Virus Isolation and Identification
Viruses were propagated in specific-pathogen-free (SPF)
chick
embryonated eggs, then these eggs were incubated for 72–96 h at 37°C.
The collected allantoic fluids were subjected to a hemagglutination
assay with 1% turkey red blood cells (TRBCs). The HA-positive samples
were further subtyped by real time RT-PCR using 16 sets of HA (H1–H16)
primers
and nine sets of NA (N1–N9) primers designed by the Chinese National
Influenza Center (CNIC).