Skip to main content
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

2021 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
This is the final report for the project 5030-32000-117-00D terminating September 30, 2021. 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 herpes virus 1 (BHV1), also known as infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza 3 virus (BPI3V), and bovine coronavirus (BCoV). The recently described bovine influenza D virus (BIDV), and bovine herpesvirus 4 (BHV4), which are emerging viruses, are also included in the list of viral agents often isolated from cases of BRDC. Significant progress has been made on each objective over the past year and is described below, followed by a summary of the significant progress made over the lifespan of the project over the past five years. Objective 1 concerns evaluation of the impact of emerging and variant viruses on the BRDC. During the fifth year of this project, work was continued on the first subobjective for Objective 1 involving diagnostics to rapidly detect viruses in cattle herds. Here, sequencing methods were refined to utilize a platform that allows complete viral genomic sequence data within 48 hours. Previously sequenced BVDV isolates were submitted for sequencing using the new rapid sequencing platform to confirm sequence depth and accuracy was maintained when utilizing the new rapid platform. The ability to refine the sequencing platform has allowed for rapid complete genome sequencing for diagnostic purposes. In the second subobjective, in vivo challenge models were used for a co-infection study that involved BVDV and BIDV. In collaboration with researchers at Oklahoma State University, colostrum deprived calves were initially challenged with BVDV to immunosuppress the calves. Approximately 21 days post-BVDV challenge a subset of calves were subsequently challenged with BIDV to evaluate the impact of virus shedding and immunological responses to a subsequent challenge. Data from this study is critical to better understand the role viral pathogens contribute to the multifactorial nature of the BRDC. Collectively for Objective 1 over the past five years of the project, significant progress has been made for all three subobjectives. Diagnostic samples were obtained and evaluated for genetic characterization and for use in in vivo studies. Furthermore, field samples were analyzed to determine the prevalence of emerging viruses and variant strains during this project. In addition to typical genetic sequencing, a rapid sequencing protocol was also leveraged. Continual improvement in sequencing technologies provide the opportunity to tailor methods for diagnostic needs such as rapid results or high throughput of samples which is useful to better understand molecular epidemiology of viruses circulating in the U.S. Additionally, genetic sequence data within Objective 1 has led to the observation that the majority of changes were found in the E2 protein of BVDV after 3-4 serial infections. This is important, as the E2 protein is the immunodominant protein that the immune system targets associated with neutralizing antibody. Data generated in support of Objective 1 is critical for providing better detection and understanding the prevalence and contribution to disease of these emerging novel bovine pathogens. Objective 2 entails the investigation of the host: pathogen interactions of viral agents associated with BRDC. The first subobjective of the investigation involves how the immune system is impacted after exposure to a dominant antigen. BVDV vaccines containing both BVDV-1 and BVDV-2 isolates have been evaluated to determine if one of the isolates induces a more predominate response. Data from these studies suggest there are dominant BVDV vaccine isolates that induce a more robust immune response. Furthermore, diagnostic data would also suggest that the BVDV vaccine strains that induce a more robust immune response as are the most frequently isolated vaccine strains in diagnostic submissions associated with clinical respiratory disease cases. This data would suggest that while some BVDV vaccine strains induce robust immune response they may also have a greater impact on the immune system as evidence by the detection in diagnostic samples. The second subobjective of research under Objective 2 involves identification of viral epitopes as determined by evaluation if BVDV E2 peptide responses. A previously evaluated synthetic BVDV E2 peptide library was further characterized in calves vaccinated with modified live or killed BVDV vaccines to identify E2 epitope-specific CD4 T-cells. Using a cell-based assay, potential E2 epitopes were identified from a pooled peptide library. Another set of calves were vaccinated with a modified live BVDV vaccine and a new and improved assay is currently being performed to enhance E2 epitope-specific signal. Data from these studies help provide specific regions of the viral genome that contribute to protective responses. The third subobjective area of research under Objective 2 was to examine expression levels of small non-coding RNAs, such as microRNA (miRNA), are involved in gene expression regulation, and determine if changes in expression levels are related to infection by viral pathogens causing BRDC. The objective was to establish a relationship between expression of non-coding RNAs and messenger RNA in animals from different challenge groups. Additionally, extraction of RNA from blood samples from a feedlot arrival vaccine experiment has been initiated. The objective is to assess differences in expression of messenger RNA in cattle that differed in vaccination strategies upon arrival at the feedlot. The goal is to determine if non-coding RNAs can be used as an alternative to antibiotics to prevent or minimize the effects of pathogens producing bovine respiratory diseases. Collectively for Objective 2 over the past five years of the project, significant progress has been made for all three subobjectives. A variety of methods were explored and developed to quantify the impact of BVDV on the immune system. These methods have led to quantification of tissue depletion and peripheral indicators of depletion and immune responsiveness. This in turn has led to the identification of dominant and immunosuppressive isolates that are being further characterized. Additionally, both T and B cell epitopes were explored in Objective 2. The Erns surface glycoprotein for BVDV was observed to have the highest reactivity in sera which is significant since this protein is not the major immunodominant protein and BRSV fusion (F) protein peptides were also demonstrated to activate bovine T cells. Given the significance of the F protein in BRSV, a vector expressing was used for vaccination and challenge studies and has demonstrated protection and a potential promising vaccine candidate. Lastly, expression levels of small non-coding RNAs have been evaluated to determine if expression levels are related to infection with bovine viral pathogens. Differential expression has been observed for different viral infection models and these differential expression patterns are being explored as preventative measures for BRDC. Data generated from Objective 2 is critical for understanding responses to bovine pathogens and implication on disease. Objective 3 focuses on intervention strategies to control viral pathogens that are known to be pathogens associated with the BRDC. A subobjective area of research was evaluated for the development of vaccines that provide better cross protection against emerging viral strains. The goal within this subobjective was to identify BVDV isolates that appear antigenically different than other BVDV isolates. Multiple BVDV isolates have been identified and have been used for vaccination studies in BVDV-naïve calves and are being used for antigenic comparisons. Multiple isolates have been determined to be antigenically different based on virus neutralization assays. Although, genetic comparison of the E2 protein of each respective virus has not lead to any identification of antigenic determinants associated with the difference observed in antigenicity. Collectively for Objective 3 over the past five years of the project, progress has been made for all three subobjectives. Recombinant Mannheimia vaccine strains expressing BVDV antigens were developed and tested in calves. While the alphavirus BVDV E2 expression system did not yield authentic immune responses in calves, a secondary approach was initiated, and antigenic epitopes are still being explored and antigenically distinct isolates have been identified using this approach. Furthermore, the cross-neutralization studies have identified isolates that appear to be more broadly cross-reactive as well. Collectively, data generated for this Objective has provided both genetic and antigenic information that is useful when choosing strains for vaccine development.

1. miRNA expression in BLV infected cows. Bovine leukemia virus (BLV) infection in cattle is omnipresent, which causes significant economic losses worldwide. MicroRNAs (miRNAs) are small non-coding RNA molecules ranging in size from 21 to 25 nucleotides in length, which regulate gene expression by altering translation. ARS researchers in Ames, Iowa, conducted a study to determine miRNA and messenger RNA (transcripts) profiles, to establish their relationship to exposure to the virus. In animals exposed to BLV, increased transcripts were related to stimulus of the immune system, while reduced transcripts were associated with antigen processing. Transcripts correlated with the expression of miRNAs were associated with virus replication. Five miRNAs and 64 transcripts were differentially expressed. The differentially expressed miRNAs targeted 17 transcripts, among which the expressions of two miRNAs were significantly negative correlated with the expressions of two transcripts. These results indicated that the differentially expressed miRNAs fine-tune most of the target genes in responding to BLV exposure. Further studies of the relationship between miRNAs and transcripts should reveal the molecular mechanisms of BLV infection and uncover the possibility to prevent the infection. Further studies are needed to identify how to prevent infection to reduce losses associated with BLV.

2. Vaccine platform for BRSV. A potential new vaccine for bovine respiratory syncytial virus and human respiratory syncytial virus has been tested by ARS researchers in Ames, Iowa, in collaboration with researchers at Rutgers University and Mount Sinai Medical School. Human (hRSV) and bovine (bRSV) respiratory syncytial viruses are closely related viruses that are among the leading causes of acute lower respiratory infection in young children and calves, respectively. Furthermore, in combination with other viral and bacterial pathogens, bRSV plays a significant role in bovine respiratory disease complex. The new vaccine utilizes an attenuated Newcastle disease virus containing a gene from bovine respiratory syncytial virus. Studies published using the vaccine in mice have shown the vaccine provides protection against lesions and viral load. A series of vaccination and challenge trials in calves have been conducted. Low dose vaccine provided incomplete protection. Vaccinated calves given a 5-fold higher dose were found to have reduced lung lesions following virulent challenge compared to non-vaccinated calves. In addition, viral load in the lungs and gene expression levels of inflammatory mediators were also reduced. After further development, the vaccine may be used by veterinarians, scientists, and vaccine manufacturers to reduce the negative effects of BRSV infection and contribution to bovine respiratory disease.

3. Susceptibility of livestock and wildlife to SARS-CoV-2. Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of the COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARSCoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts and define the mechanisms underlying cross-species transmission to humans. ARS researchers in Ames, Iowa, evaluated the susceptibility of cattle, swine, and white-tail deer to SARSCoV-2. Cattle and pigs were not susceptible, but white-tailed deer shed infectious SARS-CoV-2 in nasal secretions and on rectal swabs. Importantly, animals that were not inoculated, but were in contact with inoculation animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. These results demonstrate the zoonotic potential and public health concern as deer may serve as a reservoir for SARS-CoV-2.

4. Evaluating differences among BVDV isolates. Providing protection against bovine viral diarrhea virus (BVDV) is challenging because immunity to one BVDV strain does not always provide immunity to other BVDV strains. In addition, the ability of BVDV to infect the fetus, combined with the fact that immunity does not cross the placenta in cattle, complicates vaccine design and composition to confer fetal protection. Typically, virus neutralization (VN) is used to determine differences in immune responses among BVDV isolates, but interpretation of the data can be difficult due to the amount of data that is generated. ARS researchers in Ames, Iowa, utilized a principle component analysis that generates graphical scatter plots for visualization of VN results by reducing the dimensions of the data to more readily make comparisons between vaccine strains and BVDV field isolates. Spatial patterns in the graphs demonstrate differences in VN titers among the BVDV isolates. Some BVDV isolates had very distinct spatial patterns in the graphs that could suggest extremely antigenically divergent isolates. This analysis and graphs provide an alternative and more efficient means to analyze large VN datasets to visualize antigenic relationships between BVDV isolates and better develop BVDV vaccines that provide greater cross-protection among isolates. This information will be important for biologics companies to evaluate how similar current vaccines compare to field isolates as well as potential development of new vaccines.

5. Protective responses associated with BVDV. Differences between bovine viral diarrhea virus (BVDV) vaccine and field strains can lead to lack of efficacy associated with current vaccines. Currently, there is no data regarding potential differences in cellular immune responses between BVDV vaccine and BVDV-1b field isolates, which are not contained in current vaccines. ARS researchers in Ames, Iowa have described BVDV-1b strain differences as measured by cellular responses. This data helps provide further reasons for potential lack of efficacy issues with current vaccines as the cellular immune response is less for isolates not contained in vaccines as compared to isolates contained in current vaccines. This data can be leveraged along with antibody responses to develop vaccines that elicit a more broadly protective immune response to BVDV.

6. Interspecies transmission of Influenza D. Influenza D virus (IDV) can be repeatably detected and the prevalence in diagnostic samples from cattle is high, along with high IDV antibody concentration. Additionally, IDV is frequently detected from clinical samples from cattle exhibiting respiratory disease. While detection of IDV is high in cattle, the first IDV isolate was detected from a clinical sample collected from pigs exhibiting influenza-like illness. This is interesting given the ability of influenza viruses to transmit among species and the potential for recombination. ARS researchers in Ames, Iowa, evaluated transmission between cattle and swine as well as investigated differences in virus detection and transmission that may exist between IDV isolates originating either from cattle or swine. Similar shedding profiles were obtained for each species and viral isolate. However, interspecies transmission was found to be associated with virus origin-species; the bovine isolate when inoculated in pigs, only transmitted to cattle, not to pigs, and the swine isolate when inoculated into cattle only transmitted to pigs, and not to cattle. Together, these data show that cattle and pigs are permissive for IDV replication, but IDV transmission may be species dependent. This data is important for to better understand the zoonotic and risk associated with recombination of Influenza viruses.

Review Publications
Falkenberg, S.M., Dassanayake, R.P., Terhaar, B., Ridpath, J., Neill, J.D., Roth, J. 2021. Evaluation of antigenic comparisons among BVDV isolates as it relates to humoral and cell mediated immunological measures. Frontiers in Veterinary Science. 8. Article 685114.
Mosena, A.C., Falkenberg, S.M., Ma, H., Casas, E., Dassanayake, R.P., Watz, P., Canal, C., Neill, J.D. 2020. Multivariate analysis as a method to evaluate antigenic relationships between BVDV vaccine and field strains. Vaccine. 38(36):5764-5772.
Walz, P.H., Chamorro, M.F., Falkenberg, S.M., Passler, T., Van Der Meer, F., Woolums, A. 2020. Bovine viral diarrhea virus: an updated ACVIM consensus statement with focus on virus biology, hosts, immunosuppression, and vaccination. Journal of Veterinary Internal Medicine. 34(5):1690-1706.
Dassanayake, R.P., Wherry, T.L., Falkenberg, S.M., Reinhardt, T.A., Casas, E., Stabel, J.R. 2021. Bovine NK-lysin-derived peptides are bactericidal against Mycobacterium avium subspecies paratuberculosis. Veterinary Research. 52. Article 11.
Lippolis, J.D., Putz, E.J., Ma, H., Alt, D.P., Casas, E., Reinhardt, T.A. 2020. Genome sequence of a chronic Staphylococcus aureus isolated from a dairy cow that was non-responsive to antibiotic treatment. Microbiology Resource Announcements. 9(20). Article e00206-20.
Putz, E.J., Palmer, M.V., Ma, H., Casas, E., Reinhardt, T.A., Lippolis, J.D. 2020. Characterization of a persistent, treatment-resistant, Staphylococcus aureus infection causing chronic mastitis in a Holstein dairy cow. BioMed Central (BMC) Veterinary Research. 16. Article 336.
Casas, E., Ma, H., Lippolis, J.D. 2020. Expression of viral microRNAs in serum and white blood cells of cows exposed to bovine leukemia virus. Frontiers in Veterinary Science. 7. Article 536390.
Shepherd, B.S., Ma, H., Han, Y., Palti, Y., Gao, G., Liu, S., Wiens, G.D. 2020. Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 116 (103961).
Kaplan, B.S., Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Velayudhan, B., Li, F., Vincent, A.L. 2020. Virus strain influenced the interspecies transmission of influenza D virus between calves and pigs. Transboundary and Emerging Diseases.
Ridpath, J., Fulton, R.W., Bauermann, F.V., Falkenberg, S.M., Welch, J., Confer, A.W. 2020. Sequential exposure to bovine viral diarrhea virus and bovine coronavirus results in increased respiratory disease lesions: clinical, immunologic, pathologic, and immunohistochemical findings. Journal of Veterinary Diagnostic Investigation. 32(4):513-526.
Paredes-Sanchez, F.A., Sifuentes-Rincon, A.M., Casas, E., Arellano-Vera, W., Parra-Bracamonte, G., Riley, D.G., Welsh Jr., T.H., Randel, R.D. 2020. Novel genes involved in the genetic architecture of temperament in Brahman cattle. PLoS ONE. 15(8). Article e0237825.
Buckley, A.C., Falkenberg, S.M., Palmer, M.V., Arruda, P.H., Magstadt, D.R., Schwartz, K.J., Gatto, I., Neill, J.D., Arruda, B.L. 2021. Distribution and persistence of atypical porcine pestivirus (APPV) in experimentally inoculated pigs. Journal of Veterinary Diagnostic Investigation. 33(5):952-955.
Weber, G.M., Birkett, J.E., Martin, K., Dixon, II, D., Gao, G., Leeds, T.D., Vallejo, R.L., Ma, H. 2021. Comparisons among rainbow trout, Oncorhynchus mykiss, populations of maternal transcript profile associated with egg viability. Biomed Central (BMC) Genomics. 22. Article 448.