title: The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.
abstract: Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained.  Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) fromClostridium perfringensto allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV.
authors.affiliation: Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. S.D.Scott@kent.ac.uk.
authors.forename: Simon D
authors.initials: SD
authors.lastname: Scott
authors.affiliation: Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. rk320@kent.ac.uk.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Kinsley
authors.affiliation: Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. n.temperton@kent.ac.uk.
authors.forename: Nigel
authors.initials: N
authors.lastname: Temperton
authors.affiliation: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington LE12 5RD, UK. janet.daly@nottingham.ac.uk.
authors.forename: Janet M
authors.initials: JM
authors.lastname: Daly
keywords: equine influenza; neutralisation assay; pseudotyped virus