Download An equine-specific in vitro assay to study equine influenza

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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
• This reporter plasmid would then be used
to establish a minireplicon assay in equine
cells.
•
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 (Ederms).
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.
100%
90%
Reporter Activity (% EIV 2003)
•
80%
70%
60%
50%
40%
30%
20%
10%
0%
EIV 2003
EIV 1989
EIV 1963
Results: pEPol-Luc is
functional in equine cells
100%
90%
Reporter Activity (% EIV 2003)
• Our equine reporter
system is functional in
equine cells.
• A human reporter
system shows very
little activity in equine
cell line.
80%
70%
60%
50%
40%
30%
20%
10%
0%
Human
Human
Equine
Equine No reporter
reporter reporter no reporter reporter no
with
polymerase
with
polymerase
polymerase
polymerase
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).