Strain (accession, expression plasmid, protease for HA cleavage)
A/Texas/50/2012 (KC892952.1 pI.18 TMPRSS4)
A/Udorn/307/1972 (DQ508929.1 pI.18 TMPRSS2)
A/California/7/2004 (CY114373.1 pEVAC HAT)
A/Wisconsin/67/2005 (CY034116.1 pEVAC HAT)
A/Japan/WRAIR1059P/2008 (pEVAC HAT)
A/Switzerland/9715293/2013 (EPI814528 pEVAC HAT)
A/New Caledonia/71/2014 (EPI551570 pEVAC HAT)
A/duck/Quang Ninh/220/2014 (LC053492 pEVAC HAT)
A/ruddy turnstone/Delaware Bay/606/2017 (MH135712 pEVAC TMPRSS2)
A/Kansas/14/2017 Vaccine strain, 3c3.A (pEVAC HAT)
A/Switzerland/8060/2017 (EPI1326015 pEVAC TMPRSS4)
A/South Australia/34/2019 (EPI1607117 pEVAC HAT)
Schematic representation of the production of influenza H3 pseudotypes by plasmid transfection. (a) Using a 4 plasmid system, exogenous NA was added to the transfected cultures at least 8 h post-transfection. (b) Addition of protease, which is necessary for the production of H3 pseudotypes is optimized to increase production titres by transfecting using a ‘checkerboard’ approach with different proteases (e.g., HAT, TMPRSS4, and TMPRSS2). Protease plasmid was added at a ratio of 1:1, 1:0.5, and 1:0.25 to HA plasmid DNA for rapid optimization in a 6 well plate format. All pseudotypes were harvested after 48 h in culture. ORCID: https://orcid.org/0000-0002-7978-3815