Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).
In late October 2016, isolation was reported of highly pathogenic avian influenza (HPAI) H5N6 virus from wild migratory birds in South Korea for the first time [1] which subsequently has caused continuous outbreaks in domestic poultry. In Southeast Asia, HPAI H5 viruses have been continuously isolated from wild birds and domestic poultry since the first detection of A/Gs/ Guangdong /1/1996(Gs/GD/1996 in poultry in 1996 [2]. These viruses cause high mortality resulting in serious economic losses in the poultry industry and they spread widely. The HPAI H5N1 subtype was stably maintained for more than a decade before it started to evolve into the novel reassortant HPAI H5Nx virus in 2008 [3]. The HPAI H5N5, which was the first H5Nx subtype isolated, is a member of clade 2.3.4 while most H5Nx recently circulating worldwide, including H5N2, H5N6 and H5N8, cluster into a sublineage of clade 2.3.4 designated as 2. 3.4.4 [4]. The clade 2. 3.4.4 H5N8 influenza virus was first reported in South Korea in 2014 and subsequently spread to East Asia, Europe, and further to North America and created novel H5Nx subtypes [5][6][7]. In addition to the H5N8 viruses, the clade 2. 3.4.4 H5N6 virus that first emerged in China in 2013, spread to Laos and Vietnam in 2014/15 with evidence of sustained transmission and further geographical spread within both countries. The H5N6 virus caused fatalities in poultry and now appears to be endemic in mainland China, Laos, and Vietnam [8]. Although the H5N8 virus is contained in clade 2.3.4.4 haemagglutinin (HA) gene pools along with HPAI H5N6 viruses, it is relatively low pathogenic in mammalian hosts [5,9], and no human cases have been reported thus far. However, the avian influenza virus subtype H5N6 caused 16 human infections including six fatalities in China as of November 2016 [10].
During late October 2016, the clade 2.3.4.4 H5N6 influenza virus was first detected in faecal specimens of migratory wild birds in South Korea and has subsequently caused poultry outbreaks in South Korea from mid-November 2016 [11]. The first reported poultry cases in Chungbuk province in central South Korea were associated with nearby die-offs of wild birds leading to speculation that migratory waterfowl were the source of infection. We report here the genetic characterisation of the H5N6 viruses isolated from faecal specimens of migratory wild birds during these first outbreaks and the investigation of their pathogenic potential in chickens.

Genetic characterisation of novel avian influenza A(H5N6) viruses
Four H5N6 viruses were isolated from faecal samples obtained from migratory bird habitats in Chungbuk Province during a surveillance study conducted on 18 November 2016. Full-length genomic sequence analysis revealed that the viruses showed 99.9% to 100% nucleotide (nt) homology to one another (data not shown). One representative virus, A/Environment/   [12,13]. Molecular analysis demonstrated that the HA cleavage site of EM/W541 bears polybasic residues (RERRRKR/G) denoting a high-pathogenicity phenotype in chickens.
All four HPAI H5N6 virus isolates maintained the glutamine residue at position 226 (H3 numbering) and a glycine residue at position 228, which is suggestive of preferential binding to sialic acid receptors joined to sugar chains through an α-2,3 linkage, as is typical for avian influenza viruses. However, a characteristic of all H5N6 virus isolates was one amino acid deletion (133 site of HA1) relative to the other clade 2.3.4.4 HA genes (ex, MDk/Korea/W452/14), which is commonly found in avian influenza H5N6 viruses (Table).
The deletion at this position alters the 3D structure of the receptor binding unit causing an alteration of the HA receptor binding specificity and resulting in an increased affinity for the α-2,6 linkage [14,15]. A similar deletion has occurred and is maintained in 2.2.1.2 viruses in Egypt [14]. These viruses bear considerable zoonotic potential. In addition, the Korean H5N6 isolates had the characteristic 20 amino acid NA stalk deletion (49 to 68 sites) compared with the A/Ck/ Sichuan/NCJPL1/2014 virus, whereas a substitution associated with resistance to NA inhibitors was not noted. The isolates also possess functional polymerase basic (PB)1-F2 proteins, which have been shown to impact on host defence mechanisms and enhance pathogenicity in vivo. However, no other mammalianadaptive molecular determinants were observed in the viral genome [16]. The 2016 Korean virus isolates bear aspartic acid in place of glutamic acid at position 92 of the non-structural (NS)1 protein, which is responsible for attenuating anti-viral host interferon responses [17] and the C-terminal PDZ-binding motifs are both ESEV, which is typical for avian viruses and confers severe disease phenotype in mice [18].

Figure 9
Illustration of genotypes and reassortment events resulting in the novel avain influenza H5N6 virus isolated in South

Phylogenetic analyses
To clarify the origins of EM/W541, phylogenetic analyses were conducted with available H5Nx virus sequences and other N6 viruses from the National Center for Biotechnology Information (NBCI) GenBank database. Phylogenetic analysis of the HA genes revealed that EM/W541 was evolutionarily close to the  (Figures 7 and 8). The genotype map demonstrates that the first Korean H5N6 viruses were reassorted from at least three different subtypes (H5N6, H4N2 and H1N1) present within the natural gene pool in Eurasian avian influenza viruses ( Figure 9).

Virulence in chickens
To determine the pathogenicity of the EM/W541 in chickens, we initially measured the mean death times (MDT) and the intravenous pathogenicity index (IVPI) according to the recommendations outlined in the World Organisation for Animal Health (OIE) standards [19]. Briefly, 6.0 log10 egg infectious doses (EID50) /0.1 mL of the H5N6 virus were intravenously inoculated into ten 6-week-old chickens which were then monitored until death. The MDT was 36 hours and the IVPI was 2.66 in chickens, suggesting the EM/W541 virus should be classified as an HPAI virus according to OIE criteria [19].

Conclusions
Overall, we report the identification of a novel reassortant HPAI H5N6 virus that caused large outbreaks in domestic poultry in the late 2016 winter in South Korea [11]. This H5N6 virus is a reassortant with multiple virus subtypes (H5N6, H4N2 and H1N1) from the gene pool in Eurasian avian influenza viruses. Initial animal studies revealed that this novel H5N6 virus is highly pathogenic in chickens.
At this moment, it is hard to determine whether the presented reassortment event of the H5N6 viruses occurred in 2016 during the wild bird migration into Korea or in a previous year and in another location before migration. Further detailed broad-range molecular studies are needed to elucidate when exactly the event occurred.
The first avian influenza H5N8 (clade 2.3.4.4) virus outbreak was reported in poultry in South Korea in 2014. It rapidly spread worldwide, including to Europe and North America, by migratory wild birds [20]. This rapid and wide spread underscores the need for continuous, intensive surveillance of avian influenza viruses in wild migratory birds as it can be envisaged that these viruses may be transmitted for example to Europe, or possibly worldwide, by any migratory birds that use the same migratory flyways as the birds in the previous 2014 poultry outbreak in South Korea.

*Erratum
The cells in the first row of the