Performing Department
(N/A)
Non Technical Summary
This project seeks to answer a critical question: How do bird flu viruses like H5N1 adapt to infect cattle, and what can be done to mitigate the risk? Bird flu viruses, once thought only to affect birds, are evolving, and H5N1 infections have recently emerged in dairy cows. While not fatal, the infection causes loss of appetite, nasal discharge, reduced milk production, and virus shedding in milk, leading to economic losses for farmers and raising concerns about food safety and security. Since the first reported case in March 2024, infections in dairy cattle have continued to rise.To better understand and prevent future outbreaks, this project will examine how certain bird flu strains, particularly H5, H7, and H9, adapt to infect cattle. Influenza viruses rely on specific receptors on host cells to enter and initiate infection. While sialic acid (SA) molecules are the primary receptors, recent studies suggest that immune system molecules like MHC class-II may also act as alternative entry points. This research will investigate how these viruses bind to various cattle tissues, including the lungs, digestive system, and mammary glands, to assess their potential risk to livestock.Additionally, we will analyze genetic changes in the hemagglutinin (HA) protein, which enables the virus to attach to host cells. Understanding how these mutations affect the virus's ability to infect cattle could reveal the risk of spreading beyond the mammary gland. To ensure safety and accuracy, these experiments will be conducted using a safe, non-infectious pseudovirus system and tissues collected post-slaughter.Beyond answering these scientific questions, this project will develop a risk assessment to guide better mitigation and preparedness strategies for protecting cattle from emerging bird flu strains. The findings will evaluate the likelihood of viral spread, provide insights into viral evolution, and inform vaccine development to safeguard livestock. By proactively studying how bird flu spreads in cattle, this research will help protect animal health, strengthen food security, and ensure the resilience of the U.S. dairy and beef industries.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
The overarching goal of this project is to elucidate the receptor basis for the susceptibility and tissue tropism of emerging H5, H7, and H9 avian influenza virusesin cattle and to investigate the impact of naturally occurring mutations in clade 2.3.4.4b H5 on the pathogenicity and tissue tropism of the virus in cattle.
Project Methods
Glycan analysis: Cattle tissue glycan profile will be conducted in collaboration with the National Center for Functional Glycomics (NCFG) at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston. Our approach involves analysis of both N- and O-linked glycans employing using a Thermo Fisher TSQ Altis Mass Spectrometer coupled to a Vanquish LC system. Over 200 glycan standards are utilized to identify the chemical nature of the glycan molecules.Glycan array: Glycan array will be utilized for receptor-ligand binding analysis employing pseudovirus harboring HA mutations. In this array, a total of 562 natural and synthetic N- and O-glycans are bioprinted onto solid N-hydroxysuccinimide (NHS)- derivatized glass surface. These immobilized glycans serve as ligands. Ligand-bound pseudovirus is incubated with highly specific fluorophore-conjugated HA primary antibody that is prior validated to bind with the target pseudoviruses carrying mutant HAs. Virus receptor binding is measured using a GenePix 4300A microarray scanner (Molecular Devices, LLC) and analyzed with the GenePix Pro 7 software.Lectin histochemistry: Lectin histochemistry will be carried out on paraffin-embedded tissue sections using well established methods in our lab. Briefly, thin sections (5 μm thickness) of formalin-fixed tissue samples are deparaffinized in xylene and rehydrated through a series of ethanol treatments. Sections are then blocked using goat serum diluted to 1:40 with TBS and further blocked using a Streptavidin/Biotin Blocking Kit. The sections are subsequently incubated with fluorescein-labeled Sambucus nigra Lectin (SNA, Vector Laboratories, FL13012), Maackia amurensis Lectin II (MAL II), Unconjugated (Vector Laboratories, L-1260-2), and fluorescein-labeled Maackia amurensis Lectin I (MAL I, Vector Laboratories) at a concentration of 10 μg/ml and incubated overnight at 4°C. SNA binds specifically to sialic acid linked to galactose via an alpha-2,6 linkage (SA α-2,6-Gal), while MAL II and MAL I bind to sialic acid via an alpha-2,3 linkage (SA α-2,3-Gal) with a difference that MAL I binds to distinct glycan structures compared to MAL II. After three TBS washes, sections stained with MAL II are treated with Streptavidin, Alexa Fluor 594 conjugate for 2 hours at 4°C. Sections are subsequently mounted using ProLong™ Gold Antifade mountant with DAPI. After curing for 24 hours at room temperature, tissue sections stained with the linkage-specific lectins will be subjected to confocal microscopy.Immunostaining: Formalin-fixed tissue samples will be blocked with goat serum diluted in TBS buffer for 30 minutes at room temperature (RT). After blocking, the sections will be incubated with the target antibodies (e.g., mouse anti-bovine MHC Class II DR monoclonal antibody) at a specified dilution in TBS for one hour at RT. This will be followed by incubation with a fluorophore-labelled goat anti-mouse IgG secondary antibody at a specified dilution for two hours at RT. The sections will then be mounted with ProLong Gold antifade reagent containing DAPI for counter staining. Negative controls will be performed by omitting the primary antibody during the initial incubation. The sections will be scanned using confocal microscopy to capture detailed imagesPseudovirus Generation: We have a validated protocol to generate lentiviral vector-based replication-incompetent H5N1 pseudovirus expressing HA protein derived from a synthetic DNA sequence encoding it. In addition, we will generate pseudoviruses expressing H7 from AIV H7N9 and H9 from H9N2. Briefly, the 3rd generation lentiviral plasmids obtained from BEI resources are used to produce IAV pseudotyped virions. The HA sequence of a target strain will be retrieved from NCBI, and an expression construct is designed using SnapGene software. In certain instances, the consensus sequence of HA from multiple clades will be utilized for engineering the expression construct.Lentiviral helper plasmids, reporter plasmids, and plasmids encoding HA will be co-transfected into HEK 293T cells using Fugene6 transfection reagent (Promega, USA). The transfected cells will be treated with broad substrate-specific neuraminidase (New England Biolabs, Cat # P0720L, 100 units/mL) at a certain interval post transfection. Pseudovirus particles will be collected 48 hours post-transfection, and the viral titers will be determined by infecting the HEK-293T cells. Employing the above approach, we have already generated pseudoviruses carrying hemagglutinin from H5N1 clade 2.2 and 2.3.4.4b. We will employ this strategy for the production of H7 and H9 pseudoviruses. and H5 pseudoviruses carrying the mutations, as outlined in the following section with specific emphasis on the selection criteria for further investigation.Virus binding assay: We will utilize H5 pseudoviruses (both wild-type and mutants) to conduct tissue binding assays. We will down-select H5 mutations that show notable differences in glycan binding profiles. The choice of cattle tissues for these assays will be based on glycan distribution profiles in the tissues and the receptor binding profiles. This will help us establish the functional relevance of the mutations and determine whether the change in glycan binding pattern significantly impacts the tissue tropism of the virus. Formalin-fixed tissue samples (mammary gland, trachea, lung, intestine, and brain) will be utilized for virus binding assay. Briefly, 5 - 7 μm tissue sections are placed on glass slides, followed by deparaffinization and antigen-retrieval. Tissue sections are subsequently incubated with Influenza A pseudoviruses at 37°C for 2 hours. Inactivated diluted goal serum is used to block the sections overnight prior to incubation with mouse monoclonal anti-influenza virus H5 HA (strain-specific) for another 90 minutes. Following thorough washing, sections are incubated with a secondary goat anti-mouse IgG H&L (Alexa Fluor® 647) antibody for 40 minutes. The sections are subsequently mounted in DAPI-spiked ProLong Gold antifade mounting media. For each tissue section, a parallel negative control section is prepared wherein the virus incubation step is omitted. In addition, a serially cut consecutive deparaffinized section is stained with H&E for identification of cell types within the tissue slices. Imaging of the fluorescently labelled slides is performed using a confocal microscope (Nikon AX).Efforts: Findings from the project will be published in peer-reviewed journals, preferably in open access format. In addition, we will also communicate our findings through multiple digital platforms including those offer through University Media Relation Office.Evaluation: We have outlined a timeline of the project to accomplish the studies detailed under individual aims. Most of the acquired data will be in digital form. Machine-generated raw data will be stored as are. Wet lab-based experiments will be performed using true biological replicates and will be evaluated by the reproducibility of data along with method validation. Experiments involving subjective evaluation (e.g., microscopy) will be blinded. Virus genetic information will be collected from trusted database including NCBI and GISAID.Below are sample examples of evaluation studies planned:Glycan analysis: Biological replicates, spiking standards with analytes and measurement of recovery.Lectin histochemistry: Biological replicates, removal of glycans from the tissue and assessment of non-specificity, use of positive and negative control tissues as available.Pseudovirus Generation : Sequencing of all plasmids and comparison to the target sequence, determination of viral titer using at least 3 biological replicates.Virus binding assay: Biological replicates, utilization of tissue samples devoid of glycans, use of uniform virus dilution strategy to compare the results among different biological replicates.