2008 Annual Report 1a.Objectives (from AD-416) The goals of this project are to improve the industry's ability to detect and control viral infections of ruminants (principally cattle), with an emphasis of bovine viral diarrhea viruses (BVDV). Detection will be improved by the generation of robust field-ready tests that both detect and differentiate viral pathogens. Limiting and controlling viral infections will require research efforts to generate a more thorough understanding of viral/host interactions and identifying the mechanism(s) behind viral pathogenesis. This project has three objectives: Obj. 1. Develop innovative methods to detect infection in individual animals and to conduct surveillance of animal populations for endemic viral pathogens Obj. 2. Elucidate mechanisms involved in the development of and identify means to detect prolonged and persistent viral infections. Obj. 3. Develop tools to measure and means to limit impact of ruminant infection with viral pathogens. 1b.Approach (from AD-416) This project focuses on filling knowledge and technology gaps that hamper the detection and control of bovine viral diarrhea viruses (BVDV). Reduction of BVDV infections in the national herd requires effective surveillance programs, efficacious vaccines, and reliable means to detect both persistent and acute infections. As the BVDV control program progresses, there will be a need to differentiate between natural exposure and vaccination. Knowledge gaps to be addressed include: basic information regarding BVDV infection in wild cervids; the nature of the immunological, cellular and physiological changes accompanying the development of persistent infections; and the responsiveness of the neonate to vaccination. In addition, surveillance for newly arising BVDV variations will be maintained. Technology gaps will be addressed by: assessing new diagnostic readout methods; examining possible methods for detection of persistently infected animals in utero; and exploring marker vaccine development. Approaches to achieve these goals are to: examine the use of atomic force microscopy and Raman spectroscopy as readout methods; characterize BVDV infections in fawns and pregnant white tail deer; work with a network of laboratories to identify and characterize variant BVDV; compare fetal and associated maternal tissues from normal and persistently infected fetuses; evaluate the use of an infectious clone as a marker vaccine; and characterize the immune responses of neonates to vaccination. Some techniques to be used include phylogenetic analysis, comparative pathogenesis, genetic engineering, and serial analysis of gene expression (SAGE). 3.Progress Report In order to meet research goals described in objective 1 of our project plan we conducted collaborative research with the Nanotechnology Biosensors group of the Department of Chemistry and Chemical Engineering at the University of Utah. We also studied the impact of Bovine Viral Diarrhea Virus (BVDV) infection on reproduction in deer and characterized BVDV strains isolated from the field. Results of these activities included a prototype test, using surface enhanced Raman spectroscopy (SERS) as the readout technology, that allows detection of two viruses simultaneously. We also established a model for studying reproductive disease in deer following BVDV infection and the first histopathological study of persistently infected deer generated under experimental conditions. BVDV strains that escaped detection by commonly used commercial tests were characterized and a new species of pestivirus, isolated following outbreaks of reproductive disease in pigs, was fully sequenced. We also assessed an in vitro model for examination of BVDV infection in cells associated with the innate immune system that was developed, began construction of an infectious clone and expressed BVDV proteins using a replication-defective adenovirus vector and observed the development of viral neutralizing titers in dairy and beef cattle raised under commercial production conditions following different vaccination protocols. Projects focusing on vaccination of neonatal calves revealed that management practices have profound impact on the efficacy of vaccination. These findings have led to a new collaboration with Iowa State University, as well as, preliminary studies in full collaboration with USDA, ARS, Meat Animal Reseach Center (MARC) on developing a model to measure the genetic basis for immunocompetence in immunologically-stressed cattle. The goal of this research will be to identify bulls whose offspring are more immunocompetent following weaning and transport/commingling stress. In addition collaborated with the researchers and diagnosticians at the Elizabeth Macarthur Agriculture Institute in New South Wales, Australia to compare and characterize pestivirus isolates originating in Australia and the U.S. Research efforts supported by Cooperative Research and Development Agreement (CRADA) and trust agreements resulted in the evaluation of a new vaccine adjuvant, comparison of sensitivity and specificity of a new commercial BVDV tests to current tests in use in diagnostic laboratories and selection of new BVDV vaccine and challenge strains. We assisted the USDA Animal and Plant Health Inspection Service (APHIS) in characterizing BVDV vaccine master strains and in designing sampling protocols for the National Animal Health Monitoring System (NAHMS). This research addresses National Program 103 - Animal Health Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases. 4.Accomplishments 1. Title: Established prevalence and antigenic differences between bovine viral diarrhea virus subgenotypes isolated from cattle in the U.S. and Australia. Problem: Phylogenetic analysis has revealed subgenotype groupings within the bovine viral diarrhea virus (BVDV) 1 and BVDV2 species. Thus far twelve BVDV1 subgenotypes (BVDV1a through BVDV1l) and two BVDV2 subgenotypes (BVDV2a and BVDV2b) have been identified. The practical significance of segregation into subgenotypes is still a matter of discussion. Activity: Phylogenetic comparison of 298 pestivirus isolates of bovine origin archived at the Elizabeth Macarthur Agriculture Institute in New South Wales, Australia to 514 bovine pestiviruses isolated from U.S. between June 2007 and June 2008 revealed that the predominant subgenotype, BVDV1c, found in the Australian was not found in U.S. samples. The practical significance of differences in prevalence for subgenotypes was suggested by serological immune responses mounted by 40 co-housed feedlot cattle persistently infected with different BVDV1 strains. These animals developed differential responses to strains from the same subgenotypes as the persistent virus compared to strains belonging to other subgenotypes. Impact: These studies suggest that there are biologically important antigenic differences between BVDV strains belonging to different subgenotypes. These studies suggest that vaccine protection can be improved by basing vaccines on the BVDV subgenotypes that are prevalent in the region in which the vaccine is to be used. National Program 103 Animal Health: Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases. Problem Statement 5A: Bovine Viral Diarrhea (BVD). 2. Title: Development of model for examining changes in gene expression resulting from BVDV infection. Problem: BVDV are lymphotrophic and exist as two biotypes, cytopathic and noncytopathic, and exhibit varying levels of virulence based on destruction of lymphoid cells. The molecular mechanisms behind these different effects are unknown. Activity: To gain a better understanding of impact of BVDV infection on lymphoid cells, serial analysis of gene expression (SAGE) was used to compare gene expression in BVDV-infected cells to non-infected cells. The B-cell lymphosarcoma cell line, BL3, was used as an in vitro bovine lymphocyte model. SAGE revealed that there were some significant changes in gene expression in BVDV-infected cells. Some of the most profound and unexpected changes were increases in transcripts encoding components of the AP-1 transcriptional complex that may contribute to cell death in cells infected with cytopathic BVDV strains. Impact: Immunosuppression resulting from BVDV infection is the result of complex processes. Due to animal-to-animal variation and intricacies involved in controlling all variables it is difficult to study immunosuppression in vivo. This research generated an in vitro model for examining changes in gene expression in lymphoid cells that occur in response to BVDV infection. It provides researchers with an alternative method that does not require the use of research animals and allows greater control of variables. National Program 103 Animal Health: Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases. Problem Statement 5A: Bovine Viral Diarrhea (BVD). 3. Title: Characterization of reproductive disease and persistently infected fawns resulting from infection of white-tailed deer with BVDV. Problem: Design of effective programs geared toward the eradication of BVDV in domestic cattle requires an understanding of BVDV infections in wild ungulates frequently in contact with domestic cattle. There remain large gaps in knowledge regarding BVDV infection in non-bovine species, particularly in regards to reproductive disease and persistent infection in these species. Activity: White-tailed does were infected with BVDV, isolated from deer in the field, during the first trimester of pregnancy. Infection resulted in death or reproductive failure (abortion, readsorption, stillbirth) in 11 out of 13 naïve does. Histological and immunohistochemical examination of persistently infected fawns revealed that BVDV antigen was distributed widely throughout many tissues and cell types, most notably epithelium and vascular endothelium, consistent with that reported in cattle. In contrast to cattle, lymphocytes exhibited only very rare positive staining. These findings indicate that BVDV infection results in clinically severe reproductive disease in deer and that there may be differences in the way the virus behaves in deer compared to cattle. Impact: These findings suggest that the impact BVDV reproductive disease in deer may be under appreciated and that because the virus may be spread differently from deer than cattle different control strategies may need to be adopted. National Program 103 Animal Health: Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases. Problem Statement 5A: Bovine Viral Diarrhea (BVD). 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 2 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Ridpath, J.F., Mark, C., Chase, C.L., Ridpath, A.C., Neill, J.D. 2007. Febrile response and decrease in circulating lymphocytes following acute infection of white tail deer fawns with either a BVDV1 or a BVDV2 strain. Journal of Wildlife Diseases. 43(4):653-659. Neill, J.D., Ridpath, J.F. 2008. Increase in proto-oncogene mRNA transcript levels in bovine lymphoid cells infected with a cytopathic type 2 bovine viral diarrhea virus. Virus Research. 135(2):326-331. Kirkland, P.D., Frost, M.J., Finlaison, D.S., King, K.R., Ridpath, J.F., Gu, X. 2007. Identification of a novel virus in pigs - Bungowannah virus: a possible new species of pestivirus. Virology. 129(1):26-34. Ridpath, J.F. 2008. Bovine viral diarrhea virus. In: Mahy, B.W.J., Van Regenmortel, M.H.V., editors. Encyclopedia of Virology. 3rd edition. Oxford: Elsevier. 1:374-308.