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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

2019 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) and bovine respiratory syncytial virus (BRSV). 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 third year. Objective 1 involves evaluation of the impact of emerging and variant viruses on the BRDC. During the third year of this project, work was continued on the first subobjective for Objective 1 that involved continuation of sequencing and genome assembly of BVDV strains of interest. Here, BVDV2 isolates were sequenced because of the amount of genetic variation and antigenic differences are poorly characterized in strains circulating in cattle herds. Specific antiserum was generated against one BVDV2 strain found in cattle vaccines and was used for neutralization of assays of BVDV2. This study confirmed that the more distant genetically a virus is from the virus used in antiserum production, the less the virus was neutralized. In the second subobjective, approximately 2000 banked serum samples from the 2014-2015 Brucellosis survey were used to evaluate the prevalence of exposure to BIDV. An overall seropositive rate of 77.5 percent was determined for the samples evaluated, suggesting these animals were exposed to BIDV. Seropositive samples were found in 41 out of 42 states where cattle originated, demonstrating that BIDV circulated widely in cattle during this period. 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). The experiments involved the infection of pregnant sows with a BVDV1b strain. Serial infections were used where one sow was infected by virus isolated from the previous infection. The infections were followed by screening of dams for infection and screening fetuses and neonates for the presence of BVDV. This study revealed that BVDV changed during infection of a pregnant sow with a number of infections resulting in persistently-infected fetuses. Recovered viruses had amino acid changes in several viral proteins that appeared to be maintained after 3 to 4 serial infections. The majority of changes that affected the viral proteins were found to be in the protein that the immune system targets and may be different from those introduced by infection of a ruminant. Objective 2 entails the investigation of host: pathogen interactions of viral agents associated with BRDC. 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. A new assay was developed to incorporate both cellular and antibody responses along with measurement of intracellular BVDV. These assays are the first to measure cellular responses and the amount of intracellular BVDV as a means to evaluate immune responsiveness. This assay discovered that the natural killer (NK) cell population had antigen specific responses, a novel finding. Increased expression of IFN-gamma is observed in BVDV vaccinated calves but not non-vaccinated calves after in vitro restimulation with BVDV. Decreased amounts of intracellular BVDV is observed in the vaccinated calves as compared to non-vaccinates. 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. In the second year, expression libraries were constructed containing genomic sequences of BVDV viruses. In the third year, the libraries were screened with antisera from cattle raised against BVDV2. This antiserum treatment should result in enrichment of BVDV peptides that were recognized by BVDV2 antibodies. After isolating the elements recognized by antibodies, their identity was determined by DNA sequencing. The enrichment was concluded successfully but this subobjective is considered partially met because of delays in hiring a bioinformaticist that just recently joined the unit to conduct data analysis. The third area of research under Objective 2 involves a potential new vaccine for BRSV. The vaccine utilized an attenuated Newcastle disease virus containing a gene from BRSV. Preliminary vaccination and challenge trials in calves showed the vaccine provided incomplete protection. Results of these studies in cattle indicates that dosage of the vaccine needs to be increased for larger animal species. New trials using a 5-fold higher dose of the vaccine were done. Vaccinated calves had reduced lung lesions following virulent challenge compared to non-vaccinated calves. Viral load in the lungs was also reduced in vaccinates. Nasal washes collected from these studies are being evaluated for mucosal antibody induction by the 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. A study was conducted with the objective to determine differences in microRNA counts (miRNAs) in several tissues of calves challenged with bovine viral diarrhea viruses (BVDV), Mycoplasma bovis, or both. The calves were challenged with pathogens and were euthanized 17 days post-challenge and several immune-related and non-immune-related tissues, were collected at necropsy. There was a high relationship among immune-related tissues, whereas the association between immune-related and non-immune-related tissues was non-existent. This showed that the use of circulating miRNAs to assess disease condition or to develop intervention strategies to minimize respiratory diseases in cattle caused by BVDV or Mycoplasma bovis will be of little use. The milestones at 36 and 48 months of this subobjective were exchanged. The 48-month milestone was fully met. The 36-month milestone was delayed due to the lack of a bioinformaticist and lack of data analysis. 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 bacterial strains that express BVDV proteins. These proteins should direct an immune response that will be protective against infection by BVDV. Preliminary vaccination studies in calves using these constructs have demonstrated that they can colonize the tonsils for a considerable period of time. This will be tried soon using BVDV constructs. This subobjective is considered partially met because no animal inoculations using BVDV constructs have been conducted. The second area of research under Objective 3 is to identify BVDV strains that contain antigenic determinants that provide broader protective responses following vaccination. In addition to work characterizing BVDV2 isolates (Objective 1, subobjective 1), sequence analysis was done on BVDV1a and BVDV1b isolates for genetic characterization within these subgenotypes. This analysis revealed genetic diversity at least as great as that observed with BVDV2. Antisera were raised against three BVDV1a vaccine virus strains and were used for antigenic characterization of BVDV1 viruses obtained from Europe. These viruses do not currently circulate in the U.S. This study demonstrated that there is variable neutralization by vaccine virus strain antisera with these European isolates, raising the question of their ability to protect against these viruses should they be introduced into the United States. Sequencing of these additional viruses and antisera production was done rather than using alphavirus replicons due to the variable expression of BVDV proteins between replicons. Also, it became increasingly difficult to obtain new replicons from the supplier and completion of this work with the few replicons we were able to obtain was not possible. 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. We examined the use of a virus that does not infect cattle, feline calicivirus (FCV) that may serve as a marker for vaccination. Inactivated FCV was injected into cattle. Using a virus neutralization assay, serum from the injected cattle were shown to have significant levels of FCV antibodies, thus demonstrating antigenicity in cattle. Another method of introduction of antigenic proteins of FCV into cattle was the use of alphavirus replicons that express FCV proteins. This method was dropped because, as described above, the difficulty in obtaining the alphavirus constructs made it impossible to complete.

1. Association of numbers of small noncoding RNAs in bovine leukemia virus infection. Bovine leukemia virus (BLV) affects cattle health and productivity worldwide, causing abnormal immune function and immunosuppression. The objective of this study, conducted by ARS researchers at Ames, Iowa, was to identify specific small non-coding RNAs associated with an antibody response to BLV in Holstein cattle. Sera from 14 animals were collected to establish amount of antibody against BLV by ELISA. Seven animals were seropositive (positive group) and seven were seronegative (negative group) for BLV exposure. Leukocytes from each animal were collected and the noncoding RNAs were extracted for sequencing. Three of these RNAs were significantly different between seropositive and seronegative groups (P < 0.0067). In all cases the positive group had a lower number of these RNAs when compared to the negative group. This research will benefit researchers investigating the pathogenesis of disease and new methods to detect BLV infections in cattle.

2. Neutralization of bovine viral diarrhea viruses with vaccine virus antiserum. Bovine viral diarrhea virus (BVDV), a common pathogen of cattle, causes subclinical to severe acute disease. Two species of BVDV are recognized, BVDV1 and BVDV2 with BVDV1 divided into at least 21 subgenotypes and BVDV2 into 3-4 subgenotypes, most commonly using sequences from the 5' untranslated region (5' UTR). A study conducted by ARS researchers at Ames, Iowa, reported whole genomic sequencing of 8 BVDV2 isolates that were not BVDV2a; but represented two additional BVDV2 subgenotypes. One BVDV2 subgenotype was previously recognized only in Asia. The other seven viruses fell into a second subgenotype that was first reported in Brazil and the U.S. in 2002. Neutralization assays using antiserum raised against vaccine strain BVDV2a 296c revealed varying degrees of neutralization of genetically distant BVDV2 isolates. Neutralization titers decreased from almost 2-fold to a more than 18-fold decrease, showing that vaccine viruses produced variable protection. This study illustrated the considerable genetic and antigenic diversity in BVDV2 circulating in the U.S. and demonstrates a need for more comprehensive immune coverage than what current commercial vaccines provide.

3. Use of a new technique to study bovine viral diarrhea virus infection in bovine peripheral blood mononuclear cells (PBMCs). Bovine viral diarrhea viruses (BVDV) cause acute and persistent infections. Acute infection results in generalized immunosuppression characterized by a decrease in circulating lymphocytes as a result of depletion T cell populations. Persistent infection with BVDV is the result of immune tolerance and is generally not associated with decreased lymphocyte numbers. The health outcome of persistently infected (PI) calves varies widely; some die of mucosal disease (a rare form of disease caused by BVDV), some succumb to ill thrift and others appear normal and survive to adulthood. Detection of BVDV at the single cell level is important to the study of subpopulations of peripheral blood mononuclear cells (PBMC) during BVDV infections, however there are few methods available for the detection and quantification of BVDV at this level. A study conducted by ARS researchers at Ames, Iowa, developed a novel flow cytometry-based RNA assay using in-situ detection of BVDV. This assay was used to evaluate differences in viral distribution within subpopulations of PBMC over time in PI calves carrying one of two different species of BVDV (BVDV1 and BVDV2). Clearance of virus from T cells indicated a breakdown in immune tolerance in these calves. This was the first report of a pattern observed in the viral load in the T cell subpopulations and survival in PI calves. This research will benefit researchers investigating the pathogenesis of disease caused by BVDV.

4. New animal model for the study of respiratory syncytial virus infection. Respiratory syncytial virus (RSV) is a significant cause of pediatric respiratory tract infections. It is estimated that two-thirds of infants are infected with RSV during the first year of life and it is one of the leading causes of death in this age group worldwide. Similarly, bovine RSV is a primary viral pathogen in cases of pneumonia in young calves and plays a significant role in bovine respiratory disease complex. Importantly, naturally occurring infection of calves with bovine RSV shares many features in common with human RSV infection. ARS researchers at Ames, Iowa, provided new information to increase the current understanding of RSV infection in cattle, by providing new methods of vaccination. The recent reports in which the neonatal calf was employed for the development and testing of vaccines and therapeutics which may be applied to hRSV infection in humans. This research will benefit researchers and vaccine companies investigating better ways to vaccinate against RSV in cattle and humans.

Review Publications
Falkenberg, S.M., Dassanayake, R.P., Walz, P., Casas, E., Neill, J.D., Ridpath, J.F. 2019. Frequency of bovine viral diarrhea virus detected in subpopulations of peripheral blood mononuclear cells in persistently infected animals and health outcome. Veterinary Immunology and Immunopathology. 207:46-52.
Imus, J.K., Lehmkuhl, H.D., Woods, L.W. 2018. Resistance of colostrum-deprived domestic lambs to infection with deer adenovirus. Journal of Veterinary Diagnostic Investigation. 31(1):78-82.
Register, K.B., Olsen, S.C., Sacco, R.E., Ridpath, J., Falkenberg, S., Briggs, R., Kanipe, C., Madison, R. 2018. Relative virulence in bison and cattle of bison-associated genotypes of Mycoplasma bovis. Veterinary Microbiology. 222:55-63.
Powell, E.J., Reinhardt, T.A., Casas, E., Lippolis, J.D. 2018. The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins. Journal of Dairy Science. 101(9):8182-8193.
Dassanayake, R.P., Falkenberg, S.M., Register, K.B., Samorodnitsky, D., Nicholson, E.M., Reinhardt, T.A. 2018. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis. PLoS One. 13(5):e0197677.
Guerra-Maupome, M., Palmer, M.V., McGill, J.L., Sacco, R.E. 2019. Utility of the neonatal calf model for testing vaccines and intervention strategies for use against human RSV infection. Vaccines. 7(1):7.
Taxis, T.M., Kehrli Jr., M.E., D'Orey-Branco, R., Casas, E. 2018. Association of transfer RNA fragments in white blood cells with antibody response to bovine leukemia virus in Holstein cattle. Frontiers in Genetics. 9:236.