Production and Characterization of Influenza DI Particles

T25 flasks were seeded with a coculture of 293T cells stably expressing PB1, PB2, PA (1.4 × 10⁶ cells) and MDCK cells stably expressing PB2 and PA (0.4 × 10⁶ cells) in DMEM growth medium (Gibco). For the production of DIPs encoding two DI segments derived from IAV genomic segments 1 and 3 (S1S3 DIPs), cells were cotransfected with plasmids encoding DI RNA derived from segments 1 and 3 of PR8 origin, jointly with plasmids encoding IAV genomic segments 2 and 4 to 8 of WSN origin using the calcium phosphate method. Similarly, for production of DIPs expressing single DI segments with a deletion in segment 1 (S1 DIPs) or segment 3 (S3 DIPs), cells were cotransfected with 7 IAV genomic segments and either one DI segment derived from S1 (for S1 DIPs) or one derived from S3 (for S3 DIPs). After overnight incubation, cells were washed once with PBS and fresh DMEM infection medium (2% FBS, 1% pen/strep, without trypsin) was added. As negative control, parental MDCK and 293T cells were also transfected. Supernatants were harvested at 3, 5 and 7 days post transfection, cleared by centrifugation at 1500 × g for 10 min and stored at − 80 °C for further use. Titers for DIP supernatants were determined by focus formation assay on PB2, PA expressing MDCK cells, as described^{15 (link)}.