission and aspects underlying host range.

C. Significant Activities that Support Special Target Populations: None.

D. Progress Report: This report serves to document research conducted under a Reimbursable Agreement (1940-32000-039-01R - Agreement # 01-1940-0-0021) between ARS and the Navy Engineering Logistics Office/Department of the Navy. Comparative analysis of 103 FMDV genomes representing all seven serotypes was completed. Data reveal novel highly conserved genomic regions indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Genomic regions/signatures with potential epidemiological and forensic value were identified. Phylogenetic incongruencies observed between different viral genomic regions suggested FMDV recombination is more prevalent in nature than previously thought. A novel virus lineage containing nonstructural proteins distinct from both SAT and euroasiatic lineages was identified. Work significantly impacts understanding of FMDV genome variability, evolution and ecology.

This report serves to document research conducted under a Reimbursable Agreement (1940-32000-039-02R - Agreement # 01-1940-0-0039) between ARS and The Defense Advanced Research Projects Agency. RHKO cell libraries where both alleles of random genes are non-functional were developed and screened for resistance to African swine fever virus (ASFV) and foot-and-mouth disease virus (FMDV). Cell clones resistant to both viruses were obtained. Cell clones were assessed to define where in the virus life cycle replication was blocked. In most cases, the level of host cell restriction involved an early event that was independent of virus attachment or entry into the cell. To define molecular mechanisms of resistance, transcriptional profiling of both uninfected and infected cells is being used to identify cellular genes, cell pathways and regulatory networks affected in the resistant clones. Identification of cellular genes associated with resistance to viral infection opens completely new possibilities for disease prevention and/or control. Information will be useful for developing novel anti-viral therapeutics and for increasing natural disease resistance in livestock species.

This report serves to document research conducted under a Reimbursable Agreement (1940-32000-039-03R - Agreement # 01-1940-0-0046) between ARS and the Central Intelligency Agency. Comparative analysis of 103 FMDV genomes representing all seven serotypes was completed. Data reveal novel highly conserved genomic regions indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Genomic regions/signatures with potential epidemiological and forensic value were identified. Phylogenetic incongruencies observed between different viral genomic regions suggested FMDV recombination is more prevalent in nature than previously thought. A novel virus lineage containing nonstructural proteins distinct from both SAT and euroasiatic lineages was identified. Work significantly impacts understanding of FMDV genome variability, evolution and ecology.

This report serves to document research conducted under a Specific Cooperative Agreement (1940-32000-039-04S - Agreement #58-1940-0-0007) between ARS and the University of Connecticut. Collaborations are ongoing for the three-way consortium (USDA-ARS, University of Missouri, University of Connecticut) to address problems in foreign and domestic animal diseases, both in the design and development of new and improved vaccines as well as improved or new diagnostic tests..
1)We further developed and refined our M. gallisepticum vaccine platform [a derivative of the avirulent Rhi(GT5)] to ascertain correlates of immune protection in chickens and assessed its' efficacy as a modified live vaccine. We have gained a more complete understanding of the correlates of immune protection generated by the GT5 vaccine. These include high levels of M. gallisepticum specific IgG and IgA secreting plasma cells, the absence of inflammatory cells, especially heterophils, and a vigorous and rapid memory response (both systemically and mucosally). Additionally, we have identified a novel virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd) using signature sequence mutagenesis (SSM). A transposon insertion in the middle of the lpd coding sequence resulted in the reduced virulence of the mutant designated as Mg 7. This mutant is currently being evaluated as a vaccine candate;.
2)Porcine Reproductive and Respiratory Syndrome (PRRS): Experiments were undertaken to characterize the virus-cell interaction, persistent infection, and the role of the interferon system; and.
3)Development of Mucosal Epitope based Vaccines for FMDV: We have completed the cloning, site-directed mutagenesis and sequencing of the detoxified Pseudomonas exotoxin gene, and have engineered the cyclic FMDV G-H loop into this framework. This chimeric molecule will contain a conformationally constrained loop portion that may stimulate conformation-dependent B cells in a way that linear peptides cannot. In addition, this chimeric protein will have abundant T helper epitopes, and is well documented in its ability to cross mucosal surfaces via CD91 receptors. The ntPE/FMDV G-H loop protein has been expressed as a GST fusion protein, and studies are currently being conducted to obtain a properly folded protein. Gel electrophoresis/Western blotting has shown that the expressed protein is immunoreactive with both anti-FMDV and anti-GST antisera. Immunogenicity studies will begin once a properly folded product has been obtained.

We have completed the design of five cyclic, chimeric peptides to maximize the immune response to more broadly cross-reactive B cell epitopes. Preliminary studies conducted in mice has shown that manipulations in specific regions of the G-H loop can induce more broadly cross-reactive antibody responses to a more highly conserved B cell epitope. We have completed the cloning of two constructs encoding a B cell epitope, plus and minus a highly conserved T cell epitope from VP4. These constructs were sent to our collaborators at the AlphaVax Inc. and virus replicon particles (VRPs) produced. These VRPs will be tested in mice to determine their efficacy in inducing systemic and mucosal immunity to G-H loop epitopes.

4) Edible Vaccines: .
1)Our goal was to express the various synthetic DNA constructs encoding the codon-optimized VP1 immunodominant epitope of FMDV Taiwan serotype O in maize. We succeeded in the first round of maize transformations, and more importantly, we were able to regenerate transformed callus into mature plants in house. Gel electrophoresis/ Western blotting has confirmed that individual maize plants are expressing the fusion protein containing the B subunit of the heat labile enterotoxin of E. coli and the G-H loop epitope. These plants will be used in subsequent feeding studies of mice to determine their utility in inducing systemic and mucosal immunity; and.
2)Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. The interaction involves an adhesin called P97.We have constructed a complex gene cassette using the ACTIN promoter to express the R1 domain (15 AAKPV repeats) of P97 including the NOS terminator sequence at the 3' end. We have now generated transgenic plants from each putative transgenic callus line and produced hundreds of corn shoots. PCR and western blots have confirmed the insertion and expression of the transgenes in the putative transgenic lines. We have grown these transgenic lines and produced large amounts of seeds for animal feeding tests, which are currently ongoing.

This report serves to document research conducted under a Specific Cooperative Agreement (1940-32000-039-05S - Agreement # 58-1940-0-0008) between ARS and the University of Missouri. Collaborations are ongoing for the three way consortium (USDA-ARS, University of Missouri, University of Connecticut) to address problems in foreign and domestic animal diseases, both in the design and development of new and improved vaccines as well as improved or new diagnostic tests.

a. Mycoplasma diseases: At the end of the last reporting period, the Missouri team and collaborating institutes determined the complete genome sequence of Mycoplasma capricolum subspecies capricolum Kid. Computational and human annotation was continued throughout the current reporting period. In addition, access through collaborators to the unpublished genome sequence of Mycoplasma mycoides subspecies mycoides SC, a foreign threat agent pathogen of cattle, allowed a comparison of these key species to be initiated. The latter sequence was recently published (Genome Res. 14:221-224, 2004). This first preliminary view of a genomic "framework" for the closely related "M. mycoides cluster" of bovine and caprine mycoplasma species has revealed genomic similarities at multiple levels and underscores the need for detailed comparisons of the genomes from multiple species in this cluster, along with selective other mycoplasmal pathogens, particularly those of cattle. Multiple families of genes encoding surface proteins were identified in each genome. Each family is predicted to be structurally or antigenically analogous (but not identical) between species and is subject to high frequency phase variable expression, an important feature that has been experimentally documented. In addition to antigenic complexity that will affect strategies for development of protein based diagnostics and vaccines, the striking similarity of large genomic regions further suggested that effective nucleic acid-based diagnostics to discriminate species of the cluster will ultimately require targeted use of selected genomic differences. In addition, mobile genetic elements were identified in each genome that may reveal the role of lateral gene transfer in genomic diversification, and may provide exploitable tools for genetic manipulation of these mycoplasmas. Two additional mycoplasma pathogens of cattle, Mycoplasma bovine serogroup 7 (from the "mycoides cluster") and M. bovis, have been evaluated and experimentally characterized as high priority organisms for genome sequencing. In this reporting period, we established a collaborative agreement with TIGR to complete the M. bovis (type strain PG45) sequence, a project entering the final closure stage. Computational and human annotation will begin immediately and carry through the next reporting period. Initiation of the next genome-sequencing project will focus on M. bovine serogroup 7 and an additional, well-characterized isolate of M. mycoides subspecies mycoides SC. In parallel studies of swine mycoplasmas (M. hyopneumoniae, M. hyorhinis, M. flocculare), islands of genes that have possibly been subject to rearrangement through their linkage to common mobile genetic elements were further characterized. They appear to reflect lateral gene transfer that has shaped the genomic architecture of these pathogens, which reside in a common host niche.

b. Mycobacterium vaccines in cattle: In this project period, the PI for this component of the program moved from MU to UTMB in Galveston. The project is being completed with a final vaccine trial being undertaken presently at NADC. During the reporting period, initial trials were completed to evaluate costimulatory molecules as a means to enhance the development of memory and effector cytolytic T cells in cattle. An aerosol challenge model with Mycobacterium bovis was used to determine the potential of this approach using a subunit DNA vaccine platform. Initial studies demonstrated reduced pathology in costimulatory molecule treated animals, compared to controls receiving vector only. Results were analyzed in comparison to M. bovis BCG, the current standard for TB vaccines. Additional studies evaluated the effector pathways involved in direct killing of intracellular mycobacteria. Granulysin and perforin homologs were identified for the bovine. These studies have indicated that potent antimicrobial activities occur in various domains of the granulysin molecule, the only known lytic peptide with direct anti-mycobacterial activities. A mouse homolog for this protein has not been identified to date.

c. PRRSV: Using the published PRRSV infectious clone (J. Virol 77:3702-3711), which is derived from the virulent prototype isolate VR-2332, a series of chimeras have been constructed with the corresponding genome fragments of the attenuated vaccine strain Ingelvac ATP. In vitro growth curves demonstrated that all chimeras have similar growth properties in Marc-145 cells. However inoculation of pigs demonstrated that chimera 4 (which incorporates the 3' end of the attenuated viral genome into VR-2332) resulted in markedly reduced lung lesions at 14 days p.i. Viremic titers were also lower for this construct compared to the parental (clone-derived) strain. The in vitro interferon phenotype of PRRSV field strains has been characterized. Marked strain differences were noted although all isolates tested suppressed the interferon response in vitro.

d. Ascaris sterilization using RNA interference (RNAi): We had previously identified two expressed genes in Ascaris suum, actin and iff-1 (initiation factor five A-1), that have at least one region of a greater than 21-nucleotide exact match with the model organism, the free-living nematode Caenorhabditis elegans and no 21-nt match with mammalian DNA. After amplifying an ~500-bp fragment from each gene from reverse transcribed Ascaris ovarian RNA and adding T7 polymerase sites on both ends, dsRNAs from each were produced and tested for their ability to sterilize by RNAi either the injected worm or its offspring. Both these genes have worked to cause sterility using C. elegans as a tester strain. The degree of this effect of the Ascaris gene approaches similar effects of RNAi with the endogenous C. elegans gene. We have used C. elegans for these initial studies because parasitic Ascaris worms, although ubiquitous in domestic swine, cannot be maintained in culture in the laboratory. Several other genes have also been identified that meet the criteria used to select the two successful genes and these are currently being tested.

e. Additional viral pathogens: In this period, two exploratory projects were initiated to exploit the expertise of well-established MU faculty to study the molecular pathobiology of viral diseases affecting swine and cattle.

Porcine circovirus: The long-term goal of this pilot project is to identify and characterize the viral determinants of porcine circovirus 2 (PCV2) that contribute to the pathogenicity of this emerging swine pathogen. Critical aspects of the replication of PCV in tissue culture systems were examined. PCV RNAs were identified and quantified following infection of in PCV-free PK15 cells and in primary porcine lymphocytes. The functional significance of the PCV minor RNAs and their putative protein products in viral replication are now being assessed. We will address whether porcine parvovirus (PPV) enhances PCV2 replication and virus production in swine lymphocytes. PPV is known to increase PCV2 pathogenesis in pigs. Successful completion of the experiments proposed should give a much more detailed characterization of the expression of PCV RNAs and the role that the various nonstructural proteins play during infection.

Bovine herpesvirus 1 latency: Infection of cattle with bovine herpes virus (BHV-1) can result in respiratory disease, vulvovaginitis, balanoposthitis, abortion, and possibly neurological disease. Following replication at the initial site of infection, BHV-1 establishes a latent infection in sensory ganglia neurons. Periodically, virus is reactivated from neurons and transported via axonal transport back to the initial sites of infection. In general the molecular mechanisms that regulate latent infections of BHV-1 are not well understood and there are no in vitro models for studying regulation. The HSV-1 IE proteins ICP4 and ICP0 are key transcriptional regulators of HSV-1 genes. Studies with transgenic mice containing ICP4 and ICP0 reporter constructs have shown that neurons differentially regulate these viral promoters in the absence of viral proteins and that the ICP0 promoter was regulated in neurons in transgenic mice by stimuli know to reactivate virus in vivo, demonstrating the need for host transcription factors. The following hypothesis is being examined: BHV-1 IE gene promoter (IEtu1) is regulated by host neuronal transcription factors in vivo in the absence of viral proteins. In order to examine the regulation of the BHV-1 IEtu1 promoter in vivo, transgenic mice will be generated that contain the IEtu1 promoter fused to the E. coli ß-galactosidase coding sequence . As a control, a construct containing a late gene, gC, promoter fused to the lacZ coding sequence will be used. UV irradiation, hyperthermia, and glucocorticoids are stimuli that are known to cause reactivation of alphaherpesviruses from neurons. Experiments will be performed to determine whether the BHV-1 IE promoter responds to signals generated by these reactivation stimuli.

This report serves to document research conducted under a Specific Cooperative Agreement (1940-32000-039-06S - Agreement # 58-1940-2-0225) between ARS and the University of Nebraska. Sensitivity, specificity and diagnostic fitness of FMDV NSP assays were evaluated. In collaboration with INTA, Argentina four commercially available NSP tests were compared using serum samples representing vaccinated, vaccinated/infected and infected animals. Test sensitivities were 0.98, 0.97, 0.79 and 0.74. High sensitivities observed for two of the four tests indicate their potential usefulness in FMD control and eradication programs where discrimination between vaccinated and infected animals is essential.

This report serves to document research conducted under a Reimbursable Cooperative Agreement (1940-32000-039-07R - Agreement # 60-1940-3-0014) between ARS and the The Defense Threat Reduction Agency. FMDV genetic variability (rates of mutation) during passage in the animal host was assessed. FMDV was serially passed 13 times by contact transmission in pigs. Resulting viruses at each passage level were sequenced and analyzed to identify genetic changes. In vivo, substitutions per nucleotide per infectious cycle were found to be approximately 100-fold lower than those previously reported for virus passed in vitro with non-synonymous substitutions significantly reduced. Notably, no changes were observed for the structural protein coding region of the genome during in vivo passage. Most significantly, results indicate that extrapolation of variability data from in vitro experiments does not provide accurate estimates and that FMDV mutation rates have been overestimated. These results will permit improved phylogenetic and forensic analysis of FMDV as well as providing new insights into strain origin, routes of transmission and aspects underlying host range.

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
Rapid field-based assays to detect foot and mouth disease virus (FMDV) are not yet available. A rapid RT-PCR assay was developed and evaluated in experimentally infected animals. The assay was both highly sensitive and specific and, it detected the virus before the animals showed signs of disease. Impact: A rapid (1 - 1 % hr) field-based diagnostic assay for FMDV provides a new and powerful tool that in all likelihood will revolutionize disease management and control strategies for this highly significant viral disease.

Canarypox virus (CPV) is a increasingly important mammalian vaccine vector; however, little information regarding the canarypox genome is currently available. The 360 kpb CPV genome was sequenced and compared to fowlpox virus, a related avipoxvirus of domestic birds. Analysis revealed 325 genes, including those conserved with fowlpox virus, other novel genes in expanded gene families, and virulence and host range genes unique to CPV. Impact: Knowledge of the entire coding capacity of CPV has identified specific genes which may affect unique aspects of CPV virulence and host range, allowing more directed studies to determine gene function in the host. Engineering of improved and more efficacious CPV vaccine vectors will result.

Poxviral infections in wild animal species are increasingly documented, indicating the presence of other, yet to be characterized poxviruses circulating in wild animal species. These can be confused with FAD viruses. Two deerpox viruses isolated in the western U.S. from free ranging mule deer were sequenced. Both virus genomes of 165 kbp shared 95-6 nucleotide identity. They differed from other known poxviruses both in central conserved regions and in terminal genomic regions where they contained a unique complement of virulence and host range genes. Impact: Genomic information will permit design of improved diagnostic assays to distinguish between this virus and other exotic poxviruses associated with FAD of domestic animals.

Parapoxviruses, Orf virus (ORFV) and bovine popular stomatitis virus (BPSV) cause diseases in livestock in the United States. Significantly, these diseases are look-alike diseases, for high consequence FAD. Complete genomic sequences from two ORFV and one BPSV strain were obtained and analyzed. Genomes ranged in size from 134 to 139 Kbp encoded 130 and 131 genes, respectively, and shared colinearity over 127 genes. Genomic differences (71% amino acid identify) distinguish BPSV and ORFV as two PPV species. Genes unique to these viruses including those with putative virulence and host range functions were identified. Impact: Genomic information will permit design of improved diagnostic assays to distinguish between these viruses and other high consequence exotic poxviruses associated with FAD of livestock. In addition, viral genomic information may permit rational design of improved and very badly needed ORFV vaccines.

National Program 103, Animal Health (100%). This research, addresses Outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2 and 2.1.4 of the ARS strategic plan.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Rapid FAD diagnostic tests will be developed, evaluated and transferred to APHIS in the next 12-18 months.

Review Publications
Delhon, G., Moraes, M.P., Lu, Z., Afonso, C.L., Flores, E.F., Weiblen, R., Kutish, G.F., Rock, D.L. 2003. Genome of bovine herpesvirus 5. Journal of Virology. 77(19):10339-10347.