2.1 Ethics statements
The study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. Protocols for animal experiments were approved by Jiangsu Province Administrative Committee for Laboratory Animals (approved number: SYXK-SU-2017-0044) and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals. All experiments involving live viruses were executed in animal biosafety level 3 facilities at Yangzhou University according to the recommendations of the institutional biosafety manual.
2.2 Virus isolation and identification
During our routine surveillance in poultry in 2020, clinical samples with suspected H7N9 AIV infection from breeder farms in northern China were sent to our laboratory for disease diagnosis. The viruses were propagated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs for 48-96 hours at 35°C. Then, allantoic fluid was collected and the hemagglutinin (HA) subtype was identified by hemagglutination inhibition (HI) test and the neuraminidase (NA) subtype was confirmed by sequence analysis. After that, both viruses were purified by three consecutive rounds of plaque purification in primary chicken embryo fibroblast (CEF) cells (Hayden, Cote, & Douglas, 1980) and harvested from 10-day-old SPF embryonated chicken eggs and stored at -80℃.
2.3 Sequencing and phylogenetic analysis
Viral RNA was extracted from allantoic fluid with commercial kits (TransGen Biotech, China) according to the manufacturer’s instructions. Extracted RNA was used as template for synthesis of cDNA by using the primer Uni12 of 5′-AGCAAAAGCAGG-3′ (Hoffmann, Stech, Guan, Webster, & Perez, 2001). Then, the whole genome including basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), HA, nucleoprotein (NP), NA, matrix (M) and nonstructural protein (NS) segments were amplified by PCR with specific primers. Positive PCR products were gel purified using the QIA quick PCR purification kit (QIAGEN, Valencia, CA) and then sequenced by Sanger dideoxy method. The nucleotide sequences were assembled and edited by using the Seqman module of the DNAStar package. Multiple sequence alignments and phylogenetic analysis were performed with MEGA software, version 6.0 (www.megasoftware.net). Phylogenetic trees of all the eight genes were constructed by the neighbor-joining method with the bootstrap value of 1,000.