RNA isolation from 140 ml of each sample was carried out using a Macherey-Nagel NucleoSpin 96 virus system with RNA elution into a final volume of 60 ml. Influenza A virus was detected by real-time RT-PCR targeting the conserved matrix as describe previously [56] . All real-time RT-PCR assays were performed on a LightCycler 480 (Roche Diagnostic) in a final volume of 10 ml with 2.5 ml RNA, 0.5 mM of each primer, 0.2 mM probe and 0.4 ml enzyme mix using a Superscript III Platinum One-Step quantitative RT-PCR system (Invitrogen). The reaction was carried out with the following temperature profile: 15 min at 45uC, 3 min at 95 C, 50 cycles of 10 s at 95uC, 10 s at 55uC, 20 s at 72uC, and finally 30 s at 40uC. Positive samples were further tested for H5, H7, H9, N1 and N7 subtypes using the same real-time RT-PCR technique (primers available upon request).