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

Location: Ruminant Diseases and Immunology Research

2018 Annual Report

Objectives
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.

Approach
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), bovine parainfluenza 3 Virus (BPI3V), and bovine coronavirus (BoCV). 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 second 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 BIDV and BoCV. During the second year of this project, contacts with diagnostic laboratories and diagnosticians were continued and additional collaborative agreements were put in place, additional samples were obtained and a number of BIDV positive samples identified by PCR. Serum samples were obtained from different regions of the country to determine prevalence of BIDV in the US and association with other pathogens. All samples containing BIDV also were positive for other viral agents. A survey of serum from US cattle for the presence of BIDV antibodies was conducted. While the prevalence has been low for BIDV, there was a large number that were antibody positive. Acquiring new samples and virus isolates and establishing collaborative research with others in the field is 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. 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. This study revealed that BVDV changed during infection of a pregnant dam and resulting in a persistently-infected fetus. The majority of changes that affected the viral proteins were found to be in the protein that the immune system targets. These changes may alter immunological properties of the virus affecting vaccine efficacy. 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. During the second year of the project, a variety of methods have been evaluated to better quantify depletion of lymphoid tissues. This included evaluation of tissue and peripheral markers. Peripheral markers and tissues were evaluated from vaccinated and challenged animals to evaluate different methods for detection of depletion and the level of severity of depletion that could be detected using various methods. The peripheral markers that were evaluated were not useful indicators of depletion and others will be evaluated. 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 greater than 99 percent of the possible antibody binding sites on these viruses. The library was treated with bovine serum from cattle that had been exposed to different viruses. After isolating the elements recognized by antibodies, their identity was determined by DNA sequencing. Data analysis is ongoing. 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. A protein from BRSV, a virus commonly associated in BRDC was investigated. Laboratory tools called peptide libraries were synthesized to allow identification of sequences recognized by bovine T cells. BRSV fusion (F) protein peptides that activate bovine T cells were identified. This showed that animals immunized with BRSV recognized the peptide sequence and responded to it. Identified several immunized animals whose T cells recognized specific regions of the BRSV F protein and are further evaluating these amino acid sequences. 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. Collection of tissues was done on two experiments of cattle challenged with Mycoplasma bovis and BVDV. Total and small non-coding RNA have been extracted from all tissues collected. DNA sequencing has been completed on all samples and several small non-coding RNAs have been identified that are present at different levels in infected animals. 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. 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 a protective protein. BVDV gene fragments encoding antigenic regions of the protective proteins were synthesized and introduced into Mannheimia haemoloytica. Recombinant M. haemolytica clones of interest were identified by Western blot assay. These modified-live M. haemolytica/BVDV constructs will be used for BVDV vaccination studies. The second area of research under Objective 3 is to identify BVDV strains that contain antigenic determinants that provide broader protective responses following vaccination. We identified isolates that differed genetically from our collection as well as established collaborations to obtain other viruses of interest. These viruses are being used to evaluate cross reactivity and protection conferred by vaccination. Virus-specific antisera was produced against viruses of interest as reference sera and is being used in comparisons of cross-reactivity with a panel of viruses. Current work with the reference sera produced so far has revealed that there is cross-reactivity with more distantly related viruses but at varying degrees. BVDV isolates from other countries have been received and were sequenced in order to evaluate the similarities in viral proteins that could contribute to greater cross-protective responses. Antisera produced by expression of the major immunogenic protein of BVDV have been produced and will be used to guide further production of reference antisera for upcoming vaccination studies. 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 BVDV. We obtained three viral vectors expressing one BVDV protein each for initial tests. Each vector contained a sequence from a different subgenotype of BVDV, each conferring its own antigenic characteristics. Animals injected with a single vector were the source of antiserum against each particular subgenotype. Results from these experiments demonstrated that specific antibodies were produced against the individual proteins and that these antibodies recognized test viruses and neutralized them. The antibodies produced against a specific subgenotype of BVDV recognized that genotype more strongly. Additionally, use of a virus that does not naturally infect cattle, feline calicivirus (FCV), was tested for antigenicity in cattle. Inactivated FCV injected into cattle resulted in the production of measurable, specific antibodies.

Accomplishments
1. Determination of prevalence of pestiviruses in sheep. A serological survey was done to detect the incidence of pestiviruses in domestic sheep populations in Wyoming. Pestiviruses, which include bovine viral diarrhea virus (BVDV) and border disease virus, have been reported in cattle and sheep populations. The incidence of pestivirus infections in sheep and the potential of transfer from sheep to cattle herds is unknown. To evaluate the incidence of pestivirus infections in sheep herds, ARS researchers at Ames, Iowa, tested sera from 500 sheep in virus neutralization assays to measure the amount of antibody against pestiviruses found in individual sera. It was determined that 5.6 percent of sheep screened had measurable antibody against pestiviruses. The most common antibodies were against BVDV1 as were the highest antibody levels. This study provided critical information concerning pestivirus incidence and how infections in sheep may impact BVDV control programs in cattle.

2. Virus infection in alternate hosts drives viral genetic diversity and adaptation. Bovine viral diarrhea virus (BVDV) strains are highly diverse genetically. It is known that genetic changes are introduced to a greater degree when the infection takes place in a pregnant animal. However, it is unknown how many genetic changes are introduced in infections of pregnant, non-bovine hosts. This study was conducted by researchers at Auburn University along with ARS researchers at Ames, Iowa, to determine the rate of change of BVDV and if specific changes occurred that were maintained and that may indicate adaptive changes that allowed more efficient replication in non-bovine hosts. The experiments were conducted by infection of a pregnant heifer in the first experiment or a pregnant ewe in the second experiment with BVDV and collecting serum 5 to 7 days following infection. The serum was used to infect the next pregnant host animal and the cycle was repeated. This was done for 6 passages of the virus in pregnant animals of each species. Additionally, virus was isolated from the calf or lamb following birth. The viruses isolated following each infection and those isolated from newborn animals were sequenced. This analysis revealed that changes were introduced rapidly with more changes introduced during infection of sheep than cattle. Also, more changes became fixed in the sheep passaged viruses, indicating adaptation to the new host. These results suggested that infection of non-bovine hosts may be a significant source of genetic change in BVDV, one that has been overlooked to this point.

3. Inactivated bovine viral diarrhea virus vaccines fail to protect the fetus from persistent infection. Bovine viral diarrhea virus (BVDV) infections, if they occur early in pregnancy, can result in the birth of a persistently infected (PI) calf. This calf will spread the virus for the remainder of its life and is considered the primary source of virus in cattle herds. It is desirable to vaccinate cattle before breeding in order to prevent or limit infections of pregnant cattle. To test the efficacy of vaccines in the prevention of infections and the birth of PI cattle, a study was conducted by researchers at Auburn University and ARS researchers at Ames, Iowa, using three commercial multivalent vaccines that contained killed BVDV (1a and 2a viruses). Four groups of pregnant heifers, three vaccinated with a different vaccine and a non-vaccinated control group, were co-mingled with PI calves (2 with 1a BVDV, 2 with 1b BVDV and 3 with 2a BVDV) for 28 days. From the 104 pregnancies, 73 transplacental infections were detected. The number of pregnancies with infected fetuses varied between treatment groups, but all contained a significant number of infections. This study demonstrated the poor performance of inactivated vaccines to provide protection against fetal infection following exposure to BVDV. These results illustrate why modified live vaccines should be used in breeding stock.

4. Developed and tested novel nanoparticle vaccine for bovine respiratory syncytial virus. Bovine respiratory syncytial virus (BRSV) is a bovine viral pathogen that is commonly associated with bovine respiratory disease complex. BRSV infection in calves causes lung lesions and fever and severe cases can result in death. Nanoparticle-based vaccines have shown promise as vaccine delivery vehicles and to potentiate the immune response due to their ability to provide sustained release of pathogen molecules and to induce both antibody- and cell-mediated immune responses. ARS researchers at Ames, Iowa, in collaboration with researchers at Kansas State University and Iowa State University, have developed a novel nanovaccine delivered via the nasal route incorporates proteins from bovine respiratory syncytial virus. Calves receiving this vaccine exhibited reduced lung lesions, reduced viral burden, and decreased virus shedding compared to unvaccinated calves. This novel vaccine, with optimization, has the potential to significantly reduce the disease burden associated with BRSV as a cause of the bovine respiratory disease complex.

5. New technique for detection of virus infection in a single cell was developed. Bovine viral diarrhea virus (BVDV) can cause acute or persistent infections depending on when an animal becomes infected. BVDV can be detected in both forms of infections using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), but these are not able to detect infections at the single cell level. Detection of the single cell level is important because of the negative effects BVDV has on the immune system. A new technique, called PrimeFlow RNA assay, was used by ARS researchers at Ames, Iowa to develop a technique to detect BVDV in a bovine B-cell lymphoma cell line (BL-3). Using RNA probes that were designed to a specific region of the BVDV viral RNA, BVDV was detected in infected cells but there was no signal from non-infected cells. This was the first report of detection of BVDV at the single cell level.

Review Publications
Taxis, T.M., Casas, E. 2017. MicroRNA expression and implications for infectious diseases in livestock. Centre for Agriculture and Biosciences International. 12(26):1-20. https://doi.org/10.1079/PAVSNNR201712026.
Fulton, R.W., Neill, J.D., Saliki, J.T., Landis, C., Burge, L.J., Payton, M.E. 2017. Genomic and antigenic characterization of bovine parainfluenze-3 viruses in the United States including modified live virus vaccine (MLV) strains and field strains from cattle. Virus Research. 235:77-81. https://doi.org/10.1016/j.virusres.2017.04.009.
Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Ridpath, J.F. 2018. Evaluation of bovine viral diarrhea virus transmission potential to naïve calves by direct and indirect exposure routes. Veterinary Microbiology. 217:144-148. https://doi.org/10.1016/j.vetmic.2018.03.012.
Silveira, S., Falkenberg, S.M., Elderbrook, M.J., Sondgeroth, K.S., Dassanayake, R.P., Neill, J.D., Ridpath, J.F., Canal, C.W. 2018. Serological survey for antibodies against pestiviruses in Wyoming domestic sheep. Veterinary Microbiology. 219:96-99. https://doi.org/10.1016/j.vetmic.2018.04.019.
Cox, E.A., Ridpath, J.F., Falkenberg, S.M. 2017. Clinical report: Detection and management of bovine viral diarrhea virus Type 1b in a large dairy herd. Bovine Practitioner Journal. 51(2):153-156.
Walz, P.H., Riddell, K.P., Newcomer, B.W., Neill, J.D., Falkenberg, S.M., Cortese, V.S., Scruggs, D.W., Short, T.H. 2018. Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2. Vaccine. 36(26):3853-3860. https://doi.org/10.1016/j.vaccine.2018.04.005.
Kuca, T., Passler, T., Newcomer, B.W., Neill, J.D., Galik, P., Riddell, K.P., Zhang, Y., Walz, P.H. 2018. Identification of conserved amino acid substitutions during serial infection of pregnant cattle and sheep with bovine viral diarrhea virus. Frontiers in Microbiology. 9:1109. https://doi.org/10.3389/fmicb.2018.01109.
Runyan, C.A., Downey-Slinker, E.D., Ridpath, J.F., Hairgrove, T.B., Sawyer, J.E., Herring, A.D. 2017. Feed intake and weight changes in Bos indicus-Bos taurus crossbred steers following Bovine Viral Diarrhea Virus Type 1b challenge under production conditions. Pathogens. 6(4):66. https://doi.org/10.3390/pathogens6040066.
McGill, J.L., Kelly, S.M., Kumar, P., Speckhart, S., Haughney, S.L., Henningson, J., Narasimhan, B., Sacco, R.E. 2018. Efficacy of mucosal, polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf. Nature Scientific Reports. 8(1):3021. https://doi.org/10.1038/s41598-018-21292-2.