Performing Department
(N/A)
Non Technical Summary
Recent outbreaks of bird flu (H5N1) in U.S. cattle have raised concerns about how flu viruses spread in livestock. However, another lesser-known virus, Influenza D (IDV), is already present in cattle worldwide and evolving rapidly. While IDV was initially thought to cause only mild illness, newer strains are now leading to serious respiratory disease in calves. Even more concerning, IDV can infect other farm animals and even humans, posing a broader risk to agriculture and public health.Currently, there are no reliable antibody detection tests to track the exposure of cattle to IDV and no vaccines to protect cattle, leaving the livestock industry vulnerable. Without action, IDV could become a significant threat to cattle health, farmers' livelihoods, and the stability of U.S. agriculture.To address this, experts, the team led by the University of Pittsburgh in collaboration with scientists from South Dakota State University are working together on two critical solutions. First, we are developing new diagnostic tests to monitor IDV in cattle and other livestock. Second, we are developing an innovative nasal spray vaccine to protect cattle from IDV and prevent the disease from spreading.By proactively developing better monitoring tools and a vaccine, this research will help safeguard cattle, support US cattle industry, and strengthen the sustainability and profitability of the U.S. livestock industry.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Goals / Objectives
The goal of this project is to develop pseudovirus-based serology tests for detecting Influenza D in cattle and create a novel, safe, and effective vaccine using advanced molecular techniques for disease prevention and to protect cattle health.?
Project Methods
Pseudovirus neutralization test (pVNT):To optimize assay conditions including serum dilution and incubation times, we will use IDV antibody-positive and negative serum samples. The IDV antibody status will be determined by live virus neutralization assay (VN). Ten serum samples with no (<40), low (40 to 80), medium (80 to 160), and high (>160) antibody titers in VN will be chosen for the optimization. The serum samples will be diluted in cell culture media to achieve 1:10, 1:20, 1:40, and 1:60 dilutions. Then, 50µL of diluted serum will be mixed with 100µL of D/CA pseudoviruses leading to assay dilutions 1:30, 1:60, 1:120, and 1:180. We will use D/CA pseudoviruses for optimization, depicting the HEF of the D/CA2019 lineage, which has broadly cross-reactive epitopes. The assay includes virus control, positive serum (known positive), negative serum (fetal bovine serum), and cell control. RLU readouts will be compared between 48 and 72 hours to choose the optimum incubation time that yields 5x104 RLU in the virus control. The following formula will be used to determine the percent inhibition: 100 − (RLU of sample/RLU of virus control) *100. Ideally, the dilution of serum that yields >70% inhibition with low titer samples and <50% inhibition on IDV antibody-negative samples will be considered for pVNT assay validation using clinical samples.Validation of IDV pVNTs for the diagnosis:Cut-off determination: The serum samples will now have a positive or negative status based on VN, which is the reference test for comparison. The pVNT percent inhibition values of VN positive and negative samples will be analyzed to determine the pVNT cut-off that categorizes samples as positive and negative.Reproducibility: Identical aliquots from a panel of 20 serum samples will be tested in SDSU, PVL, ISU VDL and Pitt using the same reagents, protocols, and identical instruments to assess the accuracy of the pVNT. The repeatability will be tested within our laboratory by 3 personnel on the same panel of samples that are used for reproducibility.Analytical Specificity: The analytical specificity will be tested using influenza viruses and other viruses that are known to infect cattle causing BRDC.Determination of diagnostic sensitivity and specificity: We will tabulate the reference VN assay and pVNT results in 2x2 table and estimate the diagnostic sensitivity and specificity using the standard formula. Diagnostic sensitivity=TP/(TP + FN); Diagnostic specificity= TN/(TN + FP).Transition plan to NAHLN: The validated pVNT will be initially transferred to NAHLN laboratories in Pennsylvania, Iowa and South Dakota initially for implementation.IDV-lineage specific antibody surveillance:We will employ the pseudoviruses generated using HEF of different lineages of IDV, D/OK, D/660, D/Yama2016, D/Yama2019 and D/CA2019 for determining the lineage-specific antibody titer in the cattle serum. This will help identify the lineage that is circulating in the given location.Statistical analysis: Percentage inhibition will be calculated based on luciferase activity of cells infected with virus only, representing no neutralization. For each serum:pseudovirus combination, the measured percent neutralization will be plotted against corresponding serum dilution, and non-linear regression curves will be produced using R software or GraphPad Prism v.10.2.2 to determine the 50% neutralization titer (NT50).Antigen cartography: Creating an antibody landscape for each IDV antibody-positive serum sample will allow visualization of the antigen most strongly associated with serum antibody. Using pVNT NT50 data against each lineage, the 2-dimensional antigenic map will be computed using the Racmacs package in the R software with 1000 optimizations. Generation of HRT-18G Cell line stably expressing IDV M protein and Rescue of M-null IDV: We will use 3rd generation lentiviral vector to produce HRT-18G stable expressing IDV M protein. We will culture the HRT-18G-M cells using DMEM media supplemented with 10% FBS. The overnight culture will be co-transfected with the dual promoter-expressing plasmids pHW2000 containing IDV genome segments, PB2OK, PB1OK, P3OK, HEFCA, NPOK, MOK (M null), and NSOK. The M transcripts and M1 and DM2 proteins will be expressed in the HRT-18G-M cells, but the transcripts will not get incorporated in the virion as they lack the UTR regions. The culture supernatants will be collected on the 5th day post-transfection and filtered using a 0.45µ membrane filter.Confirmation of single-cycle replication: We will infect the ST cells at 80% confluency with the 0.1 M.O.I. of M-null vaccine virus and D/OK wild type. The virus inoculums will be prepared in DMEM containing 2% FBS and TPCK trypsin (0.5µg/mL). The inoculum will be removed after 2 hours, washed twice, and replenished with fresh media. The supernatants are collected at 0, 24-, 48-, 72-, and 96-hours post-infection. The infected ST cells will be observed for cytopathic effects; the M-null vaccine virus is expected to not induce cytopathic effects as they replicate for a single cycle and do not produce progeny virions. Meanwhile, D/OK wild type should replicate and induce cytopathic effects. The supernatant will be tested in ST cells to confirm the lack of infectious virions in the supernatant from M-null-infected ST cells.Transmission electron microscopy: We will confirm the production of intact M-null virus by transmission electron microscopy. The transfection of HRT-18G cells for M-null virus will be performed in fifty 10cm dishes. The virus-containing supernatant will be harvested and clarified by centrifugation at 2600×g, 5 min, at 4°C. The clarified supernatant will be layered over a 20% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA) and the virus concentrated by ultracentrifugation at 112,600×g for 2 hrs at 4°C in Beckman Coulter centrifuge, Fullerton, California. The concentrated virus will be purified using 10-30% gradient of Optiprep medium (Sigma) per the manufacturer's protocol. The purified virus will be absorbed onto carbon-coated nickel grids (Electron Microscopy Sciences, Hatfield, Pennsylvania). Images of negatively stained virions will be captured on a transmission electron microscope.Virus Neutralization assay (VN): We will determine the neutralizing antibody titers in vaccinated calves using VN. Serum samples will be heat-inactivated at 56°C for 30 minutes. Two-fold serial dilutions (1:20 to 1:1280) will be prepared in DMEM containing 10% FBS. For the neutralization assay, 100 µL of live IDV D/CA2019 (produced by reverse genetics) will be mixed with 50 µL of diluted serum samples and incubated for 1 hour. The mixture will then be added to a 96-well plate containing a monolayer of ST or MDCK cells (13,000 cells/well) and incubated for 72 hours. Neutralization will be assessed by measuring the cytopathic effect (CPE) and calculating the neutralization titer based on the highest serum dilution that prevents CPE.To evaluate cross-protection across multiple IDV lineages, we will employ pseudovirus neutralization tests.Measurement of M-null IDV-specific Cell-mediated immune response: The cell-mediated immune response elicited by the M-null vaccine in calves will be evaluated by intracellular cytokine staining (ICS) using flow cytometry. The cryopreserved PBMCs will be seeded in a 96-well plate (1x106 cells/well), using RPMI 1640 medium supplemented with 10% fetal bovine serum. Then the cells will be stimulated using UV-inactivated IDV (MOI 10) or live IDV virus (MOI 1) overnight. Pokeweed mitogen (Sigma Aldrich, USA) and RPMI containing 10% FBS will be used as positive and negative controls, respectively. Brefeldin A will be added during the stimulation.