Nucleic acid extraction, PCR, and sequencing.

For genetic characterization of NDV, total RNA was extracted from infectious allantoic fluid using TriPure RNA isolation reagent (Hoffmann-La Roche, Ltd., Basel, Switzerland) according to the manufacturer's instructions. The extracted RNA was used for reverse transcription with random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI) according to the manufacturer's instructions.

The amplification for partial F genes of class I and II strains are performed as described in previous studies (16, 17). Conditions for PCR of complete F genes was as follows: 95°C for 3 min, followed by 35 cycles at 95°C for 1 min, 51°C for 45 s, and 72°C for 2 min 30 s, with a final extension step at 72°C for 10 min. The primer pair sequences used are listed in Table 2. Sequencing of PCR amplicons was conducted by Majorbio (Shanghai, China).

Primers used in this study