Virus isolation and molecular screening
Swab samples were inoculated into Madin Darby canine kidney (MDCK) cells grown in 24-well plates in minimum essential medium (MEM) supplemented with 2 µg/ml phenylalanyl chloromethyl ketone-treated tryspin (TPCK-trypsin) for virus propagation on the same day and replaced with fresh culture medium on the next day. On day 3 post-inoculation (DPI), viral growth was determined by appearance of cytopathic effects (CPE) in the MDCK cells and by a hemagglutination assay (HA) of cell supernatants using 0.5% turkey red blood cells (TRBCs). The cultures which gave positive results on CPE and HA were further tested using a rapid antigen detection test, DirectigenTM EZ Flu A+B (Becton, Dickinson and Company, New Jersey, USA) to confirm influenza A virus infection. Positive cultures were then passaged on MDCK cells again for a second passage for virus isolation. Positive virus isolates were aliquoted and stored at -80°C.
Viral RNA was extracted from cell culture isolates using QIAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). The presence of SIVs was confirmed by a one-step reverse transcription PCR (RT-PCR) targeting the conserved influenza A matrix gene using PrimerScript II High Fidelity RT-PCR Kit (Takara, Japan) (Fouchier, 2000). We adapted a duplex two-step RT-PCR approach for HA and NA subtyping. Complementary DNA (cDNA) was synthesized by reverse transcription using SuperScript III Reverse Transcriptase (InvitrogenTM) with Uni12 primer (Hoffmann, 2001). Gene amplification of HA and NA gene segment was performed using AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific, Sweden) with subtype-specific primers (Supplementary table 1). PCR products were analysed with 2% agarose gel electrophoresis and virus subtypes were determined.