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.