First Detection of a Novel Reassortant Avian Influenza A(H5N6) Clade 2.3.2.1c Virus, Isolated from a Wild Bird in China

We report the first isolation of a reassortant clade 2.3.2.1c avian influenza A(H5N6) virus isolated from a wild bird sample in Jiangxi, China, in 2016. Sequence analyses indicated that this virus most likely evolved from Eurasia-derived H5N1 and H6N6 viruses through frequent interactions at the wild-domestic bird interface.

Here, we provide a report on the first detection and nearly complete genome sequence of a novel reassortant clade 2.3.2.1c H5N6 sample isolated from a wild bird in China. A total of 488 tracheal and cloacal swab samples were collected during a routine bird survey in Suichuan County, Jiangxi Province, which were preserved in a sample solution in the fridge (4°C) and subsequently shipped to a laboratory and stored frozen at Ϫ80°C. From these samples, 89 and 85 samples were taken from Streptopelia decaocto and yellow-legged button quail, respectively. 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 13 samples with hemagglutination activity using an RNeasy minikit (Qiagen, Hilden, Germany). The SuperScript III reverse transcriptase (RT) PCR kit (Invitrogen, USA) was used for the reverse transcription.
The subtype of each of the 13 positive samples was first determined using PCR of a marker gene (6,7). Seven out of 13 samples were identified as H5N6 strains. All segments of the H5N6 strains were amplified by using a Phusion high-fidelity PCR system (New England BioLabs, Ipswich, MA, USA), adhering to the manufacturer's guide (8) The amino acid sequence at the HA cleavage site is RERRRKR/GL, which is characteristic of high pathogenicity in poultry. Additionally, HA had Q222 and G224 (H3 numbering) at the receptor binding site associated with an adaptation to avian-like receptors (9). NA had an 11-amino-acid deletion (positions 58 to 68) in the stalk region, which may be associated with viral adaptation to terrestrial poultry after being introduced from water birds (10). However, there were no mutations for E627K and D701N in PB2 or at positions 26, 27, 30, 31, and 34 in the M2 protein, indicating an inefficiency to replicate in mammals (11) and sensitivity to amantadine (12).
A BLAST search in the GenBank database showed that all the eight genes of Jiangxi H5N6 virus had high nucleotide identities with viruses isolated from terrestrial poultry in China 2014 to 2015 (Table 1). These high nucleotide identities of viruses isolated from wild birds and terrestrial poultry are suggestive of frequent interactions and viral circulation between two species. Specifically, the HA gene shared 99.12% nucleotide identity with the HA gene from four H5N1 viruses. NA, PA, and NP were closely related to those of A/chicken/Jiangxi/NCDZT1123/2014 (H5N6), with identities of 99.37%, 99.24%, and 99.30%, respectively. The PB2 and NS genes shared the closest nucleotide similarity (Ͼ99.50%) with the PB2 and NS genes of A/chicken/Jiangsu/2477/ 2014 (H5N1). For the PB1 and M genes, the highest nucleotide identities were with those of A/pigeon/Zhejiang/112090/2014 (99.85%) and A/duck/Guangzhou/021/ 2014 (99.51%), respectively. Notably, phylogenetic analysis revealed that this Jiangxi H5N6 virus was a novel reassortant strain, in which the HA gene was found to belong to clade 2.3.2.1c (Fig. 1).
This detection suggests continuous reassortment and generation of emerging variants. Further investigation and active surveillance are required to detect new AIV variants.
Data availability. The genome sequence of Jiangxi clade 2.3.2.1c H5N6 virus has been deposited in GenBank with the accession numbers MN165550 to MN165557.

ACKNOWLEDGMENTS
This work was supported by the National Research Program of the Ministry of Science and Technology of the People's Republic of China (grant 2016YFA0600104) and by donations from Delos Living LLC and the Cyrus Tang Foundation to Tsinghua University.
We sincerely acknowledge the authors and submitting laboratories of the sequences deposited in the GenBank database, on which the phylogenetic analysis of this research is based.
We do not have any financial or other relationships that may pose conflicts of interest.