An equine-specific in vitro assay to study equine influenza pathogenesis Principal Investigators: P. Murcia and J. F. Marshall Researcher: Alice Coburn College of Medical, Veterinary and Life Sciences University of Glasgow Prj:009

Background and motivation Equine influenza virus (EIV) poses a threat to the horseracing industry. Molecular studies on EIV biology are required to design better vaccines and treatments. There is a lack of equine specific molecular assays to study how EIV replicates in equine cells. Minireplicon systems are powerful in vitro tools for studying viral polymerase activity and adaptation to host species We proposed to develop a minireplicon system to quantify influenza virus replication in horse cells.

Experimental design and outcomes We proposed to map the equine RNA pol I promoter into the horse genome and clone it into a reporter plasmid Completed ✓. We constructed pEPol-Luc, a plasmid carrying the Luciferase gene under the control of the equine RNA Pol I promoter. Completed ✓. We established a minireplicon assay using pEPol-Luc in an equine cell line (E-derms). This reporter plasmid would then be used to establish a minireplicon assay in equine cells.

Results: pEPol-Luc is functional in human cells pEPol-Luc effectively works in minireplicon assays in human cells. Viral polymerase complexes display different activities depending on the way the viruses evolved in horses.

Results: pEPol-Luc is functional in equine cells Our equine reporter system is functional in equine cells. A human reporter system shows very little activity in equine cell line.

Objectives Achieved Equine Pol1 promoter identified and located Equine promoter synthesized and inserted into reporter system Equine system demonstrated to work in equine cells. An equine specific minireplicon system to study influenza replication in equine cells was developed.

Implications of this work We have now a tool to study how influenza viruses replicate in horse cells. This system is entirely in vitro thus minimising the use of animals in research This system only requires the use of viral genes thus minimising the risks of using infectious virus.

Future activities To use this assay to study how influenza viruses replicate in horses at the molecular level. To use this assay to determine the risk of new influenza viruses to emerge in horses. To seek funding in order to use the equine RNA pol I promoter to improve the production of equine influenza viruses in vitro (key issue for vaccine production).