Performing Department
(N/A)
Non Technical Summary
A new strain of H5N1 influenza virus--commonly referred to as "bird flu"--has been detected in dairy cattle, which could pose a serious threat to the U.S. agricultural industry. If this virus becomes widespread in cattle, it could spread to other animals, including pigs, leading to severe consequences for livestock health, food production, and the economy. Understanding whether H5N1 can infect and spread among pigs is crucial to preventing future outbreaks and safeguarding this critical domestic industry.Our research will focus on how the H5N1 influenza virus behaves in pigs. Using a combination of animal studies and lab experiments, we will observe how the virus infects pigs, spreads within their populations, and interacts with their immune systems. By studying the virus in pigs, we aim to identify which strains pose the greatest risk to swine health and develop strategies to prevent their spread.The goal of this project is to provide farmers, policymakers, and health authorities with critical information to better manage and control the risks posed by H5N1 in swine. The insights gained will help improve surveillance, prevention, and control strategies, ultimately reducing the risk of outbreaks and protecting the agricultural economy.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The objectives of this proposal are two-fold. First, we aim to evaluate the pathogenicity and transmission potential of bovine-origin clade 2.3.4.4b viruses to swine in vivo. Second, we aim to evaluate the broad spillover potential of bovine-origin clade 2.3.4.4b viruses across diverse agricultural animal species, using highly relevant primary cells and tissues as in vitro infection models. Our long-term goal is to establish a framework for ongoing surveillance and risk assessment of influenza viruses across diverse agricultural animal populations.
Project Methods
Overview of Aim 1:This Aim evaluatesthe spillover potential of bovine-infecting H5N1 clade 2.3.4.4b viruses into lactating sows. We will assess the infection and transmission dynamics through two experimental approaches: intranasal (Aim 1.1) and intramammary (Aim 1.2) routes of exposure.Aim 1.1 - Intranasal infection:Lactating sows (n=4 infected, n=2 controls) will be intranasally inoculated with 2x106 TCID50/mL of A/bovine/Ohio/2048/2006 virus (H5N1 clade 2.3.4.4b). Postpartum sows will receive inoculation one week after parturition. Samples (nasal, oral, fecal swabs) will be collected daily from days 1-14 post-inoculation (DPI), and milk will be collected at DPI 1, 3, 5, 7, 10, and 14.Aim 1.2 - Intramammary infection:Lactating sows will be intramammary inoculated with 2x106 TCID50/mL H5N1 virus in the gland cistern of lactating mammary glands. Milk, blood, and tissue samples (mammary gland, lung, trachea, nasal turbinates, intestines) will be collected at necropsy on days 7 and 14.Experimental timeline and virus handling:Sows will be housed at the PAAR facility two weeks prior to parturition. Once post-partum, animals will be inoculated, and samples will be collected at the intervals mentioned. We will use standard techniques for swab and tissue collection, with sample processing for viral RNA quantification and TCID50 assays to evaluate infectious virus.Data analysis and evaluation:We will quantify viral RNA through RT-qPCR and infectious virus using TCID50 assays. Data will be analyzed using regression analysis to correlate viral load with clinical outcomes. Pathological analysis of tissues will include histological examination by licensed pathologists. This approach will yield a comprehensive understanding of spillover dynamics, with clear measures of viral replication, transmission, and pathology.Efforts and impact:Results from this work will provide insights into the spillover risk posed by H5N1 clade 2.3.4.4b viruses. Findings will be disseminated through workshops, extension outreach, and scientific publications. The project's success will be evaluated through milestones such as the clinical evidence of disease, routes of transmission,and viral persistence in sows and piglets.Overview of Aim 2:This aim evaluates the spillover potential of bovine-infecting H5N1 clade 2.3.4.4b viruses in bovine and swine cell culture models, with a focus on replication dynamics and host immune responses in the respiratory tract and mammary gland.Aim 2.1 - Infections in primary cell culture models:Bovine and swine primary airway epithelial cultures (PAEC) and mammary epithelial cultures (MEC) will be inoculated with bovine-infecting H5N1 clade 2.3.4.4b viruses (n=5) and avian (n=3) controls. Infections will be conducted at low MOI, with viral replication measured over time (1, 8, 24, 48, 72, and 96 hours) by plaque assays. Samples from the apical surface of PAEC and media from MEC will be collected to determine viral titers.Aim 2.2 - Host response analysis:Gene expression of proinflammatory cytokines (e.g., IL-6, TNF-α) and antiviral genes (e.g., IFN-β, MX1) will be evaluated by RT-qPCR in infected cultures. Host immune response will be assessed at 24 and 72 hours post-infection, comparing responses across cell types and species. Cytokine induction will be correlated with viral replication to assess host immune activation.Experimental timeline:Primary bovine and swine airway and mammary epithelial cells will be collected from necropsied animals and cultured as described. After infection with the H5N1 clade 2.3.4.4b viruses, samples will be collected at specified time points for viral titration and gene expression analysis.Data analysis and evaluation:Viral RNA will be quantified by RT-qPCR, and infectious virus will be titrated using plaque assays. Host immune responses will be analyzed through relative gene expression comparisons. Statistical analysis will be performed using one-way ANOVA and regression analysis to correlate viral load and immune response.Efforts and impact:Results will provide insights into the replication potential and immune response of bovine H5N1 clade 2.3.4.4b viruses in bovine and swine cell/tissue models. Findings will be disseminated through scientific publications and outreach, contributing to the understanding of viral spillover risk. Indicators of success include viral load dynamics, host immune response, and the identification of high-risk viruses.