Performing Department
(N/A)
Non Technical Summary
Recent outbreaks of bird flu (H5N1) in dairy cattle have affected several hundreds of dairy herds across multiple U.S. states, and more cases are continuing to emerge. Unlike in birds and other animals, where the infection often targets the lungs or brain, in cattle, the virus behaves very differently. It mainly affects the udder, causing mastitis (mammary gland inflammation) with only mild respiratory signs. This unusual outcome makes H5N1 infection in dairy cattle scientifically puzzling and deeply concerning for the dairy industry.Infected cows produce thick, yellowish, and clotted milk resembling colostrum, showing a sudden drop in milk production. Even more troubling, they shed very high levels of the virus in their milk. This contamination has been linked to cow-to-cow spread through milking machines and even cases of death in cats after consuming infected raw milk, pointing to serious animal and food safety risks. Moreover, human infections following direct exposure to infected cattle have now been documented, raising urgent concerns about the zoonotic potential of this virus and the broader public health risk it poses.Our research aims to understand why and how H5N1 behaves so differently in cattle, using cutting-edge laboratory techniques that do not involve live animals. Instead, we use cells grown in the lab, including mammary gland, lung, and blood vessel cells, to closely mimic how the virus infects a live animal. These models allow us to study how the virus spreads and how the cells respond, including the body's natural inflammatory and antiviral defenses. All of this work is conducted under strict biosafety protocols in high-containment laboratories, ensuring both safety and scientific precision. Understanding the mechanism behind this unique disease pattern in dairy cattle is essential to protecting our milk supply, food safety, and the agricultural economy. By identifying how the virus behaves in cows, we can develop better strategies to detect, control, and prevent future outbreaks, helping dairy producers and animal and public health officials stay ahead of this evolving threat.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Goal:To investigate the mechanisms underlying the pathogenicity, tissue tropism, virus dissemination, and clinical outcomes of H5N1 clade 2.3.4.4b virus infection of cattle.Overarching Hypothesis:"We hypothesize that the permissiveness, infection patterns, and cellular gene expression profiles in cattle respiratory, mammary, and endothelial cells elucidate the mechanisms underlying the pathogenicity, tissue tropism, virus dissemination, and clinical outcomes of H5N1 infection in cattle."We propose two independent aims. In Aim 1, we will investigate the permissiveness and relative replication of the H5N1 virus in cattle nasal, tracheal, endothelial, and mammary epithelial cells to understand the basis for the tissue tropism and systemic spread of the virus. In Aim 2, we will characterize the cellular and molecular responses of H5N1 virus-infected cattle cells to elucidate the mechanisms driving clinical outcomes of H5N1 infection in cattle.
Project Methods
To investigate the permissiveness, pattern of infection, replication kinetics of H5N1 in cattle cells, we will use Primary tracheal epithelial cells (PTEC) isolated from fresh cattle tissue, Primary bovine airway epithelial cells, Bovine turbinate (BT) cell line and EBTr cell line, Bovine Aortic Endothelial Cells (BAOEC), Bovine Mammary Luminal Epithelial Cells (BMLEC).Lectin binding assay will be performed on the cells using FITC-labeled Sambucus Nigra Lectin (SNA) (Vector Labs) and biotinylated Maackia Amurensis Lectin II (MAL II), (Vector labs). Streptavidin conjugated to Alexa Fluor 594 will be used to stain the MAL II lectins. Images of the stained cells will be acquired initially using Confocal Microscope. For the quantification, automated image acquisition will be performed using a Cytation 7 multimode reader to capture 50 fields of view (FOV) per well in each fluorescent and brightfield channel at 20x magnification. Gen5 software (BioTek) will be used to quantify total cells and lectin-positive cells. The lectin binding will provide the comprehensive landscape of SA α-2,6-Gal and SA α-2,3-Gal receptor distribution in the cattle cell types.Virus binding assay:We will use pseudoviruses to explore the H5N1 binding preferences of the cattle cell types. The H5 clade 2.3.4.4b pseudoviruses will be produced using 3rd generation lentiviral plasmids obtained from BEI resources (catalog # NR-53816). Lentiviral helper plasmids, reporter plasmids, and plasmids encoding H5 will be co-transfected into HEK. The pseudoviruses with infectivity titer between 104 and 105 RLU/mL were used for the virus binding assay.The cattle cells will be cultured in Nunc glass bottom dishes, fixed using acetone-methanol, and treated with H5N1 pseudoviruses following blocking with goat serum. A monoclonal mouse anti-hemagglutinin (H5) antibody will be used for staining. The fluorescently stained cells will be imaged using Confocal Microscope. We will quantify the number of cells bound to pseudoviruses normalizing to the total number of cells.Immunofluorescence:To visualize if there are differences in the virus entry in different cell types, we will probe the H5N1 virus nucleoprotein using anti-nucleoprotein antibodies at 8 hours post-infection. AlexaFluor 488 anti-mouse antibody will be used for imaging. Automated image acquisition and quantification of total cells and Np-positive cells will be performed as described for lectin cytochemistry. These results will provide the estimation of the proportion of cells that get infected.Comparative replication kinetics: The cattle cells will be cultured using the culture media recommended by the manufacturer or previous publications. The influenza A H5N1 stock viruses will be used to inoculate the cells with a multiplicity of infection (MOI) of 0.1 to 1. Cell supernatants will be harvested at 1-, 6-, 12-, 24-, 48-, 72-, and 96 hours post-inoculation (hpi). The virus production over the experiment time will be determined by real-time RT-PCR and virus infectivity titration (TCID50).Real-time RT-PCR will be performed using primers targeting M and HA (H5) genes and AgPath-ID™ One-Step RT-PCR Reagents using the protocol and cycling conditions recommended by USDA-National Veterinary Services Lab. Viral genome copy equivalents will be quantified using a standard curve and expressed as copies/mL of supernatant.To characterize the cellular responses of H5N1-infected cells, we will quantify the cell viability, apoptosis and necrosis.Quantification of cell viability: To determine the cell viability following infection, the cells will be seeded in the 96-well plate to obtain 70-90% confluency overnight. Then infected with the H5N1 virus at MOI of 0.1 to 1 and incubated for 24 to 48 hours. We will measure the viability of H5N1-infected cattle cells using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). The percentage of viability is calculated as (OD of infected wells - OD of cell-free wells)/OD of mock control wells - OD of cell-free wells) x100.Apoptosis assays: To investigate the relative apoptosis between the cattle cell types, we will perform TUNEL (Terminal Deoxynucleotidyl Transferase-mediated dUTP?Nick-End) staining. The infected cells will be fixed using 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and treated with the labeling reaction mixture containing recombinant Td transferase, fluorescein isothiocyanate-conjugated nucleotide, and labeling buffer following the manufacturer's protocol (DeadEnd™ Fluorometric TUNEL System, Promega). To detect viral antigens after TUNEL staining, we will use anti-NP mouse monoclonal antibody and secondary antibody. Automated image acquisition will be performed using a Cytation 7 multimode reader. We will normalize the number of apoptotic cells with cells expressing NP viral protein, by calculating the % of NP-expressing cells and % of apoptotic cells.Caspase 3/7 assay: For the caspase assay, the cells will be seeded in a white-walled optical bottom 96-well plate and infected as mentioned for the MTS assay. The Caspase-Glo® 3/7 Assay System will be added in each well and the luminescence will be read after 30 minutes using a Synergy 2 multimode plate reader (Biotek). Fold change in apoptosis will be calculated using the formula: (Luminescence of treated wells - Luminescence of cell)/(Luminescence of control wells - Luminescence of cell-free wells). This assay will include the positive control containing apoptosis inducer, and the negative control containing pancaspase inhibitor z-VAD-FMK.Necrosis and cytotoxicity assay: To measure the lactate dehydrogenase released during necrosis and cytotoxicity, we treat the 5uL infected cell supernatant with LDH-Glo™ Cytotoxicity Assay reagent as per the manufacturer's instructions. The luminescence will be recorded after 1 hour of incubation at room temperature as mentioned in the caspase assay.To investigate the gene expression profiles of infected cells we employ RNAseq and qPCR.RNAseq: Various cattle cells seeded in the 12-well plates will be infected with H5N1 virus at the MOI of 0.1 to 1. The cell supernatants and adherent cells will be collected at 1, 12, 24, 48- and 72-hpi. We will treat the cattle PBMCs and MDMs with infected supernatants. The supernatants will be collected to determine the TCID50 and cytokine and chemokine quantification. Following the manufacturer's protocol, the adherent cells will be collected in Buffer RLT Plus. Total RNA from the cell lysates will be extracted for cDNA library preparation. Library prep and RNA sequencing will be performed in-house using the Illumina Stranded mRNA Prep workflow and NextSeq2000. The sequencing reads will be analyzed through the Genomics and Systems Biology core. The differential gene expression in cattle cell types will be depicted as the heat map.Innate immune profiling using qRT-PCR: The cDNA will be used to conduct quantitative gene expression analysis of IAV M gene and the innate immune genes, CSNK1D, CXCL10, IFI44, IFI6, IFITM1, IRF4, ISG15, OAS1, OAS2, SERPING1, STAT2, STAT1, MASP, CSNK1D, CSF2RA, TNF-α, MCP-1, RANTES, IP-10, IL-1β, IL-6, IFN-αβ, C1QC and C3. We will also confirm the up- or down-regulation of selected genes determined in RNAseq using qRT-PCR. Gene expressions will be normalized to the expression of one or more optimal reference genes.Quantification of cytokines/chemokines: We will use the supernatantsfor the quantification of a panel of cytokines and chemokines using the MILLIPLEX® Bovine Cytokine/Chemokine Magnetic Bead Panel 1 - Immunology Multiplex Assay (BCYT1-33K Millipore Sigma). The concentrations of cytokines/chemokines (pg/mL) will be compared between the cattle cell types at different time points.