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United States Department of Agriculture

Agricultural Research Service

2011 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. Replacing 5348-32000-018-00D October, 2006.

3.Progress Report
Non-nested and real-time PCRs for routine diagnosis of Sheep Associated malignant catarrhal fever (SA-MCF) were validated and a multiplex real-time PCR for detection and differentiation of MCF viruses in clinical samples was developed. Although lacking an in vitro propagation system for OvHV-2, MCF was successfully and reliably reproduced in bison, cattle and rabbits by aerosol transmission using a pool of infectious virus collected from naturally shedding sheep. Using these systems, disease-susceptibilities among sheep, cattle and bison were determined, revealing significant differences in susceptibility among these species. Virus-host interactions in a carrier host (sheep) and clinically susceptible species (bison) were defined. Studies in sheep documented that a brief period of viral replication occurred in the lung that concurred with significant increases in immune response gene transcription and was followed by systemic dissemination of predominantly latently-infected cells in sheep. OvHV-2 replication also occurred in bison lung during initial infection. However, evidence of a vigorous, consistent local or regional immune response during pulmonary viral replication in bison was not detected, as was documented in sheep. Subsequent systemic dissemination of OvHV-2 was followed by a lytic gene expression profile in nearly all tissues preceding onset of clinical signs. Expression of the ORF25 gene, a marker for OvHV-2 lytic replication was correlated with lesion severity in bison. These data suggest that OvHV-2 changes cell tropism during initial replication in lung of both carrier and clinically susceptible hosts, and the ultimate differences between subclinical infection and disease in these species depends on how their immune functions are modulated by the virus. In vivo cell types supporting OvHV-2 lytic replication in sheep were discovered and the data showed that OvHV-2 predominantly replicates in nasal turbinate cells during transmission, while it exclusively replicates in the lung during entry. Lung epithelial cells were identified as the primary target cells for viral replication during entry and epithelial cells of nasal turbinate cells during transmission from sheep. However, attempts to propagate OvHV-2 in vitro with various cell lines, including sheep epithelial cells, were unsuccessful. To determine if rabbits are a suitable model for OvHV-2 vaccine development, OvHV-2 replication and host response was characterized in rabbits and compared to pathogenesis in bison. These studies revealed that early viral replication, subsequent systemic dissemination of OvHV-2, and host responses were similar in rabbits as compared to bison, indicating that rabbits are a suitable research model for SA-MCF, including vaccine development and pathogenesis studies. To prepare for the next 5-year research plan on immunological intervention of OvHV-2-induced MCF in bison, the genomic sequence of AlHV-2, a non-pathogenic MCF virus from African antelope (hartebeest or topi) was obtained. AIHV-2 will be used to develop a bacterial artificial chromosome clone as a vaccine backbone. New Project 5348-32000-032-00D start date 10-1-11.

1. Rabbits are a suitable animal model for sheep-associated MCF. Although MCF can be experimentally induced in rabbits, there is little information about whether viral replication and host response in rabbits are the same as in bison. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and the University of Wyoming, Laramie, Wyoming, determined the kinetics of OvHV-2 replication and evaluated host immune response gene expression following infection in rabbits, in comparison with bison. We determined when and where viral replication and dissemination occur in rabbits, and we found that there is a similarity in OvHV-2 replication and host response between rabbits and bison, suggesting that rabbits are a suitable research model for sheep-associated MCF. The study confirms that rabbits can be used as a model for MCF vaccine development and disease study prior to using bison. Development of a vaccine for MCF in bison will have a significant impact on the growing bison industry in the U.S. because MCF in bison is one of the most important infectious diseases resulting in bison death and financial losses. It will also impact the sheep industry, since sheep ranchers will no longer have to maintain separation of their animals from bison; allowing sheep and bison to occupy the same rangeland is economically beneficial to both industries.

2. Developed an alternative management control for MCF in bison. MCF can be devastating for bison that are associated with sheep due to their high susceptibility to the disease, and separation of bison from sheep is the only control strategy currently available for MCF. However, most bison producers prefer to have bison co-grazing with sheep to control rangeland weeds, instead of using herbicides, in order to maintain bison meat quality. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, and the University of Wyoming, Laramie, Wyoming, launched an alternative control strategy for MCF in bison by production of MCF-virus-free sheep that share rangeland with bison. A flock of forty lambs were raised, confirmed as MCF virus-free, and are ready to be placed with bison in the same rangeland. Success in co-grazing bison with MCF virus-free sheep without MCF risk would be a significant alternative control strategy for MCF in bison before a vaccine becomes available.

Review Publications
Li, H., Cunha, C.W., Gailbreath, K.L., O'Toole, D., White, S.N., Vanderplasschen, A., Dewals, B., Knowles Jr, D.P., Taus, N.S. 2011. Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits. Veterinary Microbiology. 150(3-4):270-7.

Last Modified: 11/27/2015
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