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.