Novel reassortant clade 2.3.4.4 avian influenza A (H5N8) virus in a grey heron in South Korea in 2017

We report the identification of a novel reassortant clade 2.3.4.4 H5N8 virus from a dead grey heron in Korea in 2017. Outbreaks of clade 2.3.4.4 H5 HPAIVs have been reported worldwide, and they have evolved into multiple genotypes among wild birds. Phylogenetic analysis indicated that this virus likely originated from Qinghai Lake and Western Siberia and further evolved through reassortment with Eurasian LPAI during the 2016 fall migration of wild birds. Enhanced surveillance and comparative genetic analysis will help to monitor the further evolution and dissemination of clade 2.3.4.4 HPAIVs.

2016, and they have since caused numerous outbreaks in domestic poultry and wild birds in Korea [13,14]. In January 2017, during HPAI surveillance of wild birds in Korea, we isolated a group B H5N8 clade 2.3.4.4 virus from the carcass of a grey heron (Ardea cinerea). In this study, we sequenced this H5N8 virus, A/grey heron/Korea/ W779/2017(H5N8) (W779), and conducted a comparative phylogenetic analysis to trace its origin and understand its genetic relationship to other group B H5N8 clade 2.3.4.4 viruses identified in Eurasia and Africa in 2016-2017.
The dead grey heron from which we isolated A/grey heron/Korea/W779/2017(H5N8) was found in Jeonju, South Korea (35°52'4.15"N, 127°6'28.08"E), on January 27, 2017. Tissue samples were collected from the carcass and homogenized with phosphate-buffered saline (PBS) to a 10% (wt/vol) final concentration. The swabs were collected into 1 ml of antibiotic-containing PBS (gentamicin, 250 μg/mL). After centrifugation, 100 μl of medium from each tissue and swab sample was inoculated into the allantoic cavity of a 10-day-old specific-pathogen-free (SPF) embryonated chicken egg. The allantoic fluid was harvested after 72 h of incubation at 37 °C. AIV was identified by a hemagglutination assay and an H5 subtype-specific reverse transcription polymerase chain reaction (RT-PCR) assay [15]. The isolate was identified as H5N8 AIV by subtyping RT-PCR [16]. The complete genomes were sequenced using influenza A universal primers as described previously [17]. Nucleotide sequences were deposited in the GenBank database (accession nos. MF155629-MF155636). For phylogenetic analysis, by reference sequences were selected based on sequence homology as determined by GISAID EpiFLU BLAST searches (http://platform.gisaid.org), including clade 2.3.4.4 H5 HPAIVs identified in 2016-2017 and related low-pathogenic strains [3]. For Bayesian phylogenetic analysis and estimation of the time to the most recent common ancestor (tMRCA), Bayesian analysis was performed for all eight gene segments using BEAST version 1.8.4. We employed a Hasegawa-Kishino-Yano (HKY) substitution model with four gamma categories and specified an uncorrelated lognormal relaxed clock and GMRF Bayesian skyride tree prior for each segment. The Markov chain Monte Carlo (MCMC) method was employed with 50 million chain lengths to draw inference under this model. BEAST output was analyzed with TRACER v1.4 (https://beast.bio.ed.ac.uk/tracer) with 10% burn-in. Maximum-clade-credibility (MCC) trees with median heights were generated for each dataset using TreeAnnotator version 1.8.4 and visualized using the program FigTree v1.4.2. Estimated tMRCA values were obtained from MCC trees with common ancestor heights.
We considered the W779 virus to be an HPAIV on the basis of the deduced amino acid sequence at the HA proteolytic cleavage site (PLREKRRKR/G). In the HA, the receptor-binding sites maintained Q226 and G228 (H3 numbering), which is suggestive of preferential binding to the sialic acid-2,3-NeuAcGal, as is typical for avian influenza viruses [18]. The established markers of NA inhibitor resistance (E119A, H274Y, and N294S, N2 numbering) and amantadine resistance (V27A and S31N, M2 protein) were not found [19,20]. In addition, phenotypic markers related to increased potential for transmission and pathogenicity to mammals such as markers in PB2 (E158G, E627K, and K701N), PB1 (Y436H), and PA (T515A) were also not found [21,22].
We estimated that ancestors of the novel H5N8 HPAIV identified in Korea emerged among wild birds during May-September 2016, based on the tMRCA for each gene segment of H5N8 HPAIV identified in Korea during December 2016-February 2017 (Table 1), which corresponds to timing of HPAIV detection in Qinghai Lake, Western Siberia, and Russia. Based on these data, we speculate that the W779 virus is a descendant of group B H5N8 viruses identified in waterfowl in Siberia (or nearby high latitudes) during May-September 2016 that acquired PA and NP segments from a gene pool of Eurasian LPAIV before detection in Korea  Previous studies also suggested that group B clade 2.3.4.4 H5N8 viruses identified from stopover and wintering regions of wild birds (Europe, India, and Russia) in fall and winter 2016 were genetically close to H5N8 viruses identified from breeding regions in Western Siberia during spring 2016 [9][10][11][12]. The W779 virus had a different genome constellation from other reassortant H5N8 viruses identified in Europe, Russia and India during 2016-2017, suggesting that the W779 virus evolved by an independent reassortment event from other reassortant H5N8 viruses (Supplemental Figure 1).
It is believed that wild birds have played an important role in dissemination of Gs/Gd HPAIV, as seen in the spread of clade 2.2 H5N1 HPAIV from Qinghai Lake   [8]. Based on previous reports, possible long-distance dissemination of virus via wild bird migration, and our phylogenetic analysis in this study, we suspected that migratory wild birds introduced the novel reassortant H5N8 HPAIVs into Korea. Since 2014, outbreaks of clade 2.3.4.4 H5 HPAIV have been reported in various geographic regions, and they have evolved into multiple genotypes [5]. Enhanced surveillance and comparative genetic analysis will help to monitor the further evolution and dissemination of clade 2.3.4.4 HPAIVs.