Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Veterinary Medicine
Non Technical Summary
Affections of the respiratory tract in calves lead to significant economic losses for cattle producers. Respiratory disease in cattle may be caused by a plethora of viral and bacterial pathogens combined with environmental factors and host characteristics. Decreased performance and mortality also lead to potential genetic losses and food security concerns. Viral infections are usually the initiating pathogens, and disease progression is associated with secondary viral and bacterial involvement. Among primary viral pathogens, RNA viruses, including bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV), are well-recognized players in the development of bovine respiratory disease. In contrast, the role of the recently identified bovine influenza virus type D (IDV) in the bovine respiratory disease complex (BRDC) is a matter of current investigation.Although inactivated and modified live vaccines (MLV) are widely used for BVDV and BRSV in the US, both pathogens persist as a significant problem. BRD is associated with stressful management practices frequently in young animals. At this age, in addition to the immature immune system, maternally derived antibodies may interfere with vaccine efficacy and hamper the fitness of the available control measures. Currently, alternative approaches to decrease losses from respiratory disease in calves are being sought after, including the identification of biomarkers toward increased disease resistance/protection and immunomodulation strategies to develop efficacious preventive or treatment approaches.Modulation of the immune system to promote resistance to respiratory viral infections have vast use potential. However, there is a lack of comprehensive studies investigating both innate and adaptive immunity in cattle following exposure to BRD pathogens. Therefore, our goal is to use cutting-edge technology to analyze innate and adaptive host responses to BVDV, BRSV, and the recently discovered IDV in cattle. The work will be accomplished using RNA sequencing to compare the gene expression during single or multiple infections with BRD RNA viral pathogens. Flow cytometry, histology, serology, and other laboratory assays will be used as additional tools to assess relevant aspects of pathogenesis, innate, and specific responses to these pathogens. Advances in understanding host-pathogen interaction will undoubtedly contribute to developing and refining control strategies for respiratory viruses in cattle, especially immunomodulation. Overall, the project may contribute toward reducing costs related to respiratory diseases and support food security and animal welfare.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Although the availability of killed and MLV vaccines for most BRD pathogens, including BVDV and BRSV, respiratory disease in cattle remains a significant burden for the US cattle producers. BRD involves multiple pathogens affecting young animals with the relatively immature immune system, and it is also linked to stressful conditions, all of which impact vaccines' efficacy and suitability as preventative measures to BRD. Therefore, new lines of investigation have pursuit immunomodulation as a measure of control, aiming to improve overall health and resistance to infections, supporting animal welfare, and limiting the impact of BRD on the cattle industry. However, both immunomodulatory and more suitable vaccinology approaches depend on the better understanding of host responses to pathogens (both deleterious and protective). Local, primary, and secondary lymphoid organ responses are largely unknown for these critical RNA viruses and cattle in general.Due to technology limitations, past evaluations of the innate immune responses were mainly conducted using biased approaches, mainly by RT-qPCR for a limited number of genes somewhat arbitrarily selected in each study. The global and parallel study of innate and adaptive immune responses, locally and in lymphoid organs affected by BRD pathogens using cutting-edge technologies will reveal essential knowledge about the immune response landscape to these RNA viruses in cattle. Ancillary and corroborative techniques (ELISA, viral neutralization, flow cytometry, histological and other immune assays) will be employed to increase and strengthen the obtained data. The information obtained in this project may provide the basis for developing and refining products focusing on cattle immunomodulation and identifying possible genomic makers related to successful infection control.Specific objectives:Evaluate the gene expression profile to BVDV and BRSV using anin-vitromodel in multiple time points following infection.Typical and high virulence BVDV strains will be inoculated in primary bovine cells (nasal and lung) and PBMCs.BRSV will be inoculated in primary bovine nasal cells.RNA-seq will be conducted in cells in serial time points post-inoculation.Transcriptome findings will be validated using RT-qPCR.Evaluate local in-vivo host responses to BVDV and BRSV in the lung and in lymphoid tissues of calves previously inoculated with BVDV, BRSV, and IDV alone, or BVDV followed by IDV inoculation.Lung, retropharyngeal and submandibular lymph nodes will be submitted to RNA-seq and transcriptome analyses.Transcriptome findings will be validated using RT-qPCR, and aspects of adaptative immunity evaluated by flow cytometry when suitable.Correlate the in-vivo transcriptome data with the in-vitro (Objective 1) findings.Conduct a global correlation of results obtained, including innate and adaptive immune responses, with clinical and virological aspects (levels of viremia and shedding, disease severity, and pathological findings).
Project Methods
Viruses and cellsWe have several strains of typical virulence BVDV-1 and BVDV-2 and a high virulence BVDV-2. For Objective 1, we will use the high virulence BVDV-2 1373 and the typical virulence BVDV-2 RS886, which is the strain used in the animal studies described earlier. The viruses will be amplified in both primary bovine turbinate and primary bovine lung cells. These cells are maintained in cell culture media (MEM) supplemented with L-glutamine, antibiotic-antimycotic, and 10% of fetal bovine serum at 37°C in a humid atmosphere of 5% CO2. BVDV will also be inoculated in PBMCs of naïve calves. The BRSV-375 was received from a USDA collaborator and has been passed in-vivo by our group. This strain is usually described as a suitable virus for challenge studies, including vaccine trials. BRSV is also amplified in primary bovine turbinate cells.The IDV was isolated from a bovine respiratory sample in Texas. The HRT-18G (ATCC CRL-11663) cells are used for virus amplification using RPMI media supplemented with L-glutamine, antibiotic-antimycotic (ST), and 10% FBS. Swine testicle cells are used for IDV titration. All viral titrations are performed using the Reed and Muench method.For Objective 1, we will use a multiplicity of infection (MOI) of 1 for all BVDV and BRSV virus inoculations in different cells. Cells will be harvested in serial time points, for example, 1, 6, and 12 hours post-infection. Cells will be placed in RNALater® or equivalent and stored at -80 until RNA extraction. RNA extraction will be conducted using commercially available kits and standard procedures. RNA-seq will be conducted either in-house at the OSU Genomics core or at a service provider.Bovine samplesOur group currently has three ongoing animal projects involving the inoculation of calves with BVDV, IDV, BRSV alone, or sequential infection with BVDV and IDV. A total of 41 calves will be used to complete these studies. In all our studies, serial collection of PBMCs is being conducted, and cells are being preserved in liquid nitrogen. Nasal and rectal swabs are also being collected and stored at -80ºC. During necropsies, several organs are being collected and stored at -80ºC. The tissue list includes the thymus, retropharyngeal lymph node, submandibular lymph node, tracheobronchial lymph node, mesenteric lymph node, tonsils, spleen, lung, and trachea. We are also collecting and storing bronchoalveolar lavage. This archive of tissues will be available for the proposed studies. We are conducting necropsy during the acute- (day 13) and convalescent-phase (day 35) of infection for the BVDV and IDV projects. The BRSV study was designed to evaluate the disease progression during the acute phase. Animals were divided into two groups, and a necropsy will occur around days 7 and 10 post-infection.Gene expression analysesGene expression will be assessed using RNA-seq transcriptome analyses. Cell culture samples or tissue collected during necropsy will be subjected to RNA sequencing to explore the transcriptional network with a particular interest in the analyses of transcripts related to immune responses and tissue regeneration. RNA will be isolated from the tissues using commercially available kits and standard procedures. We expect to retrieve RNA with RIM (RNA integrity number) over 7. However, samples with lower RIM may be used. Library preparation and sequencing will be performed in-house at the Genomics core or a service provider. Sequencing will be performed, and at least 15 million reads per sample are targeted. We expect to retrieve at least 90% of the reads with a quality score over 30 (Q30 - the probability of incorrect base call was 1 in 1000).FastQC will be used to trim the paired-end reads. Reads will be aligned to the reference genome (Bos taurusreference annotation ARS-UCD1.2) using STAR. Gene and transcript expression levels will be conducted using HTSeq, while identification of genes with different expression levels between groups and statistical significance will be conducted with maSigPro package. All the listed tools are part of the OmicsBox Blast2GO Pro Transcriptomics module. Other equivalent app suites may be used at the time of analyses, for example, the Illumina DRAGEN suite apps. We employ a null hypothesis of the genes not being differently expressed than 1.5 X fold and specify an overall false discovery rate (FDR) of 0.1 (e.g. 10%). Raising the stringency of the test supports the identification of gene expression changes that are more likely to represent significant biological changes. Based on the results identified by RNA-seq, we will select some genes and design a RT-qPCR assay toward further validation of the transcriptome data. Gene ontology and orthology will be conducted using the free app Reactome (reactome.org).Cell-mediated immune responsesHumoral and cellular immune responses will be evaluated by serological (virus neutralization) and cell-mediated immune assays (flow cytometry-based lymphocyte proliferation- and intracellular cytokine staining assays). Neutralizing antibody levels for BVDV and BRSV in serum and BAL samples will be defined using virus neutralization (VN) assay, while hemagglutination inhibition (HI) will be used for IDV. Both VN and IDV will be conducted using standard OIE protocols.Pathogen-specific cell-mediated responses to the pathogens by PBMCs or alveolar lymphocytes (BAL) will be assessed for all major bovine T cell subsets (CD4, CD8, γδ T cells). Afterin-vitrorecall stimulation with the specific pathogen, proliferation (CellTrace dilution assay), cytokine expression (INF-gamma, TNF-alpha), and activation markers (CD25, CD44, CD62L) will be assessed. Antibody clones that will be used are CD3 (MIA11), CD4 (ILA11), CD8 (BAQ111A), TCR1 δ chain (GB21A) (all from the Washington State University MAb Center), mouse anti-bovine IFN-γ-RPE (clone CC302, Bio-Rad), and TNF-α (clone MCA2334; Bio-Rad). Panels have also been established to assess the expression of T cell activation markers by T cells: CD44 (BAT31A), CD62L (BAQ92A), CD25 (CACT116A), and CD27 (anti-human, clone: M-T271). All antibodies required for the flow cytometry assays are commercially available and have been previously optimized (Maggioli et al., 2016, 2015) by Dr. Maggioli (Co-PD). Different markers may be included in the analysis, based on transcriptomic and or proteomic analysis, depending on reagent availability. Cells will be acquired using BD LSR II (BD Biosciences) cytometer at the Immunology Core at the Veterinary School, and analyses will be conducted using FlowJo (Tree Star Inc.). Statistical analysis between groups will be performed using ANOVA (GraphPad Prism software, Tree Star Inc).Contingencies RNA integrity is always critical for RNA-seq studies, and obtaining high-quality RNA from different tissues may pose a challenge. Although we are following appropriate steps toward sample storage, RNA quality may still be an issue for transcriptome analyses. If that occurs, because of the increased protein stability compared to RNA, we plan to conduct proteomics analyses instead or in addition to transcriptomic analysis. Dr. Steven Hartson, Director of the Proteomics Core will assist in establishing the protocols and the pipeline analyses.