Strain (accession, expression plasmid, protease for HA cleavage)
A/South Carolina/1/1918 (AF117241.1 phCMV1 TMPRSS4)
A/Puerto Rico/8/1934 (AF389118.1 pI.18 TMPRSS4)
A/New Caledonia/20/1999 (EU103824.1 phCMV1 TMPRSS4)
A/duck/Italy/1447/2005 (HF563054.1 pI.18 TMPRSS4)
A/Solomon Islands/3/2006 (EU124177.1 pI.18 TMPRSS4)
A/Brisbane/59/2007 (CY163864.1 pI.18 TMPRSS4)
A/California/7/2009 (CY121680.1 pI.18 TMPRSS4)
A/Texas/05/2009 (GQ457487.1 pI.18 HAT)
A/England/195/2009 (GQ166661.1 pEVAC TMPRSS4)
A/Bolivia/559/2013 (EPI466837 pEVAC TMPRSS4)
A/swine/Guangxi/1/2013_12_26_4 (KJ725056 pEVAC TMPRSS4)
A/Michigan/45/2015 (EPI662594 pEVAC TMPRSS4)
A/Slovenia/2903/2015 (EPI768541 pEVAC TMPRSS4)
A/Brisbane/02/2018 (EPI1383389 pEVAC TMPRSS4)
A/swine/Henan/SN10/2018_02__4 (MN416619 pEVAC TMPRSS4)
A/swine/Beijing/0301/2018_03__4 (MN416589 pEVAC TMPRSS4)
Schematic representation of the production of influenza H1 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 H1 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