RNA Isolation and RT-PCR for Virus Detection

RNA isolation and RT-PCR were performed as described previously (14 (link), 15 (link)). Briefly, RNA from swabs and tissue suspensions was isolated by using a MagNaPure LC system with the MagNaPure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, the Netherlands). Real-time RT-PCR assays were performed on an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) by using the TaqMan EZ RT-PCR core reagents kit (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands) according to the manufacturer’s instructions. For each run, the samples were prepared and processed in parallel with several negative and positive control samples. Virus titers were determined by serial 10-fold dilution of the homogenized tissue samples and swabs on MDCK cells, as described elsewhere (14 (link), 15 (link)). Virus titrations were performed in triplicate.