2007 Annual Report 1a.Objectives (from AD-416) This Project is comprosed of four objectives designed to address key areas of research related to malignant catarrhal fever (MCF) and its causative viruses, particularly ovine herpesvirus 2 (OvHV-2). Discovery of new viruses in the MCF virus group necessitates extension of current nucleic acid based diagnostic tests with an emphasis on developing rapid and reliable assays for use by veterinary diagnosticians. In addition this project seeks to define basic virus-host interactions at the molecular level in order to identify how OvHV-2 causes disease so that control strategies, including immunological based methods such as vaccination, can be developed to protect clinically susceptible ruminants. These four objectives include. 1)extend nucleic acid based diagnostic tests to include newly discovered members in the MCF virus group;. 2)define host-virus interactions in sheep and develop an in vitro propagation system for OvHV-2;. 3)define OvHV-2 gene expression within MCF lesions; and. 4)develop an immunological control strategy for MCF in clinically susceptible ruminants. 1b.Approach (from AD-416) Extend current nucleic acid-based tests for clinical diagnosis of MCF by validating the recently developed real-time PCR using a large set of defined field samples from animals with clinical MCF and by developing DNA microarray-based PCR for detection and differentiation of MCF group viruses. MCF pathogenesis will be studied in three phases:. 1)characterize lesion development in bison during preclinical and clinical stages;. 2)determine OvHV-2 genes that are highly expressed during preclinical and clinical stages using a gene expression microarray containing all 73 OvHV-2 open reading frames; and. 3)define the role of OvHV-2 gene products in MCF lesion development by developing an infectious OvHV-2 bacterial artificial chromosome (BAC) clone and testing the pathogenicity of infectious OvHV-2 BAC clones in bison, with the deletion of genes associated with lesion development. In developing an immunological control strategy for MCF in clinically susceptible ruminants, we will first characterize bison MHC class I and class II haplotypes and determine any association between MHC specific alleles and MCF resistance/susceptibility. For analysis of immune responses to OvHV-2 and development of vaccines for protection of animals from MCF losses, we will determine if animals that survive initial low dose infection with OvHV-2 are resistant to clinical MCF after a subsequent high dose challenge using our recently established animal model, bison. We will also determine which immune response dominates in the animals that survive the challenge, and finally evaluate vaccine candidates for protection against MCF in clinically susceptible ruminants. BSL-1; 9/04/06. Replacing 5348-32000-018-00D October, 2006. 4.Accomplishments 1) Validation of non-nested and real-time PCR for sheep-associated MCF diagnosis Although highly sensitive, the use of nested PCR as a routine method to detect OvHV-2 DNA for confirmation of sheep-associated MCF in diagnostic laboratories is problematic because the nested amplification format has the potential for significant amplicon contamination leading to false positive. Recent data revealed that there were high levels of OvHV-2 DNA in the blood and tissues of various animals with sheep-associated MCF, which meant that the detection of OvHV-2 DNA in clinically affected animals might not require the use of the most sensitive methods, such as nested PCR. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and at the University of Wyoming, Laramie, Wyoming, validated a non-nested PCR and a real-time PCR for diagnosis of sheep-associated MCF in clinical samples using a large set of blood and tissue samples collected from various animals, including cattle and bison with naturally-occurring and experimentally-induced sheep-associated MCF. The data confirmed that both non-nested and real-time PCR retained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples and can replace the existing nested PCR for diagnosis of sheep-associated MCF in diagnostic laboratories. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity. 2) Detection of OvHV-2 lytic replication in vivo by RT-PCR Ovine herpesvirus 2 (OvHV-2), the etiological agent of sheep-associated malignant catarrhal fever (SA-MCF), has never been propagated in vitro and little is known about OvHV-2 replication in vivo, either in its natural host or in clinically affected animals. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and at the University of Wyoming, Laramie, Wyoming, developed a reverse transcriptase PCR (RT-PCR) targeting a late lytic gene (ORF 25) that encodes the major capsid protein, and determined OvHV-2 lytic replication sites in sheep experiencing intensive virus-shedding events and in cattle and bison with SA-MCF. The data strongly suggest that OvHV-2 replication is localized to the respiratory tract of shedding sheep, predominantly in the turbinate, while it occurs in virtually all tissues of cattle and bison with SA-MCF. These findings represent an important initial step in understanding viral pathogenesis in these hosts, and in potentially establishing an in vitro propagation system for the virus. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity. 3) Characterization of MCF lesion development during preclinical and clinical stages in bison Understanding of MCF pathogenesis is critical to design effective control strategies for the disease. A basic framework of MCF lesion development, including the lymphocyte subtypes participating in the lymphoproliferative and inflammatory lesions, especially in preclinical stages, is a key step to understanding the pathogenesis. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at the University of Wyoming, Laramie, Wyoming, and at Washington State University, Pullman, Washington, developed a bison experimental model using aerosol transmission of a standardized pool of nasal secretions from shedding sheep, a route that mimics natural transmission. The data showed that the levels of OvHV-2 DNA in peripheral leukocytes of bison experimentally infected with virus are a reliable predictor of developing MCF, which allows characterization of lesion development and analysis of which viral genes are associated with the lesion development during preclinical stages. This is the first research on pre-clinical MCF in any species and identification of viral genes that are associated with lesion development will greatly contribute to our understanding of MCF pathogenesis. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity. 5.Significant Activities that Support Special Target Populations Improving MCF diagnostics has a significant impact in enhancing the effectiveness of an MCF control program especially for game farms, zoological collections and American bison producers. 6.Technology Transfer Number of new commercial licenses granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 8 Review Publications Taus, N.S., Oaks, J.L., Gailbreath, K.L., Traul, D., O'Toole, D., Li, H. 2006. Experimental aerosol infection of cattle (Bos taurus) with ovine herpesvirus 2 using nasal secretions from infected sheep. Veterinary Microbiology. 116(1-3):29-36. Vikoren, T., Li, H., Lillehaug, A., Jonassen, C.M., Bockerman, I., Handeland, K. 2006. MALIGNANT CATARRHAL FEVER IN FREE-RANGING CERVIDS ASSOCIATED WITH OVHV-2 AND CPHV-2 DNA. Journal of Wildlife Diseases. 42(4):797-807. Taus, N.S., Herndon, D.R., Traul, D., Stewart, J.P., Ackermann, M., Li, H., Knowles Jr, D.P., Lewis, G.S., Brayton, K.A. 2007. Comparison of ovine herpesvirus 2 genomes isolated from domestic sheep (Ovis aries) and a clinically affected cow (Bos bovis). Journal of General Virology. 88(1):40-45. Traul, D., Li, H., Dusgupta, N., O'Toole, D., Eldridge, J.A., Besser, T.E., Davies, C.J. 2007. Resistance to malignant catarrhal fever in American bison (Bison bison) is associated with MHC class IIa polymorphisms. Animal Genetics. 38(2):141-146 Traul, D., Taus, N.S., Oaks, J.L., O'Toole, D., Rurangirwa, F.R., Baszler, T.V., Li, H. 2007. Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples. Journal of Veterinary Diagnostic Investigation. 19:405-408