Genetic characterization of an H5N6 avian influenza virus with multiple origins from a chicken in southern China, October 2019

Background Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China. Results Nucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified. Conclusions Our results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-021-02903-z.

East, Africa, and Europe, undergoing a number of genetic reassortments [8][9][10][11]. In 2014, the first H5N6 strain was isolated from backyard poultry flocks in Guangdong province, China [12]. Subsequently, HPAIV H5N6 became one of the major subtypes of the H5 clade 2.3.4.4. To date, since the first case of human infection with HPAIV H5N6 in Sichuan province in April 2014 [13], more than 23 cases of infection and 9 deaths were recorded (www.who.int/influenza/human_animal_ interface). During the past several years, the HPAIV H5N6 subtype has continuously evolved and has replaced H5N1 as one of the main HPAIV subtypes in domestic poultry in China [13][14][15][16]. A more recent study suggested that the positive rate of H5N6 in live poultry markets (LPM) is 7.87%, which became the second most dominant subtype currently circulating in LPMs in China [16]. Moreover, human infection of HPAIV H5N6 subtype even had a higher mortality than H5N1 subtype based on the clinical statistical data thus far [17]. Therefore, the HPAIV H5N6 subtype contributes as a major threat to domestic poultry and human health.
In this study, we report the genetic and phylogenetic characteristics of a HPAIV H5N6 strain isolated (October 2019) from chicken farms in Dongguan, Guangdong province, China.

Results
Primers targeting the conserved region of M segment were used to detect the influenza A virus in the liver and spleen of chickens (Supplementary Figure 1) Figure 2B) were successfully amplified and sequenced by cloning to the pMD 18-T vector. The results revealed that the AIV in this study belongs to a H5N6 subtype, which was designated as A/ chicken/Dongguan/1101/2019 (H5N6)(DG/19). DG/19 contained six basic amino acid residues at HA cleavage site (RERRRKR↓GLF) ( Table 1), which suggested a high pathogenicity phenotype in chickens.
The nucleotide sequences of DG/19 were aligned with the reference strains deposited in Global Initiative on Sharing Avian Influenza Data (GISAID, www.gisaid.org) and GeneBank by ClustalW. Subsequently, the phylogenetic trees for HA (Fig. 1a) and NA (Fig. 1b)  We further analyzed the glycosylation sites by NetN-Glyc 1.0 server, the data revealed that the DG/19 has eight potential glycosylation sites at positions 27, 39, 70, 140, 180, 301, 497, and 556 within the HA protein (Fig.  2a). The glycosylation site at the 158 site has been lost due to the T160A substitution. Meanwhile, a total of five potential glycosylation sites at positions 57, 60, 65, 141, and 196 within the NA protein were identified (Fig. 2b). In addition, the 3D structure model of HA of DG/19 was predicted by the SWISS-MODEL server and the predicted local similarity to the target with the highest sequence similarity (6PCX) was shown in Fig. 2c.
Further analysis of internal genes revealed that the PB2 gene segment had high identity with those recent  Figure 3. These results suggest DG/19 is a reassortant of H5N1, H5N2, H5N8, and H6N6 AIVs (Fig. 2d), which might happen through the East Asian-Australasian flyway [18].

Discussion
Southern China has been considered as a hot spot for the generation of novel AIVs. In this study, we reported the genetic characterization of a recent H5N6 HPAIV strain isolated from chickens in southern China. In addition, novel mutations such as D36N, which is one of the six common amino acid substitutions of H2 subtype AIV from mallards to swine [19], are emerging from H5N6 HPAIVs. These observations indicate a potential mammalian adaptation of H5N6 AIV. Fortunately, our results showed that DG/19 possessed 226Q and 228G (H3 numbering) in the HA, which suggested a typical avian receptor preference and were consistent with previous studies [16]. However, DG/19 has 222Q and 227R in the HA, which has been shown to facilitate virus binding to fucosylated sialosides [20]. Sialyl Lewis X  [13,16].
In addition, a number of novel mutations in the HA of H5N6 HPAIV were identified, including Q142H, N89S, and D193N. It has been suggested that the D193N mutation of H10N7 virus promoted virus binding to α2,6linked sialic acid receptors without an impair of binding to α2,3-linked sialic acid receptors [23]. However, the biological effects of those mutations need further investigation.
The NA protein of DG/19 has a 11-aa stalk deletion at residues 59-69, which has been shown to enhance virulence in mammals [24]. Moreover, the L473V substitution was observed in PB1 of DG/19, which was associated with the improved replication of AIV in mammalian cells [25].

Conclusion
In this study, we report the genetic characteristics of a recent HPAI H5N6 (DG/19) strain from chickens in southern China. Our results revealed HPAIV H5N6 strain circulating in south China is potentially a

Surveillance of avian influenza virus in domestic poultry
The routine surveillance of AIV was performed in domestic poultry in Guangdong, China. In October 2019, an outbreak of AIV was reported in a chicken farm in Dongguan, Guangdong province. The liver and spleen samples collected from chickens were homogenized 3 cycles by Precellys Evolution Super Homogenizer (France) at 6000 rpm for 15 s. The viral RNA extraction and reverse transcription was performed as previously described [18,26]

Virus sequencing
All 8 gene segments of DG/19 were successfully amplified by Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA) with a set of universal primers described by Hoffmann et al. [27]. The temperature cycle parameters were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 53°C for 30 s, and 72°C for 2 min, with a final extension at 72°C for 10 min. The PCR products were purified by using Gene JET Extraction Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Subsequently, the purified PCR segments were incubated for 15 min at 72°C with Premix Taq (Takara, Japan), then subcloned into the pMD-18 T vector and sequenced by Sanger sequencing.

Phylogenetic analysis
A total of 95 representative H5N6 strains from Global Initiative on Sharing Avian Influenza Data (GISAID, www.gisaid.org) or Influenza research database (IRD, www.fludb.org) were selected for molecular evolutionary analyses. The full length of HA (1701 bp) and NA (1380 bp) genes were aligned by ClustalW and phylogenetically clustered by maximum likelihood method with 1000 bootstrap replicates in MEGA7.

Structural and N-linked glycosylation site prediction
The 3D structure model of HA of DG/19 was analyzed by the SWISS-MODEL server (https://swissmodel. expasy.org/) [28]. In brief, the HA sequence of DG/19 was blasted through the SWISS-MODEL library and the template 6PCX with the highest sequence similarity was selected for the model building. In addition, the potential of N-linked glycosylation sites based on the consensus N-X-S/T motif was calculated as previously described [26].
Additional file 1: Figure S1. Clinical pictures of chickens in this study.