Performing Department
(N/A)
Non Technical Summary
Virus replication was mainly confined to the mammary tissue, and high levels of viral loads were detected in the milk.The results from this study will fill the knowledge gap in our understanding of HPAI H5N1 viral disease in agricultural animals and will have a significant positive impact on the maintenance of the overall health of US agricultural (and tribal) animals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overarching goal of this project is to better understand HPAI H5N1 viral pathogenesis, tissue tropism, and transmission in agricultural ruminant animals. There is limited to no H5N1-related knowledge available regarding infection in small ruminants such as goats and sheep, or mammary-associated infections. In this proposed work, we will determine the (i) susceptibility of various ruminant mammary gland epithelial cells (MGECs), including those from cows, goats, sheep, and bison udder tissue to the bovine HPAI H5N1 virus and its receptor specificities, and (ii) the viral pathogenesis and tissue tropism in a small ruminant model (goats) via intramammary and oronasal inoculations. Our proposed objectives will address critical knowledge gaps in the H5N1 disease biology and meet the mission goals of the Program Area of the USDA-NIFA. This animal model will also be important for studying other aspects of influenza A virus (IAV) infection in ruminants such as viral pathogenesis and evolution, and for the development of therapeutics.?
Project Methods
We propose to determine the susceptibility of various ruminant mammary gland epithelial cells (MGECs) to the bovine HPAI H5N1 virus and their receptor specificities (Aim 1) and establish a small ruminant model (goats) to study H5N1 viral pathogenesis (Aim 2).Aim 1. Determine the susceptibility of various ruminant mammary gland epithelial cells (MGECs) to the bovine H5N1 virus.To establish the primary cell cultures of MGECs, udder tissues from confirmed healthy animals (bovine, bison, sheep, and goats) will be used. Small tissue slices will be cultured in T75 tissue culture flasks using 1x DMEM containing 20% FBS and 2x antibiotic and antimycotic mix in a cell culture incubator at 37 oC, 5% CO2, and 95% humidity for one week. Primary cell cultures will be further validated for epithelial cell types by immunofluorescence assay (IFA) for cytokeratins, which are known to express mainly in the epithelial cells. We will also employ other markers, including milk proteins (casein) and tight junction (TJ) proteins, to confirm correct MGECs.Next, we will induce milk production in MGECs by adding necessary growth hormones (insulin, epidermal growth factor, prolactin, and temperature (39 oC) in the growth medium and confirm the induction of casein & TJ before and during viral infection studies. Expression of milk materials and TJs formation are considered mimics of functional lactating mammary glands.HPAI H5N1 clade 2.3.4.4b genotype B3.13 will be infected with milk-induced and non-induced MGECs at 0.01 MOI. Monolayers of MGECs from cows, goats, sheep, and bison will be grown in 12-well plates and infected. After 24 and 48 hours of infection, the virus in the culture medium and the cells will be determined by plaque assay using MDCK cells and RT-qPCR using the M gene of influenza A. We will evaluate cell susceptibility and viral replication efficiency for each MGEC type. We will also determine the type of sialic acid (SA) receptors expressed in these cells under both growth conditions.IAV uses host glycans, SAs, that are linked to subterminal sugar galactose with specific linkages that determine specific virus binding and entry into the cells. The haemagglutinin of avian influenza virus preferentially binds to SA a-2,3-galactose linked receptors, whereas human strains bind to SA a-2,6-galactose linked receptors. The bovine H5N1 replicated robustly in the cow mammary gland and secreted into the milk. To determine SA receptors on MGECs, we will use lectin and virus binding assays.We will use Cy5-labeled SNA, FITC-Mal I, and biotin-labeled Mal II lectins (Vector Laboratories) that are specific for each receptor type. SNA lectin (Sambucus nigra agglutinin) detects SA with a-2,6-gal; Mal-I and Mal-II lectins (Maackia amurensis) are used to detect SA with a-2,3-gal-b (1-4) N-acetylglucosamine (GlcNAc) and SA a-2,3-gal-b (1-3) N-acetylgalactosamine (GalNAc) linkages, respectively. The MGECs from each animal species cultured in 8-chamber glass slides under milk-inducing and non-inducing conditions for 24 h will be analyzed for lectin binding specificity as recommended by the manufacturer. Cells devoid of lectin incubation will serve as controls.Complementary to the lectin binding assay, we will also employ a virus binding assay specific to SAs. To verify SA type (a-2,3-galactose and SA a-2,6-galactose) on MGECs, specific type of IAV will be infected MGECs and assayed by IFA. For example, the human or swine H1N1 virus binds to SA a-2,6-gal, and the avian H2N3 virus binds to SA a-2,3-gal.Aim 2. Develop a small ruminant model (goats) to study H5N1 B3.13 viral pathogenesis. While several reports are available for H5N1 infected dairy cattle, none exist on H5N1 pathogenesis in goats. In this proposal, we will develop lactating and non-lactating goat models to understand H5N1 pathogenesis through a) intramammary and b) oronasal routes of infections, respectively.a). Goat Intramammary inoculation. A total of 10 lactating goats, approximately 14-16 months of age and at the early stages of their first or later stage of lactation, will be used for this study. Six goats will receive virus challenge, 2 goats will serve as sentinels to evaluate transmission, and 2 goats will serve as mock-infected controls. H5N1 clade 2.3.4.4b genotype B3.13 will be used for animal challenge in a BSL3+-Ag and associated BSL3 laboratory facilities at the Biosecurity Research Institute (BRI), Kansas State University (KSU), Manhattan KS in compliance with the Institutional Animal Care and Use Committee (IACUC) and Federal Select Agent Program regulations and approvals. During the acclimation period, animals will be screened for current or recent IAV infections including H5N1 and other respiratory infections using blood and nasal swab samples.Animals will be acclimated at the KSU Large Animal Receiving Center facility for two weeks, moved into BSL-3Ag containment, and acclimated for a week before inoculation. During the acclimation period, the goats will be milked once per day and documented for each animal. Six goats will be inoculated each with 1 ml of ~1x106 TCID50/goat, 0.5 ml/teat of H5N1 B3.13 virus by an intramammary route in both teats using a teat canula. After inoculation, the virus will be moved upward into the teat sinus with a gentle massage. For the mock infected goats, equal volumes of culture medium, as used for viral challenge above, will be used for inoculation. Two sentinel goats will be co-mingled with virus-inoculated goats to monitor virus transmission. The remaining mock-infected goats will be housed separately in the LARC and serve as baseline negative controls.Animals will be monitored daily per the Animal Monitoring Plan (AMP) until the end of the study on 14 dpi. Milk will be collected from each animal from all three groups (negative control, contact, and infected groups) daily using the milking machine (or manually as necessary) and evaluated for various tests, including the H5N1 virus by RT-qPCR, and the IAV-specific ELISA targeting the viral nucleoprotein (NP) or H5 hemagglutinin. Nasal, oral, rectal swabs, and blood for viremia and serum will be collected from all animals for analysis.b). Goat oronasal inoculation. We propose to use non-lactating goats for the oronasal inoculation and monitor if this route of inoculation infects the respiratory tract or spreads systemically. We will use a similar number of animals, groups, and ages as described for the goat intramammary inoculation. Six goats in experimental group 1 will be administered each with 1x106 TCID50 (5x105 TCID50/ml) H5N1 in 2 ml via intranasal/oral route of inoculation as follows: 0.5 ml per nostril (both nostrils) using an atomization device and 1 ml orally using a syringe. Forty-eight hours post-infection, contact sentinel goats will be co-mingled with principal-infected goats. Animal monitoring, sample collection, and necropsy will be similar as mentioned above.Post-mortem examinations will be conducted by an experienced veterinary pathologist for gross lesions prior to extensive sampling of tissues to determine the scope and extent of impacted tissues (tissue tropism) and any correlation with subsequent H5N1 virus detection. Tissue samples will be fixed in 10% neutral buffered formalin, processed, and stained with H&E. Samples include mammary, respiratory, gastrointestinal, and reproductive tract, in addition to central and peripheral lymphoid tissue (bi-lateral), CNS, and eye lids. Bronchoalveolar lavage fluid, fluids from the anterior eye chamber, abdominal, paracardial, thoracic cavity, synovial fluid, cerebral spinal fluid, bile, and bladder will also be collected. Necropsy sample processing will be based on clinical symptoms, available data on viral RNA shedding and viral titer from various samples, and swabs. IAV-specific immunohistochemistry will be performed to detect H5N1 NP antigen in the formalin-fixed tissues. Necessary positive and negative controls will be included.