Viral RNA Extraction and RT-PCR
The HA-positive allantoic fluid was used for viral RNA extraction using a High Pure Viral Nucleic Acid Kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). Reverse transcription was performed with the Uni12 primer (5ʹ-AGCAAAAGCAGG-3ʹ) and the RevertAid First-Strand cDNA Synthesis Kit (Fermentas, Canada). cDNAs of the eight segments were then amplified with gene-specific primers designed by the CNIC. All experiments were conducted in accordance with a previous published study[15].