<li>Compare the murine and equine cytokine expression profiles in response to equine-1 (H7N7) and equine-2 (H3N8) influenza viruses. <li> Elucidate the capability of influenza virus to evade cellular innate immune responses at the level of Toll-like receptors of dendritic cells. </ol>
NON-TECHNICAL SUMMARY: The equine-1 (H7N7) influenza virus possesses the most characteristic molecular feature of the high-pathogenicity avian influenza viruses, i.e. the furin-cleavable connecting peptide between the subunits of the viral surface, protein, hemagglutinin. And indeed equine H7N7 viruses are lethal in chickens and mice-but not in horses. The reason for the difference in pathogenicity of these viruses in the different species is unclear. Influenza pathogenesis is the outcome of interactions between the host innate immune responses-the first line of defense-and virus-coded factors, the properties of which are not uniform among influenza virus strains. Also, new knowledge is accumulating about the host genes and proteins that are collectively responsible for host innate immune responses. The purpose of this proposed research is to decipher the basis for the differential pathogenicity of equine-1 influenza virus in different species, and to elucidate further the interaction of influenza factors with some newly recognized players in the innate response.
APPROACH: Three model systems will be used: ex vivo in equine PBMCs; in vivo in mice; and in vivo in horses. We hypothesize the innate responses of the 2 species will differ. Viruses will include eq/Prague/56, the prototype equine H7N7 strain; eq/KY/2002, a wild-type equine H3N8 virus and the WT equivalent of the mutant NS-1 viruses; and eq/NY/73, a natural reassortant virus between the H7N7 Prague/56 lineage and H3N8 KY/02 lineage. We will also compare cytokine production using live viruses to that of UV-inactivated viruses, which can enter host cells but not replicate. All viruses will be grown in eggs and purified by density gradient ultracentrifugation. Virus titers will be determined by TCID50 assay. Equine PBMCs will be harvested from peripheral blood of random, naive horses. Cells will be washed and plated. Wells will be infected with viruses at MOI (TCID units/cell) either low (0.1) or high (5). At low MOI, we expect to observe secondary cytokine production by neighbor cells responding to signals from infected cells; at high MOI production will be almost exclusively by infected cells. Concanavalin A will be used as positive control. Cells will be harvested at 0, 2, 4, 6, 12, 24 hr p.i. for high-MOI experiments and 0, 12, 24, 36, 48 hr p.i. for low-MOI experiments. Cells will be used for intracellular staining and flow cytometry analysis, or lysed and mRNAs extracted for real-time PCR. Cell supernatants will be harvested for cytokine bioassays. Balb/c mice (6 groups per virus, 4 mice per group) will be infected by nosedrop with 1000 TCID units of each of the 3 viruses. At 0, 12, 24, 36, 48, and 72 hr p.i., a group of 4 mice will be bled from the retroorbital socket to obtain plasma, then euthanized. Each groups samples will be pooled. Whole blood will be used to assess systemic cytokine levels. Lungs will be processed 2 ways: <ul>
<li>(1) frozen and thin-sliced for histopathologic staining to reveal pathologic changes in tissue architecture, or immunostained for influenza NP protein to reveal regions of virus growth. <li>(2) Lung tissues will be minced and homogenized. Homogenates will be titrated for virus content by TCID50 assay. Finally, trachea and bronchi will be excised and flushed with sterile normal saline solution to obtain mononuclear cells; these will be lysed and mRNAs obtained for real-time PCR analysis, or stained for flow-cytometry analysis. Influenza-seronegative horses (n=4) will be infected with influenza viruses by established procedures. Daily from Day 0 to Day 8, we will collect nasopharyngeal swabs to measure virus shedding; PaxGene tubes of venous blood to obtain lysates of PBMCs for cytokine mRNAs; plasma and serum samples for cytokine flow cytometry and bioassays. Rectal temperatures will be taken daily as a proxy of pathogenicity. Each day from Day 0 to Day 4, 2 of the horses will be used to obtain bronchio-alveolar lavage samples by intubation of sterile normal saline followed by its suction-recovery. The recovered fluid is rich in lung macrophages, neutrophils and other mononuclear cells. BAL samples (whole cells, lysates, and supernatants) will be used to quantitate cytokine mRNAs and proteins. </ul>
PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: Specific aim 1: Comparison of cytokine responses to h7n7 and h3n8 equine influenza viruses. We have evaluated cytokine responses to equine h7n7 influenza in both horses and balb/c mice. We confirmed that infection kills the mice whereas it produces a non-fatal influenza illness in horses with relatively low fever and other clinical signs, with normal recovery. For comparison we also have evaluated cytokine responses to equine h3n8 influenza in horses. Clinical signs were subjectively more severe but not statistically significantly so. Equine cytokine responses in response to infection were analyzed by rt-pcr of mrna from whole blood samples collected daily. While minor differences were observed between the virus subtypes with respect to pro-inflammatory cytokines including interferon-gamma, tumor necrosis factor, and interleukin-6, a large difference was seen with mx (myxovirus resistance factor) which was heightened more following h7n7 infection than following h3n8 infection. The mouse h3n8 experiments are in progress. Specific aim 2: Evasion of cellular innate immune responses at the level of dendritic cells. Our work to date has been to develop the techniques to reliably culture equine monocyte-derived dendritic cells (eqmodc), verify their phenotype, and examine their susceptibility to equine influenza virus. Blood was collected from healthy influenza-seronegative horses and pbmc were isolated from heparinized blood by ficol-hypaque centrifugation. Pbmc were plated and incubated for 4 hours at 37c with 5% co2 to permit monocytes to adhere to the plate. Thereafter, plates were gently washed with pbs twice to remove floating and loosely adhering cells. Complete rpmi media were supplemented with eqgm-csf and eqil-4. On day 3, half the media was replaced with fresh media. On day 6, microscopic observation revealed dendritic cell-like phenotypes. Dendritic cells were isolated by density gradient centrifugation. Dendritic cells were analyzed for their purity based on phenotypic characterization (surface marker expression) by flow cytometry. Cells positive for either cd86 or cd1w2 or both and negative for cd14 were characterized as dendritic cells. Cell purity was around 60%. Eqmodc were resuspended at the rate of one million cells per ml of serum free rpmi media and plated in 24 well plates. Eqmodc were infected with equine influenza strain ny/73 (h7n7) at multiplicity of infection of 5 eid50 units/cell. Cells were incubated at 37c with 5% co2. Flow cytometry using anti-influenza np monoclonal antibody confirmed infectivity of eqmodc by ny/73 virus. Similar results were obtained using ky/02 (h3n8) virus. Therefore, indirect immunofluorescence assay shows eqmodc can be infected by both subtypes of equine influenza. <BR>PARTICIPANTS: Faculty: Thomas Chambers, David Horohov Technicians: Lynn Tudor, Vet Science animal annex staff Graduate students: Saikat Boliar, Liang Zhang, Tracy Sturgill <BR>TARGET AUDIENCES: Equine virologists, equine veterinary biologics companies
IMPACT: 2007/01 TO 2007/12 <BR>
n this first year of the project, main impact has been (1) on understanding the pathogenicity of equine H7N7 infection under experimental conditions, which has never been done before (all experimental challenges with equine influenza here and elsewhere have been with the H3N8 subtype). (2) development of techniques for reliable culture of equine monocyte-derived dendritic cells. This is new knowledge of practical value to equine infectious disease researchers. Better understanding of the causes of viral pathogenicity in the horse will lead to more effective practical therapies. As cytokines are largely responsible for the mediation of adjuvant responses, better understanding of influenza-induced cytokine responses will aid in the development of more effective vaccines.