Research Accomplishment Reports 2007

Innate Immune Responses to Influenza Virus Infection

T. Chambers, D. Horohov
Department of Veterinary Sciences

 

Project Description

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.

Impact

In 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.

Publications

Adams, A.A., Breathnach, C.C., Sturgill, T., Chambers, T., and Horohov, D.W. (2007). Characterization of the immunological and physiological response of aged horses to equine influenza infection. 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil (ER091).

Boliar, S., Barnett, C., and Chambers, T.M. (2007). Characterization of temperature sensitive (ts) phenotype of Flu Avert I.N. equine influenza vaccine. Proceedings of the 26th Annual Meeting of American Society for Virology, P30-11, p. 285.

Adams, A.A., Breathnach, C.C., Sturgill, T., Chambers, T., and Horohov, D.W. (2007). Characterization of the immunological and physiological response of aged horses to equine influenza infection. 88th Conference of Research Workers in Animal Disease, Chicago, Illinois (#114).

Chambers, T.M. (2007). What in the World is Going on with Equine Influenza?, Equine Disease Quarterly, 16(4): 2-3.