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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Research Project #432021

Research Project: Identification of Disease Mechanisms and Control Strategies for Viral Respiratory Pathogens of Ruminants

Location: Ruminant Diseases and Immunology Research

2017 Annual Report

Objective 1. Determine the impact of variant and emerging viruses on the development and control of respiratory disease in ruminants, such as conducting molecular epidemiology studies to determine respiratory viruses currently circulating in U.S. herds and identifying the molecular determinants that drive strain prevalence and host-range specificity. Subobjective 1A – Conduct molecular epidemiology studies to determine respiratory viruses currently circulating in U.S. herds. Subobjective 1B - Identify the molecular determinants that drive strain prevalence and host-range specificity. Objective 2. Elucidate the host-pathogen interactions associated with the Bovine Respiratory Disease Complex, including identifying host factors associated with viral infection that predispose to respiratory disease complex, identifying T and B cell epitopes that drive protective immunity against respiratory viral pathogens, and characterizing functional genomics of the host associated with susceptibility to respiratory infection. Subobjective 2A – Identify host factors associated with viral infection that predispose to respiratory disease complex. Subobjective 2B – Identify T and B cell epitopes that drive protective immunity against respiratory viral pathogens. Subobjective 2C - Characterize functional genomics of the host associated with susceptibility to respiratory disease. Objective 3. Develop intervention strategies for controlling viral respiratory infections of ruminants, including developing vaccine platforms that can be delivered to stressed cattle, developing vaccines that provide better cross-protection against emerging field strains, and developing a DIVA vaccine and companion diagnostic test kit to enable eradication of BVDV in U.S. herds. Subobjective 3A – Develop vaccine platforms that can be delivered to stressed cattle. Subobjective 3B - Develop vaccines that provide better cross-protection against emerging field strains. Subobjective 3C - Develop a DIVA vaccine and companion diagnostic test kit to enable eradication of BVDV in U.S. herds.

Bovine respiratory disease (BRD) is a major cause of monetary losses in the cattle industry. The aim of the research in this project is to provide scientific information to better understand the viral pathogenesis of BRD. In particular, the disease dynamics of host-pathogen interactions responsible for the BRD will be investigated. Agents of interest include bovine viral diarrhea virus (BVDV), bovine parainfluenza 3 virus (BPI3V) and bovine respiratory syncytial virus (BRSV). This research is a multidisciplinary approach to address the broad and ambitious goal of controlling viral diseases of cattle, with a priority on respiratory viral pathogens. The approach used here is consistent with the multifactorial nature of bovine respiratory disease. Bovine respiratory disease (BRD) is multifactorial in origin as it results from an interplay of infection by multiple viral and bacterial pathogens, stress, immune dysfunction and environmental factors. The first aspect of this project addresses the impact of variant and emerging viruses. Screening to determine the incidence of variant and emerging viruses will require the development of surveillance tools and methods to measure impact. This will lead to a greater understanding of all viruses that play a role in BRD. A major thrust here is evaluation of currently marketed vaccines and whether there is a need to modify them to protect against emerging/variant viruses. There is a need to identify emerging/variant viruses that interact with the host in producing BRD. A second area addresses the understanding of host/pathogen interactions, specifically to determine how respiratory viral pathogens interact with the host to moderate innate and adaptive immune responses. This includes interaction by and between BVDV, BPI3V and BRSV and emerging/variant viruses. It is established that most BRD involves interactions of multiple agents, both viral and bacterial, thus experiments involving multiple agents will be conducted to look at this interplay and how each contributes to BRD. The third part of this project involves defining events that promotes the production of a strong, protective immune response (both innate and acquired immunity). Results from this will reveal targets or points of intervention that can be utilized in the development of robust vaccines and management regimen that reduces the impact of BRD. The knowledge gained here will be used for the design of new vaccines, including subunit vaccines, or for providing greater knowledge for the selection of virus strains used in vaccines. This part of the project will evaluate the practical applications of information generated in the form of improved vaccines or vaccination strategies. The ultimate, cumulative goal of this research is to promote the generation of the best protective immune responses possible in cattle to reduce BRD.

Progress Report
The goal of this project is to find novel means to address and reduce the impact of Bovine Respiratory Disease Complex (BRDC) on domestic cattle herds. The viral agents of concern are bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BHV1), also known as infectious bovine rhinotracheitis virus (IBR), bovine parainfluenza 3 Virus (BPI3V), and bovine coronavirus (BCV). Emerging viruses, the recently identified bovine influenza D virus (BIDV), and bovine herpesvirus 4 (BHV4), are also included in the list of viral agents often isolated from cases of BRDC. Significant progress was made on each objective of this project in its first year. Objective 1 concerns evaluation of the impact of emerging and variant viruses on the BRDC. This objective contains two subobjectives with a total of three areas of research. The first two areas concerned surveillance for emerging viral pathogens that may be unrecognized components of BRDC and determining the pathogenic potential of these pathogens. Agents targeted here were HoBi (an emerging pestivirus), BIDV and bovine rhinitis virus. This being the first year of this project, contacts with diagnostic laboratories and diagnosticians were established and collaborative agreements have been put in place. In discussions with diagnosticians, another emerging virus, BHV4 was reported to be isolated more frequently and was added to our list of emerging viral agents. Both samples and isolated virus strains were received from outside sources and the investigatory phase with these samples has begun. Acquiring new samples and virus isolates and establishing collaborative research with others in the field will be an ongoing process throughout the life of the project. The third area of research under Objective 1 was to investigate the effect of genetic change (impacting amino acid sequence) of the structural proteins of BVDV and the impact on diagnostic test accuracy and vaccine efficacy. Earlier reports have demonstrated that passage through pregnant animals results in more rapid genetic change. In collaboration with other researchers, non-bovine species have been infected with bovine viral diarrhea virus (BVDV). These experiments involve the infection of pregnant animals followed by screening of dams for acute phase virus and screening fetuses and neonates for the presence of BVDV. Non-bovine species (goats and white-tailed deer) were infected and viruses have been isolated from dams and offspring. Objective 2 entails the investigation of host: pathogen interactions of viral agents associated with BRDC. This objective is composed of three subobjectives with 4 areas of research. The first area of investigation involves evaluation of the bovine immune response in the face of exposure to exposure to viruses that suppress and deplete the immune system. Banked tissue samples from previous studies were examined for proteins that can be used for indicators of tissue depletion. It was determined that the previously banked frozen tissues were not optimal for evaluating these markers and other methods were tested. Other tissues and cells from more recent animal studies were tested using other preservation methods for the lymphoid depletion markers and these methods will allow downstream examination of preserved tissues and cells. The second area of research under Objective 2 involved identification of B cell epitopes (amino acid sequences that are recognized and bound by antibodies) of proteins from viral pathogens. A survey of viruses known to infect cattle was done and all protein sequences from these pathogens were obtained from available public databases and analyzed. This information was used to design a laboratory tool called an expression library that can be used to screen antisera from cattle raised against various bovine viral pathogens that covers >99% of the possible antibody binding sites on these viruses. The third area of research under Objective 2 involves identification of regions of viruses that trigger protective cellular immunity (small protein sequences that stimulate T cells) in cattle experiencing BRDC. To begin this work, a protein from one of the viruses involved in the syndrome was investigated. Laboratory tools called peptide libraries were synthesized to allow identification of sequences recognized by bovine T cells. Significant progress was made identifying BRSV F protein peptides that activated bovine T cells, an indication of recognition of the peptide sequence. We identified several immunized animals whose T cells recognized specific regions of the BRSV F protein and are now homing in on a particular region of the F protein in pursuit of a protective vaccine. The fourth area of research under Objective 2 was to examine expression levels of small non-coding RNAs and determine if changes in expression levels are related to infection by viral pathogens causing BRDC. Molecules circulating in live cattle, known as transfer RNA-derived RNA fragments (tRFs), have been suggested to be regulators of gene expression in mammals. Establishing the difference in type and quantity of tRFs between healthy and diseased cattle could produce information needed to understand how genes in the animal are turned on or off, and how the animal’s immune system responds to viral infection. Over a series of experiments, various tissues and sera were collected from both control animals and those infected with viruses. RNA has been extracted and processed from these tissues, and analysis of tRFs is underway. Objective 3 focuses on intervention strategies to control viral pathogens that are known to be components of BRDC. This objective contains three areas of research, the first to develop vaccine platforms that can be used in stressed animals, the second to develop vaccines that provide better protection against the viruses and the third to develop a differentiation of infected from vaccinated animals (DIVA) vaccine to be used in an eradication program to eliminate BVDV in the United States. The first area of research under Objective 3 involved development of Mannheimia bacterial strains that express BVDV proteins found on the surface of the viral particle. These proteins will direct an immune response that will be protective against infection by BVDV. The first phase of this study was completed and involved the design and synthesis of DNA segments that had BVDV sequences encoding the protective protein. These DNA segments were optimized for Mannheimia to achieve the greatest level of production of the BVDV protein. The design of the sequences of the DNA segments were based on different genetic groups (genotypes) of BVDV to allow the broadest immune response to provide protection against various field strains of BVDV. The second area of research under Objective 3 is to identify BVDV strains that contain antigenic determinants that provide broader protective responses following vaccination. To begin this work, we identified isolates of interest from our collection as well as established collaborations to obtain other viruses of interest. These viruses will be used to evaluate cross reactivity and protection conferred by vaccination. New type-specific antisera are being prepared as reference sera used to compare cross-reactivity of these viruses. Previously published BVDV isolates from other countries have been received, propagated, and sequenced in order to evaluate the similarities that could contribute to greater cross-protective responses. Sequences from regions of interest from the isolates have been produced and will be used in further production of reference antisera and in upcoming vaccination studies. A monoclonal antibody has been chosen from the existing data that binds all isolates we have received. The third area of research under Objective 3 is to develop reagents to be used in a DIVA vaccine to aid in an eradication program for BVDV. The ability to differentiate infected from vaccinated animals is important to determine infection rate and herds that are positive for viable BVDV. We have obtained three viral vectors expressing one BVDV protein for initial tests. The three vectors contain BVDV sequences that are used to produce a specific protein following injection into cattle. Each vector contains a sequence from a different subgenotype of BVDV, each conferring its own antigenic characteristics. An additional vector containing sequences from a separate region of BVDV (an indicator of infection by live virus) is being constructed and evaluated. Animals injected with a single r vector are the source of antiserum against each particular subgenotype. Results from these experiments will be used to test the vectors in an animal disease study.

1. Bovine Viral Diarrhea Virus (BVDV) is often associated with respiratory disease in cattle. MicroRNAs (miRNAs) have been proposed as indicators of exposure to respiratory pathogens. A study was conducted by ARS researchers at Ames, Iowa to identify miRNAs in Holstein cattle that had been challenged with a field strain of BVDV. This study identified two miRNAs that could potentially be used as indicators for exposure to BVDV. These results are important for future studies where the utility of these indicators is determined. Markers identified here could potentially find use in rapid screening tests and in eradication programs for BVDV where determining differences between vaccinated and naturally infected animals is important.

2. No evidence of HoBi virus infection in U.S. cattle. Four species of ruminant pestiviruses are present in the United States; bovine viral diarrhea virus 1 and 2 (infect primarily cattle), border disease virus and pronghorn virus (infect sheep and deer). The HoBi virus has not been found in the United States. ARS researchers at Ames, Iowa examined 2000 cattle sera to determine if antibodies against any of these pestiviruses were present. This study showed that 92.4% of the sera had antibodies against pestiviruses. Because of the possibility of antibodies recognizing more than one pestivirus, it was not possible to determine the pestivirus that caused the infection. Additionally, it was found that a significant number of animals did not have what was considered a protective level of antibodies. This study showed that infection with pestiviruses is a common occurrence; however, antibodies produced by infection by HoBi virus were not present indicating that the HoBi virus is not present in the United States.

3. Primary cell lines from different breeds of cattle equally support growth of two bovine pestiviruses. Bovine respiratory disease complex is very common and potentially detrimental to the health of afflicted cattle. Host factors appear to play a role in susceptibility to respiratory disease as breed differences appear to have an effect on disease outcome. ARS researchers at Ames, Iowa examined the growth of bovine viral diarrhea viruses and HoBi virus in testicular cells isolated from different breeds of cattle. The growth of each virus used in the study was compared after one passage in the different testicular cells. The results of this study demonstrated that there was not a difference in growth between the different breeds of cattle but rather differences in growth was shown between different animals. This showed that other factors in the host response to infection impact disease outcome in infected animals and that individual animals can respond differently to infection.

4. Bovine leukemia virus (BLV) causes economic loss through reduced productivity, especially reduced milk production. Earlier studies showed this virus to be widespread, especially in dairy cattle. ARS researchers in Ames, Iowa conducted a study examining 2000 sera from all regions of the United States for the presence of antibodies against BLV. This study revealed that 38.6% of the sera tested was positive for BLV antibodies. Breaking the data down by region, 32.5% of the positive sera came from the Pacific Northwest and 54.3% were from the northeastern states. This study showed that BLV infections are widespread through the United States and there indicates a higher rate of infection in dairy cattle than in beef cattle. This study showed which cattle populations were at greater risk for infection with BLV.

Review Publications
Pecora, A., Perez-Aguirreburuald, E.S., Malacari, D.A., Zabal, O., Bauermann, F.V., Ridpath, J.F., Dus-Santos, M.J. 2016. Hobi emergent pestivirus: Its impact in animal health and importance as a contaminant in biotechnological products. Agricultural Research Journal.
Downey-Slinker, E.D., Ridpath, J.F., Sawyer, J.E., Skow, L.C., Herring, A.D. 2016. Antibody titers to vaccination are not predictive of level of protection against a BVDV type 1b challenge in Bos indicus - Bos taurus steers. Vaccine. 34:5053-5059. doi: 10.1016/j.vaccine.2016.08.087.
Hause, B.M., Huntimer, L., Falkenberg, S.M., Henningson, J., Lechtenberg, K., Halbur, T. 2016. An inactivated influenza D virus vaccine partially protects cattle from respiratory disease caused by homologous challenge. Veterinary Microbiology. 199(2017):47-53.
Weber, M.N., Bauermann, F.V., Canal, C.W., Bayles, D.O., Neill, J.D., Ridpath, J.F. 2016. Temporal dynamics of ‘HoBi’-like pestivirus quasispecies in persistently infected calves generated under experimental conditions. Journal of Virology. 227(2017):23-33: doi: 10.1016/j.virusres.2016.09.018.
Newcomer, B.W., Neill, J.D., Galik, P., Riddell, K.P., Zhang, Y., Passler, T., Velayudhan, B., Walz, P.H. 2017. Serologic survey for antibodies against three genotypes of bovine parainfluenza 3 virus in unvaccinated ungulates in Alabama. American Journal of Veterinary Research. 78(2):239-243.
Bauermann, F.V., Ridpath, J.F., Dargatz, D.A. 2017. Bovine leukemia virus seroprevalence among cattle presented for slaughter in the United States. Journal of Veterinary Diagnostic Investigation. doi: 10.1177/1040638717702183.
Bauermann, F.V., Ridpath, J.F., Dargatz, D.A. 2017. A serosurvey for ruminant pestivirus exposure conducted using cattle sera collected for brucellosis surveillance in the United States. Journal of Veterinary Diagnostic Investigation. 29(1):76-82. doi: 10.1177/1040638716680251.
Gomez-Romero, N., Basurto-Alcantara, F.J., Verdugo-Rodriguez, A., Bauermann, F.V., Ridpath, J.F. 2017. Genetic diversity of bovine viral diarrhea virus in cattle from Mexico. Journal of Veterinary Diagnostic Investigation. 29(3):362–365. doi: 10.1177/1040638717690187.
Stanton, J.B., Swanson, B., Orozco, E., Munoz-Gutierrez, J.F., Evermann, J.F., Ridpath, J.F. 2017. Immortalized sheep microglial cells are permissive to a diverse range of ruminant viruses. The Veterinary Quarterly. 37(1):52-56. doi: 10.1080/01652176.2017.1297550.
Weber, M.N., Bauermann, F.V., Romero, N.E., Herring, A.D., Canal, C.W., Neill, J.D., Ridpath, J.F. 2017. Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed. Veterinary Research Communications. 41(1):1-7. doi: 10.1007/s11259-016-9666-5.
Casas, E., Kehrli Jr., M.E. 2016. A review of selected genes with known effects on performance and health of cattle. Frontiers in Veterinary Science. 3(113). doi: 10.3389/fvets.2016.00113.
Taxis, T.M., Bauermann, F.V., Ridpath, J.F., Casas, E. 2017. Circulating microRNAs in serum from cattle challenged with Bovine Viral Diarrhea Virus. Frontiers in Genetics.
Ridpath, J.F., Neill, J.D., Palmer, M.V., Bauermann, F.V., Falkenberg, S.M., Wolff, P.L. 2017. Isolation and characterization of a novel cervid adenovirus from white-tailed deer (Odocoileus virginianus) fawns in a captive herd. Virus Research. 238:198-203. doi: 10.1016/j.virusres.2017.06.020.
Pecora, A., Perez-A, M.S., Malacari, D.A., Zabal, O., Sala, J.M., Konrad, J.L., Caspe, S.G., Bauermann, F.V., Ridpath, J.F., Dus Santos, M.J. 2017. Serological evidence of Hobi-like virus circulation in Argentinean water buffalo. Journal of Veterinary Diagnostic Investigation. 1(4). doi: 10.1177/1040638717720246.