Isolation of two novel reassortant H3N6 avian influenza viruses from long‐distance migratory birds in Jiangxi Province, China

Abstract Two novel reassortant avian influenza A (H3N6) viruses were isolated from swan goose in Poyang Lake, Jiangxi Province, China, in 2014. Phylogenetic analyses indicated that these viruses are most likely derived from the Eurasian‐originated H3Ny (N3, N6, N8) and H5N6 viruses circulating among wild and domestic birds. It is noteworthy that H9N2 viruses have contributed PB1 gene to these novel H3N6 viruses. Our findings provide phylogenetic evidence to elucidate the ongoing viral reassortment in the wild bird population in southern China. Active surveillance of avian influenza viruses in Poyang Lake is warranted.

. Additionally, H9N2 has undergone a constant change of genetics and pathogenicity since 1994 onward (Bi et al., 2011). By contributing their internal genes, H9N2 viruses have facilitated the emergence of novel reassortants such as H5N6, H7N9, and H10N8 (Bi et al., 2016;Liu, Li, et al., 2015;Liu, Xie, et al., 2015;Pu et al., 2015). This frequent reassortment raises the concern about its potential to have continuous reassortment with other subtypes such as H3. In the interim, constant surveillance in wild bird population has been carried out. Although substantial phylogenetic diversity has been proposed (Zhang, Li, Zhu, Chang, & Xu, 2019;author's unpublished results), the extent of reassortment and the potential emergence of novel strains are likely to be underestimated due primarily to the limited surveillance in wild birds.

| MATERIAL S AND ME THODS
To elucidate a complete landscape of AIV ecology in wild birds, we undertook a routine bird ring survey in Poyang Lake, Jiangxi Province, in 2014-2016. Details of surveillance and isolation of AIVs have been presented in our previous studies (Zhang et al., 2019). Briefly, tracheal and cloacal swab samples were collected from migratory birds and domestic ducks and chickens. Virus isolation using these specimens was conducted in 9-to 11-day-old specific pathogen-free embryonated chicken eggs. The viral RNAs were extracted from allantoic fluid of samples with hemagglutination activity using RNeasy Mini Kit (Qiagen), and reverse transcription was carried out using the SuperScript III Reverse Transcription-PCR (RT-PCR) Kit (Invitrogen). The subtype of these positive samples was determined using PCR of a marker gene (Tsukamoto et al., 2009;Lee, Chang, Shien, Cheng, & Shieh, 2001).
All segments of these samples were amplified using a Phusion High-Fidelity PCR System (New England Biolabs) (Hoffmann, Stech, Guan, Webster, & Perez, 2001) and sequenced as individual amplicons using Applied Biosystems Automated 3730xl DNA Analyzer.
To investigate the phylogenetic relationship of Jiangxi H3N6 viruses with other AIV subtypes, we selected representative sequences by using the clustering algorithm and BLAST search. For each of the eight genes, we initially retrieved all the sequences available from GenBank and GISAID repositories. To reduce the size of this dataset, we firstly removed identical sequences by keeping the sequence with the earliest data. With the remaining sequences, we constructed the maximum-likelihood tree and used the clustering algorithm to select representative sequences. Additionally, we used BLAST search to identify sequences with >90% identity. Sequences selected by clustering algorithm and BLAST search were integrated as a dataset of reference sequences (Tables A1-A8). Molecular phylogenetic analyses were conducted using the maximum-likelihood method based on the Kimura 2-parameter model (Kimura, 1980) with 1,000 bootstrap replicates in MEGA 6.0 software (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). Nucleotide substitution models were compared and selected based on BIC (Table A9).
Phylogenetic relationship between H3N6 and other subtypes was investigated based on the topology of phylogenies, which was then verified by estimating the evolutionary divergence. sites. Given the free-range manner and limited biosafety measures of poultry raising in Poyang Lake, sharing common habitats such as the same waterbody and outdoor areas might have created opportunities for poultry to acquire viruses shed by migratory birds.

| RE SULTS AND D ISCUSS I ON
Through frequent exposure and continuous reassortment, viruses isolated from wild and domestic birds share a high genetic relatedness which suggests a two-way viral transmission at the wild-domestic interface.

| CON CLUS IONS
In summary, two H3N6 reassortants were isolated from wild birds in Jiangxi Province, presumably originated from Eurasian H3Ny, H5N6, and H9N2 viruses. The findings suggested the complexity of reassortment of multiple AIV subtypes. Therefore, active surveillance in wild birds could improve the early warning system for an avian influenza outbreak. We sincerely acknowledge the authors and submitting laboratories of the sequences deposited in the GenBank database, on which the sequence analyses of this research were based.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C A L S TATEM ENT
All animal work was approved by the Beijing Association for Science   TA B L E A 6 (Continued)