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

Location: Ruminant Diseases and Immunology Research

2020 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 primary 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 fourth year. Objective 1 involves evaluation of the impact of emerging and variant viruses on the BRDC. During the fourth year of this project, work was continued on the first subobjective for Objective 1 that involved completion of sequencing and genome assembly of BVDV strains of interest. Here, multiple BVDV1b isolates were sequenced because the amount of genetic variation and antigenic differences are poorly characterized in strains circulating in cattle herds. Specific antiserum was generated against twenty BVDV1b strains found in cattle vaccines and was used for neutralization assays of several BVDV subgenotypes. This study confirmed that there was cross-reaction across subgenotypes but that there were distinct groupings of virus with different neutralization characteristics. This represents important findings in relating genetic variation to antigenic differences in BVDV and has implications in future vaccine design. In the second subobjective, a novel cell mediated immune assay has been developed and is being utilized to determine isolates that may induce a more robust immune response and hence potentially be more pathogenic. The assay utilizes detection of cytokine expression in specific cell populations. Increases in a specific cytokine has been associated with exposure to BVDV and increased expression of this cytokine is observed with more virulent or pathogenic strains. In addition, the assay is being used to determine if the cellular responses measured in the assay are specific to BVDV exposure, or if these responses are generalized immune changes observed after exposure to BVDV that can be measured after exposure to non-BVDV antigens. 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 viruses that suppress and deplete the immune system. Approximately 30 BVDV isolates have been used to inoculate naïve calves to evaluate immunological responses to each respective virus. Immune response measures are used to determine isolates that have robust cell mediated and humoral responses, as well as determine isolates that may be more immunosuppressive. Data from these inoculated calves is being utilized to select isolates that can be used in studies, for vaccination/challenge studies, co-infection, dominant antigen, and isolates that may induce increased immune suppression for co-infection studies. This data suggests that there is a variability in immunosuppression among isolates and robust immune response appears to be associated with immunosuppression, but not increased protection. 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. To this end, bacteriophage peptide expression libraries were constructed containing genomic sequences of BVDV viruses. A BVDV2-specific peptide (short protein) library was screened with bovine antisera raised against BVDV2. This antiserum treatment should result in enrichment of BVDV peptides that were recognized by BVDV2 antibodies. Analysis of the sequences present in the enriched population revealed, surprisingly, that the protein with the strongest recognition was the Erns surface glycoprotein, a major viral protein. It is not known at this time whether the humoral response to this protein is protective or is this protein serves as a decoy, drawing a significant portion of the antibody response to focus on this protein. The third area of research under Objective 2 involved identification of the BVDV E2 peptides that promote a specific immune response. A peptide library was synthesized based on strain NADL E2 protein domain1 and 2 that are major antigenic regions of this protein. Peptides were mixed to make pools. BVDV strains NADL and 296c E2 (full-length protein) were expressed in E. coli for use in the studies. To measure T cell function, an assay was performed to measure protein secretion from T cells to determine if there were functional changes. Although responses were observed with BVDV modified live vaccinated animals as compared to controls and with BVDV killed virus-vaccinated calves with a peptide pool, the background responses were high. Therefore, based on appropriate criteria, E2 peptide-specific CD4 T cells remain to be identified. The fourth area of research under Objective 2 was to examine expression levels of small non-coding RNAs, such as microRNA (miRNA) that are involved in gene expression regulation, and determine if changes in expression levels are related to infection by viral pathogens causing BRDC. Bovine leukemia virus (BLV) infection in cattle causes significant economic losses. Transcriptome profiles were generated by sequencing total RNA libraries prepared from leukocytes from BLV-infected and noninfected control calves. RNA-Seq revealed 64 differentially expressed transcripts (DETs). Some up-regulated DETs associate with gene ontology (GO) terms of response to stimulus and immune system processes showed negative regulation of viral entry or release from host cells, while some down-regulated DETs related to antigen processing and presentation. The differentially-expressed microRNAs targeted 5741 transcripts, 18 of them were DETs. The target transcripts showed a wide range of biological processes and molecular functions. A further study of the miRNAs and the genes might reveal the molecular mechanisms of BLV infection and find possible ways to prevent the infection. 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. Recombinant expression of BVDV antigenic fragments in Mannheimia haemolytica serotype 6 was constructed and the protein expression was confirmed. Recombinant M. haemolytica expressing vaccine delivery platform without the antigen was tested in calves for colonization and immune response. Vaccine isolates were efficiently colonized in the upper respiratory tract and antibodies against vaccine delivery platform were detected in the serum, nasal wash and tear samples. A vaccine efficacy study in calves is planned. 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. Given that the replicon particles, initially used in antisera generation, did not induce an authentic humoral response to the E2 region of BVDV, the whole virus was used for antisera generation and antigenic comparisons. Antisera to approximately 30 BVDV isolates has been generated, as well as to BVDV strains contained in commercially available vaccines. Genetic comparisons of the E2 region of BVDV, which is a major immunodominant region of the virus, was used to select the isolates for antisera generation and subsequent antigenic comparisons. Data generated suggests that genetic relatedness within the E2 region of BVDV, is not associated with antigenic relationships. This is important because this indicates that genetic information should not be used exclusively in the choice of strains for inclusion in vaccines. The third area of research under Objective 3 is to test BVDV E2-expressing alphavirus replicons in cattle and test levels of E2-specific antibodies in treated cattle. As a first step, these replicons had earlier been tested in sheep to examine expression and host antibody response. It was determined at that time that because of extreme variability in both expression of E2 proteins and in the antibody response to the E2 proteins, this replicon approach would not be pursued in cattle.

Accomplishments
1. Development of a novel method that measures the immune response from vaccines. The response to vaccination of cattle with commercial vaccines is typically judged by the amount of circulating antibody. However, antibodies are only a part of the protective response to neutralize the virus. Cell mediated immunity (CMI) is another part of the immune system important for killing virus-infected cells and is thought to be the difference in efficacy between live and killed virus vaccines. Previously, CMI has been difficult to measure, being tedious and time consuming. Here, a novel method was developed by ARS researchers at Ames, Iowa, that more accurately measured CMI in BVDV vaccinated calves. The method, called PrimeFlow assay, allows for assessing CMI measurements in specific cell populations. The cells were stained and the cells that are positive for the cytokines associated with CMI; interferon-gamma (IFg) and interleukin 2 (IL-2) was determined. A novel finding was that a specific cell population, natural killer cells (NK), were responsible for a large amount of the IFg production, which has not been shown previously. This method allowed not only the ability to follow CMI responses but also to understand the antiviral properties of the CMI response. This is important in evaluating BVDV strains for inclusion in vaccines by measuring the strength and breadth of the CMI response generated. Comparing the CMI response will allow biologics companies other methods to evaluate protective responses and more efficacious vaccines to be produced.

2. Determination of prevalence of bovine influenza D virus in the US. Characterization of disease potential of emerging viral pathogens is important to maintain the safety and efficiency of our domestic livestock herds. Influenza D virus (IDV) is a recently characterized viral pathogen of cattle with some potential for transmission to swine. It is found by veterinary diagnostic laboratories most commonly associated with other viral pathogens. It is not clear how widespread this virus is and where it is found in the U.S. A survey was conducted by ARS scientists at Ames, Iowa, using cattle sera that was collected from 1,992 animals across the country in 2014 and 2015. Overall, there was a 77.5 percent positive rate for IDV in the tested sera, with regional rates varying between 47.4 and 84.6 percent. Antibody positive samples were found in 41 of 42 states from which samples were obtained. This high antibody positive rate shows that there is a need for studies to determine severity of disease caused by IDV as well as how the virus is spread and its potential to cause disease in both cattle and swine.

3. Emerging bovine viral diarrhea virus strains in the US. A bovine viral diarrhea virus (BVDV) strain was isolated in California that was shown to be in the BVDV1 species but was not of the BVDV1a or BVDV1b groups that were known to be in the U.S. Characterization of this virus was important to maintain vigilance for emerging pestiviruses and to determine the effectiveness of current bovine vaccines in protecting against these viruses. ARS scientists at Ames, Iowa, in collaboration with researchers at the University of California, Davis, revealed that this virus was a novel BVDV1 strain that had not been previously reported in the U.S. Genetic analysis of the virus sequence showed that this virus was a BVDV1i virus that had only been reported in Europe and South America. Further, tests were conducted using antisera raised against BVDV strains that are found in commercial bovine vaccines. This showed that the antibodies in these antisera recognized this BVDV1i strain but at a reduced level. This research suggests that current vaccines can provide some protection against emerging BVDV1 but further studies are warranted to confirm this observation.

4. Infection of non-bovine hosts with bovine viral diarrhea virus. Bovine viral diarrhea viruses (BVDV) are known to infect a wide range of ruminants with reports of infection of swine. Earlier studies by ARS researchers at Ames, Iowa, in collaboration with scientists at Auburn University have shown that infections of pregnant non-bovine hosts resulted in nucleotide changes in the genomic RNA of the virus that appear to be adaptive in nature, allowing more efficient replication in these novel host animals. In a further study by both groups where pregnant swine were infected with a BVDV1b, adaptive changes were identified in the viruses recovered from piglets after birth. These changes appeared rapidly and were primarily located in the viral surface proteins. This indicated that these changes may have allowed more efficient infection of the non-ruminant host. Additionally, these changes contributed to the genetic and perhaps, antigenic variability of the virus. Infection of non-bovine hosts may be a significant source of genetic variability of BVDV. This information is important in understanding how BVDV changes and impacts efficacy of currently marketed vaccines.

5. Development of a new vaccine for bovine respiratory syncytial virus (BRSV). BRSV is a major viral pathogen of young calves causing severe respiratory disease. Current vaccines are protective but degree of efficacy variable. ARS researchers in Ames, Iowa, in collaboration with researchers from Iowa State University have previously published research on a novel vaccine comprised of nanoparticles (NP) containing two proteins from BRSV. Calves receiving a single, intranasal dose of the BRSV-NP vaccine were partially protected from BRSV challenge, with reduced viral loads in the lung, reduced virus shedding and significantly reduced lung pathology compared to unvaccinated calves. Protection was found to be associated with an increase in mucosal antibody responses and in specific cellular immune responses. Furthermore, these researchers have now shown that vitamin A deficiency has a negative impact on the measured responses to the vaccine. These studies show an important impact of nutritional status on mucosal immunity and respiratory viral infections. This information will be important in development of new treatments for BRSV infections in calves.

Review Publications
Silveira, S., Falkenberg, S.M., Kaplan, B.S., Crossley, B., Ridpath, J.F., Bauermann, F.B., Fossler, C.P., Dargatz, D.A., Dassanayake, R.P., Vincent, A.L., Canal, C.W., Neill, J.D. 2019. Serosurvey for influenza D virus exposure in cattle, United States, 2014-2015. Emerging Infectious Diseases. 25(11). https://doi.org/10.3201/eid2511.1902532.
Neill, J.D., Crossley, B.M., Mosena, A.C., Ridpath, J.F., Bayles, D.O., Killian, M.L., Falkenberg, S.M. 2019. Genomic and antigenic characterization of a cytopathic bovine viral diarrhea virus 1i isolated in the United States. Virology. 535:279-282. https://doi.org/10.1016/j.virol.2019.07.020.
Dassanayake, R.P., Falkenberg, S.M., Stasko, J.A., Shircliff, A.L., Lippolis, J.D., Briggs, R.E. 2020. Identification of a reliable fixative solution to preserve complex architecture of bacterial biofilms for scanning electron microscopy evaluation. PLoS One. 15(5):e0233973. https://doi.org/10.1371/journal.pone.0233973.
Dassanayake, R.P., Falkenberg, S.M., Nicholson, E.M., Briggs, R.E., Tatum, F.M., Sharma, V.K., Reinhardt, T.A. 2019. Synthetic bovine NK-lysin-derived peptide (bNK2A) does not require intra-chain disulfide bonds for bactericidal activity. PLoS One. 14(6). https://doi.org/10.1371/journal.pone.0218507.
Hwang, S., Dassanayake, R.P., Nicholson, E.M. 2019. PAD-bead enrichment enhances detection of PrPSc using real-time quaking-induced conversion. Bioscience Reports. 12:806. https://doi.org/10.1186/s13104-019-4842-7.
Silveria, S., Falkenberg, S.M., Dassanayake, R.P., Walz, P.H., Ridpath, J.F., Canal, C.W., Neill, J.D. 2019. In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay. Virology. 536:101-109. https://doi.org/10.1016/j.virol.2019.07.029.
Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Walz, P., Casas, E., Ridpath, J.F., Roth, J. 2020. Measuring CMI responses using the PrimeFlow RNA assay; a new method of evaluating BVDV vaccination response in cattle. Veterinary Immunology and Immunopathology. 221:110024. https://doi.org/10.1016/j.vetimm.2020.110024.
Kuca, T., Passler, T., Newcomer, B.W., Neill, J.D., Galik, P.K., Riddell, K.P., Zhang, Y., Bayles, D.O., Walz, P.H. 2020. Changes introduced in the open reading frame of bovine viral diarrhea virus during serial infection of pregnant swine. Frontiers in Microbiology. 11:1138. https://doi.org/10.3389/fmicb.2020.01138.
Hofstetter, A.R., Sacco, R.E. 2019. Oxidative stress pathway gene transcription after bovine respiratory syncytial virus infection in vitro and in vivo. Veterinary Immunology and Immunopathology. 219(2020):109956. https://doi.org/10.1016/j.vetimm.2019.109956.
Eder, J.M., Gorden, P.J., Lippolis, J.D., Reinhardt, T.A., Sacco, R.E. 2020. Lactation stage impacts the glycolytic function of bovine CD4+ T cells during ex vivo activation. Nature Scientific Reports. 10(4045). https://doi.org/10.1038/s41598-020-60691-2.
McGill, J.L., Kelly, S.M., Guerra-Maupome, M., Winkley, E., Henningson, J., Narasimhan, B., Sacco, R.E. 2019. Vitamin A deficiency impairs the immune response to intranasal vaccination and RSV infection in neonatal calves. Scientific Reports. 9(15157). https://doi.org/10.1038/s41598-019-51684-x.
Putz, E.J., Putz, A.M., Jeon, H., Lippolis, J.D., Ma, H., Reinhardt, T.A., Casas, E. 2019. MicroRNA profiles of dry secretions through the first three weeks of the dry period from Holstein cows. Scientific Reports. 9(19658). https://doi.org/10.1038/s41598-019-56193-5.
Powell, E.J., Eder, J.M., Reinhardt, T.A., Sacco, R.E., Casas, E., Lippolis, J.D. 2019. Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony stimulating factor (PEG-gCSF) therapy during a chronic Staphylococcus aureus infection in lactating Holsteins. Journal of Dairy Science. 102(10):9268-9284. https://doi.org/10.3168/jds.2019-16448.
Taxis, T.M., Bauermann, F.V., Ridpath, J.F., Casas, E. 2019. Analysis of tRNA halves (tsRNAs) in serum from cattle challenged with bovine viral diarrhea virus. Genetics and Molecular Biology. 42(2). https://dx.doi.org/10.1590/1678-4685-GMB-2018-0019.