Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

ABSTRACT

During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential.

IMPORTANCE The H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

INTRODUCTION

Influenza A viruses are encoded by eight segments of negative-sense RNA (vRNAs), which enable rapid evolution via an error-prone RNA-dependent RNA polymerase and gene transfer by reassortment of vRNAs during coinfections. Human infections with zoonotic influenza A virus subtypes continue to be a global concern. A highly pathogenic avian influenza (HPAI) A(H5N1) virus caused its first human infection in Hong Kong in 1997 (1). To date, more than 800 human cases in 16 countries, with an overall fatality rate of 53%, have been reported since 2003 (2). The H5 hemagglutinin (HA) vRNA continues to evolve into diverse clades and subclades (3, 4). In early 2014, a novel subtype of HPAI A(H5N8) virus of subclade 2.3.4.4 caused poultry outbreaks in South Korea and subsequently spread to China, Japan, the Russian Federation, and Europe. Another novel HPAI A(H5N6) virus subtype of the same H5 subclade caused multiple outbreaks in Southeast Asia and resulted in one fatal human infection in China in April 2014 (5).

At the end of 2014, commercial turkey farms in southern British Columbia, Canada, reported increased mortality in their flocks. Subsequent investigation revealed the presence of an HPAI A(H5N2) virus containing five Eurasian-lineage vRNA segments of A(H5N8) origin and three vRNA segments from North American-lineage viruses (6). During this time, U.S. authorities also detected an HPAI A(H5N2) virus (northern pintail/Washington/40964/2014) with the same vRNA constellation as the Canadian virus, an HPAI A(H5N8) virus with all vRNAs of Eurasian lineage (gyrfalcon/Washington/41088-6/2014), and an A(H5N1) virus (American green-winged teal/Washington/195750/2014) composed of four Eurasian- and four North American-lineage vRNAs (including a North American neuraminidase [NA] vRNA) (7). These virus reassortants are collectively referred to as H5Nx viruses and have dispersed throughout the Pacific, Central, and Mississippi flyways. As of September 2015, the U.S. Department of Agriculture (USDA) reported 219 detections across 15 states, affecting more than 48 million domestic birds (www.aphis.usda.gov). The fast movement of the HPAI H5N8 virus across Eurasia and North America, and its ability to reassort with circulating viruses, generates serious concerns among the poultry industry and the public health community.

Two surface proteins of the influenza A virus, hemagglutinin and neuraminidase, play essential roles during virus entry into host cells and release from those cells (8). Influenza A viruses attach to cells through HA binding to terminal sialic acids of glycoproteins on the surfaces of respiratory epithelial cells. The host range of influenza A viruses is dictated mainly by their affinity for different sialosides; avian viruses preferentially bind to sialic acid linked to galactose via an α2-3 linkage, and human viruses preferentially bind to sialic acid linked to galactose via an α2-6 linkage (9, 10). NA catalyzes the hydrolysis of terminal sialic acid residues from cell receptors and from newly formed virions and therefore helps release the virus from cells for the spread of infection, as well as prevention of the aggregation of virus particles (11). The indispensable role of NA during the virus life cycle also makes it an important target for antiviral therapeutics (12). In order to understand the molecular characteristics of the HPAI H5Nx viruses and their pandemic potential, the HA and NA proteins from A/Sichuan/26221/2014 (H5N6) (5), A/gyrfalcon/Washington/41088-6/2014 (H5N8), A/northern pintail/Washington/40964/2014 (H5N2) (6), and A/American green-winged teal/Washington/195750/2014 (H5N1) (7) were structurally and functionally analyzed. Specifically, we expressed HA and NA proteins from these H5Nx viruses and determined their 3-dimensional atomic structures by X-ray crystallography. In addition, the receptor binding of these recombinant HAs (RecHAs) was analyzed by both glycan microarray and biolayer interferometry (BLI). Finally, the enzymatic activities of the N8, N2, and N1 NAs, as well as their sensitivities to neuraminidase inhibitors (NAIs), were tested. Changes observed in the HAs close to the receptor binding site (RBS) in recent strains were also studied. While our results indicate that these H5Nx viruses currently pose a low risk to humans, it is important to carry out surveillance and pandemic risk assessment of continually evolving H5Nx viruses.

MATERIALS AND METHODS

Cloning and expression of recombinant HAs and NAs.

The cDNAs for the ectodomains of the H5Nx HA (residues 1 to 503 in mature protein numbering) and NA (residues 80 to 470) were synthesized (GenScript USA Inc.) as codon-optimized genes for insect cell expression and were subcloned into the baculovirus transfer vector pAcGP67B (BD Biosciences). To aid in purification, the HA had an additional C-terminal thrombin site, followed by a foldon trimerization sequence from bacteriophage T4 fibritin and a His tag, incorporated into the final construct (13). The recombinant NA (RecNA) protein contained an N-terminal His tag, a tetramerization domain from the human vasodilator-stimulated phosphoprotein (14), and a thrombin cleavage site (15). Both secreted proteins were recovered from the culture supernatant and were purified by metal affinity chromatography and size exclusion chromatography (SEC). For structural analyses, proteins were further subjected to trypsin cleavage and were repurified by SEC. Trypsin-treated RecHA and RecNA were buffer exchanged into 10 mM Tris-HCl–50 mM NaCl (pH 8.0) and 10 mM Tris-HCl–50 mM NaCl–1 mM CaCl₂ (pH 8.0), respectively, and were concentrated for crystallization trials.

Crystallization and data collection.

Initial crystallization trials were set up using Oryx4, a crystallization robot for the sitting drop procedure (Douglas Instruments Ltd., Berkshire, United Kingdom). The conditions under which crystals were observed were optimized at 20°C using a modified method for microbatch screening under oil (16). All crystals were flash-cooled at 100 K, and data sets were collected and were processed with the DENZO-SCALEPACK suite (17). More-specific information for each target is included in Table 1.

TABLE 1.

Data collection and refinement statistics for the H5Nx crystal structures

Condition or parameter	Setup or value for the following influenza virus (PDB code) protein:
	A/Sichuan/26221/2014 (5HU8) H5 HA	A/gyrfalcon/Washington/41088-6/2014 (5HUF) H5 HA	A/American green-winged teal/Washington/195750/2014 (5HUG) N1 NA	A/Northern pintail/Washington/40964/2014 (5HUK) N2 NA	A/Sichuan/26221/2014 (5HUM) N6 NA	A/gyrfalcon/Washington/41088-6/2014 (5HUN) N8 NA
Exptl conditions
Protein concn (mg/ml)	18	14	20	16	12	15
Crystallization conditionsa	0.1 M imidazole (pH 7), 22.5% PEG (cutoff, 0.3 to 10 kDa; Polypure)	0.1 M sodium citrate–citric acid (pH 5.5), 20% (wt/vol) PEG 3000	0.2 M magnesium chloride, 0.1 M HEPES–NaOH (pH 7.5), 30% (vol/vol) PPG P400	0.1 M sodium citrate–citric acid (pH 5.5), 40% PEG (vol/vol) 600	0.2 M sodium acetate, 0.1 M Tris-HCl (pH 8.5), 16% (wt/vol) PEG 4000	0.16 M calcium acetate, 0.08 M sodium cacodylate–HCl (pH 6.5), 14.4% (wt/vol) PEG 8000, 20% (vol/vol) glycerol
Cryoprotectant	None	None	None	20% PEG 200	20% glycerol	None
Data collection statisticsb
Beamline collected	APS, 22-ID	APS, 22-BM	APS, 22-ID	APS, 22-ID	APS, 22-ID	APS, 22-ID
Space group	P212121	P212121	P4212	P212121	P21	I4
Cell dimensions (Å)	89.51, 104.42, 215.69	125.33, 252.39, 70.62	91.64, 91.64, 108.97	116.23, 122.79, 176.85	117.34, 76.28, 117.21	90.65, 90.65, 110.81
Cell angle (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90.2, 90	90, 90, 90
Resolution range (Å)	50–2.45 (2.54–2.45)	50–2.81 (2.91–2.81)	50–1.85 (1.92–1.85)	50–2.45 (2.54–2.45)	50–1.60 (1.66–1.60)	50–2.30 (2.38–2.30)
Rsym (%)	10.9 (87.0)	11.7 (71.9)	11.7 (45.7)	12.6 (64.9)	9.2 (36.5)	7.5 (17.0)
I/σ	25.8 (2.0)	12.3 (1.6)	27.9 (5.4)	24.9 (3.6)	22 (3.3)	47.8 (24.4)
Completeness (%)	100.0 (100.0)	96.5 (96.2)	99.8 (100.0)	98.3 (95.8)	99.9 (100.0)	99.8 (99.9)
Redundancy	7.3 (6.7)	5.2 (4.9)	6.9 (5.8)	6.2 (6.4)	3.7 (3.7)	6.3 (7.0)
Refinement
No. of reflections
Total	74,675	53,618	40,235	91,882	272,309	19,878
Test	7,154	5,033	3,949	8,819	26,255	1,900
Rwork/Rfree	19.1/22.7	22.8/25.6	16.4/19.5	15.7/18.4	19.8/22.0	20.1/23.7
No. of atoms	12,524	12,219	3,232	12,932	13,870	3,110
RMSD
Bond length (Å)	0.014	0.012	0.021	0.018	0.018	0.013
Bond angle (°)	1.69	1.71	1.90	1.94	1.69	1.57
MolProbity scores (%)c
Favored regions	94	90	97	96	97	95
Outliers	0.4	1.6	0	0	0	0

^aPEG, polyethylene glycol; PPG, polypropylene glycol.

^bNumbers in parentheses refer to the highest-resolution shell.

^cSee reference 27.

Structure determination and refinement.

The HA and NA structures were determined by molecular replacement with Phaser (18). For the HA, the influenza virus A/Anhui/5/2005 (H5N1) structure (PDB code 4MWQ) was used as a search model (19). The sequence for the model was then mutated to the correct sequence, rebuilt by Coot (20), and refined with REFMAC (21) using both transition/libration/screw (TLS) refinement (22) and Phenix.refine (23).

The N1, N2, N6 and N8 structures were solved using NA structures from the influenza A/Brevig Mission/1/1918 (H1N1) (PDB code 3B7E), A/RI/5+/1957 (H2N2) (PDB code 3TIA), A/chicken/Nanchang/7-010/2000 (H3N6) (PDB code 4QN4), and A/harbor seal/Massachusetts/1/2011 (H3N8) (PDB code 4WA3) viruses, respectively, as search models (15, 24–26). The NA structures were refined using the same strategy as the HA structures. All final models were assessed using MolProbity (27), and statistics for data processing and refinement are presented in Table 1.

Glycan binding analyses.

Glycan microarray printing and recombinant HA analyses have been described previously (13, 28–32). Table 2 lists the glycans used in these experiments as well as a tabulated qualitative assessment of binding for each protein analyzed. For kinetic studies, biotinylated receptor analogs, Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcb-biotin (3SLNLN-b) and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcb-biotin (6SLNLN-b), were obtained from the Consortium for Functional Glycomics (www.functionalglycomics.org) through the resource request program. Glycans were precoupled to streptavidin-coated biosensors (ForteBio, Inc.), and the binding of recombinant HA, diluted to 5.4 μM trimer in kinetics buffer (phosphate-buffered saline [PBS] containing 0.02% Tween 20, 0.005% sodium azide, and 100 μg/ml bovine serum albumin), was analyzed by biolayer interferometry (BLI) using an Octet Red instrument (ForteBio, Inc.) according to the manufacturer's instructions. Data were analyzed using the system software and were fitted to a 1:1 binding model.

TABLE 2.

Glycan microarray for H5Nx HAs

Glycan no. by category	Structurea	Bindingb
		A/northern pintail/ Washington/ 40964/14	A/Sichuan/ 26221/14	A/gyrfalcon/ Washington/ 41088-6/14
Sialic acid
1	Neu5Acα	NB	NB	NB
2	Neu5Acα	NB	NB	NB
3	Neu5Acβ	NB	NB	NB
α2-3 sialosides
4	Neu5Acα2-3(6-O-Su)Galβ1-4GlcNAcβ	+++	+++	+++
5	Neu5Acα2-3Galβ1-3[6OSO3]GalNAcα	+++	+++	+++
6	Neu5Acα2-3Galβ1-4[6OSO3]GlcNAcβ	+++	+++	+++
7	Neu5Acα2-3Galβ1-4(Fucα1-3)[6OSO3]GlcNAcβ-propyl-NH2	+++	+++	+++
8	Neu5Acα2-3Galβ1-3[6OSO3]GlcNAc-propyl-NH2	+++	+++	+++
9	Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4)GlcNAcβ	+++	+++	+++
10	Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-6)GalNAcα	+++	+++	+++
11	Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ	+++	+++	+++
12	Neu5Acα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[Neu5Acα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ	+++	+++	+++
13	Neu5Acα2-3Galβ	+++	+++	++
14	Neu5Acα2-3Galβ1-3GalNAcα	+++	+++	+++
15	Neu5Acα2-3Galβ1-3GlcNAcβ	+++	+++	++
16	Neu5Acα2-3Galβ1-3GlcNAcβ	+++	+++	+++
17	Neu5Acα2-3Galβ1-4Glcβ	+++	+++	+++
18	Neu5Acα2-3Galβ1-4Glcβ	+++	+++	+++
19	Neu5Acα2-3Galβ1-4GlcNAcβ	+++	+++	+++
20	Neu5Acα2-3Galβ1-4GlcNAcβ	+++	+++	+++
21	Neu5Acα2-3GalNAcβ1-4GlcNAcβ	NB	NB	++
22	Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ	+++	+++	+++
23	Neu5Aca2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ	+++	+++	+++
24	Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ	+++	+++	+++
25	Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ	+++	+++	+++
26	Neu5Acα2-3Galβ1-3GalNAcα	+++	+++	+++
27	Galβ1-3(Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-6)GalNAcα	NB	+	NB
28	Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ	+++	+++	+++
29	Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ	+++	+++	+++
30	Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ	+++	+++	+++
31	Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ	+++	+++	+++
32	Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ	+++	+++	+++
33	Neu5Acα2-3Galβ1-3[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ	+++	+++	+++
34	Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ	+++	+++	+++
35	Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ	NB	NB	NB
36	Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ	NB	NB	NB
37	Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ	NB	NB	NB
38	Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ	NB	NB	NB
39	Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ	NB	NB	NB
40	Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ	NB	NB	NB
α2-6 sialosides
41	Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ	NB	NB	NB
42	Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	NB	NB
43	Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	NB	NB
44	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	NB	NB
45	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-6]-Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	+	NB
46	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]GalNAca	NB	NB	NB
47	Neu5Acα2-6Galβ1-4GlcNAcβ1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-6]GalNAca	NB	NB	NB
48	Neu5Acα2-6GalNAcα	NB	NB	NB
49	Neu5Acα2-6Galβ	NB	NB	NB
50	Neu5Acα2-6Galβ1-4Glcβ	NB	NB	NB
51	Neu5Acα2-6Galβ1-4Glcβ	NB	NB	NB
52	Neu5Acα2-6Galβ1-4GlcNAcβ	NB	NB	NB
53	Neu5Acα2-6Galβ1-4GlcNAcβ	NB	NB	NB
54	Neu5Acα2-6GalNAcβ1-4GlcNAcβ	NB	NB	NB
55	Neu5Acα2-6Galβ1-4GlcNAcβ1-3GalNAcα	NB	NB	NB
56	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ	NB	NB	NB
57	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3GalNAcα	NB	NB	NB
58	Neu5Aca2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ	NB	NB	NB
59	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ	NB	NB	NB
60	Galβ1-3(Neu5Acα2-6)GlcNAcβ1-4Galβ1-4Glcβ-Sp10	NB	NB	NB
61	Neu5Acα2-6[Galβ1-3]GalNAca	NB	NB	NB
62	Neu5Acα2-6Galβ1-4GlcNAcβ1-6[Galβ1-3]GalNAca	NB	NB	NB
63	Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6[Galβ1-3]GalNAca	NB	NB	NB
Mixed α2-3 and α2-6 biantennaries
64	Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	NB	NB
65	Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ	+++	+++	+++
66	Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα	+++	+++	+++
67	Neu5Acα2-3(Neu5Acα2-6)GalNAcα	NB	NB	NB
N-Glycolylneuraminic acid glycans
68	Neu5Gcα	NB	NB	NB
69	Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ	NB	NB	NB
70	Neu5Gcα2-3Galβ1-3GlcNAcβ	NB	NB	NB
71	Neu5Gcα2-3Galβ1-4(Fucα1-3)GlcNAcβ	++	+++	+++
72	Neu5Gcα2-3Galβ1-4GlcNAcβ	NB	NB	NB
73	Neu5Gcα2-6GalNAcα	NB	NB	NB
74	Neu5Gcα2-6Galβ1-4GlcNAcβ	NB	NB	NB
α2-8-linked sialosides
75	Neu5Acα2-8Neu5Acα	NB	NB	NB
76	Neu5Acα2-8Neu5Acα2-8Neu5Acα	NB	NB	NB
77	Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ	NB	NB	NB
78	Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ	NB	NB	NB
79	Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ	NB	NB	NB
80	Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ	NB	NB	NB
81	Neu5Acα2-8Neu5Acβ-Sp17	NB	NB	NB
82	Neu5Acα2-8Neu5Acα2-8Neu5Acβ	NB	NB	NB
β2-6-linked and 9-O-acetylated sialic acids
83	Neu5Acβ2-6GalNAcα	NB	NB	NB
84	Neu5Acβ2-6Galβ1-4GlcNAcβ	NB	NB	NB
85	Neu5Gcβ2-6Galβ1-4GlcNAc	NB	NB	NB
86	Galβ1-3(Neu5Acβ2-6)GalNAcα	NB	NB	NB
87	[9NAc]Neu5Acα	NB	NB	NB
88	[9NAc]Neu5Acα2-6Galβ1-4GlcNAcβ	NB	NB	NB
Asialoglycans
89	Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ	NB	NB	NB
90	Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ	NB	NB	NB
91	Galβ1-4GlcNAcβ1-2Manα1-3[Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAcβ	NB	NB	NB
92	GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ	NB	NB	NB
93	GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ	NB	NB	NB
94	Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ	NB	NB	NB
95	Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ	NB	NB	NB
96	Galβ1-3GalNAcα	NB	NB	NB

^aNeu5Ac, sialic acid; Neu5Gc, N-glycolylneuraminic acid; OSO₃, sulfate; Gal, galactose; Fuc, fucose; Glc, d-glucose; GlcNAc, N-acetyl-d-glucosamine; GalNAc, N-acetyl-d-galactosamine; Man, d-mannose; 9NAc, 9-O-acetyl.

^bSignificant binding of samples to glycans was estimated qualitatively based on the relative strength of the signal for the data shown in Fig. 3. Fluorescence intensity is indicated as follows: +++, >3,000; ++, 2,000 to 2,999; +, 1,000 to 1,999; NB, <1,000 (no binding observed).

NA activity and drug susceptibility assays.

RecNA activities were assessed using the fluorescent compound 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA; Sigma-Aldrich Inc.) as the substrate (33). Briefly, RecNA protein was mixed with MUNANA in a reaction buffer containing 32.5 mM 2-(N-morpholino)ethanesulfonic acid (MES), 4 mM CaCl₂, and 50 μg/ml of bovine serum albumin. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by the addition of a stop solution containing 25% ethanol and 0.1 M glycine. The fluorescence of the enzyme-cleaved product was measured using a Synergy H1 hybrid multimode microplate reader (BioTek) with excitation and emission wavelengths of 360 nm and 460 nm, respectively. To determine the enzyme kinetics of RecNA, measured as a function of the amount of the substrate, a series of 2-fold-diluted RecNAs were mixed with 200 μM MUNANA, and a sigmoidal curve of NA activity was generated. The NA concentration corresponding to the midpoint of the linear section of the curve was chosen for the kinetics assay. Different concentrations of MUNANA in a series of 2-fold dilutions were mixed with 31.25 ng/ml, 20 ng/ml, 156.3 ng/ml, and 19.5 ng/ml of the N1, N2, N6, and N8 RecNAs, respectively. Reaction parameters (K_m, V_max, and k_cat) were calculated by fitting the data to Michaelis-Menten equations using GraphPad Prism software (GraphPad Software, Inc.).

The susceptibilities of RecNAs to neuraminidase inhibitors were tested in the fluorescent neuraminidase inhibition (NI) assay by the use of the NA-Fluor influenza neuraminidase assay kit (Life Technologies), as described previously (26). The NA inhibitors zanamivir (GlaxoSmithKline), oseltamivir carboxylate (Roche Diagnostics GmbH), peramivir (BioCryst Pharmaceuticals), and laninamivir (Biota) were kindly provided by the respective manufacturers. The IC₅₀ value (the drug concentration required to inhibit enzyme activity by 50%), expressed as a nanomolar concentration, was determined using JASPR curve-fitting software, version 1.2 (26). Mean IC₅₀s and standard deviations (SD) were calculated based on the results of three independent tests, each conducted in triplicate.

Protein structure accession numbers.

The atomic coordinates and structure factors of influenza virus A(H5N6) HA, A(H5N8) HA, A(H5N1) NA, A(H5N2) NA, A(H5N6) NA, and A(H5N8) NA are available from the RCSB PDB under accession codes 5HU8, 5HUF, 5HUG, 5HUK, 5HUM, and 5HUN, respectively.

RESULTS AND DISCUSSION

Overall structure of the H5Nx HAs.

In order to understand the structural features of HA from the novel HPAI virus H5Nx subtypes and to compare them to human influenza virus H5 HA, the 3-dimensional HA structures of trimeric ectodomains from A/gyrfalcon/Washington/41088-6/2014 (H5N8), isolated in the first case of the poultry outbreak in the United States in December 2014, and A/Sichuan/26221/2014 (H5N6), isolated from a human patient with a fatal infection in China in April 2014, were determined by X-ray crystallography at 2.4 Å and 2.8 Å resolution, respectively (Table 1). The HA protein is synthesized as a single-chain precursor (HA0) during viral replication and is subsequently cleaved by host proteases into the functional/infectious HA1/HA2 form. The recombinant HAs (RecHAs), overexpressed in a baculovirus expression system, were designed to be produced in the HA0 form. However, both RecHAs were found to be partially digested into the HA1/HA2 form after purification, due to the presence of polybasic sequences at the HA1/HA2 cleavage site in both HAs. This polybasic sequence is the central virulence determinant of HAs from HPAI viruses, because it increases the susceptibility of HA to a range of cellular proteases and therefore changes the tissue tropism from localized epithelial infection to systemic infection (34, 35). The RecHAs were trypsin digested into the functional HA1/HA2 form before the crystallization trial. In the final structures, the N terminus of HA2 of H5N6 HA has poor electron density, while in H5N8 HA, the N-terminal loop of HA2 is tucked into the fusion pocket.

The overall structure of the HA monomer for both viruses is composed of a globular head containing the receptor binding site (RBS), a membrane-proximal domain that includes a central helical stalk, and the HA1/HA2 cleavage site (Fig. 1A and B) (13, 29–32, 36–45). Both HAs have seven potential N-linked glycosylation sites (NXS/T), at amino acid residues 10, 11, 23, 38, 165, and 286 on HA1 and at residue 154 on HA2 (residue 483 in HA0 numbering). For H5N6 HA, residue 193 (NPT) was predicted not to be glycosylated due to the presence of proline at the X position in the sequon (46). In the final structures, interpretable carbohydrate electron density was observed at three sites on HA1 (Asn23, Asn165, and Asn286) for both HAs and at Asn154 on HA2 for H5N8 HA only (Fig. 1A).

FIG 1.

Structure of H5Nx HA. (A) HA monomer for H5N6 HA (cyan) and H5N8 HA (green). The occupied glycosylation sites, A23, A165, and A286 on HA1 and B154 on HA2, are labeled and are shown as sticks. (B) Comparison of the H5N6 HA RBS (green) with equivalent, overlapping structures from H5N8 HA (cyan) and Anhui HA (magenta). The three structural elements making up the binding site—the 130-loop, the 190-helix, and the 220-loop—are labeled. Conserved residues are shown as green sticks. The amino acids discussed in the text are shown as orange sticks. All structural figures were generated with MacPyMol (75).

Comparison of the HA monomers from the H5N6 and H5N8 viruses to that of a clade 2.3.4 HA of influenza virus A/Anhui/1/2005 (H5N1) (PDB code 4KWM) (37) reveals highly similar structures for the viruses, with the Cα atoms superimposing to give root mean square deviations (RMSD) of 1.47 Å and 1.28 Å, respectively. There are only 16 amino acid differences between H5N6 and H5N8 HAs, and their structures are almost identical, with an RMSD of 0.72 Å.

H5Nx antigenic sites.

While human seasonal influenza virus A(H1N1) HAs have four distinct antigenic sites (Sa, Sb, Ca, and Cb [47]), five antigenic sites (A, B, C, D, and E) have been described for human seasonal influenza virus A(H3N2) HAs (48). Antigenic and escape mutation analyses of older influenza virus A(H5N1) HAs identified antigenic residues that map onto regions similar to these A(H1N1) and A(H3N2) antigenic sites (19, 49). Based on structural alignment, positions equivalent to both A(H1N1) and A(H3N2) antigenic sites were identified on current U.S. H5Nx HAs and are highlighted in Fig. 2. Relative to the older Anhui/1/2005 clade 2.3.4 virus HA used in the alignment, a number of amino acid substitutions are found within these antigenic sites, suggesting that these viruses are antigenically distinct. The sequence identities of the positions equivalent to influenza virus A(H1N1) antigenic sites on the U.S. influenza virus A(H5Nx) HAs with corresponding positions on the Anhui virus HA ranged from 69 to 85%, while those for positions equivalent to influenza virus A(H3N2) antigenic sites ranged from 66 to 91%. The lowest sequence identities were found for A(H3N2) antigenic sites A (66%) and B (68%), both of which are close to the receptor binding site (Fig. 2). Analysis by a hemagglutination inhibition assay revealed a low reactivity of sera raised against these U.S. H5Nx viruses to the clade 2.3.4 Anhui/1/2005 virus, prompting the WHO to initiate the development of candidate vaccine viruses (50).

FIG 2.

Structure-based sequence alignment of H5Nx HA. The secondary structure (ss) is highlighted in cyan for the β-sheet and in magenta for the α-helix. The locations equivalent to H1 and H3 antigenic sites are labeled with the antigenic site designation (Sa [1], Sb [2], Ca [3], or Cb [4] for H1 antigenic sites and A, B, C, D, or E for H3 antigenic sites). Residues around the RBS are shaded.

Receptor binding analyses of the H5Nx HAs.

The HA RBS contributes to the host range (51) and is at the membrane distal end of each HA monomer (Fig. 1A). The consensus RBS in all influenza A HAs is composed of three structural elements: a 190-helix (residues 184 to 190), a 220-loop (residues 215 to 224), and a 130-loop (residues 126 to 135) (Fig. 1B). In addition, the highly conserved residues Tyr91, Trp149, His179, and Tyr191 form the base of the pocket (Fig. 1B). Double mutations in the HA receptor binding domains of A(H1N1) (Glu190Asp and Gly225Asp) and A(H2N2)/A(H3N2) (Gln226Leu and Gly228Ser) subtypes have been shown to be critical for the adaptation of these avian viruses into human pandemic viruses (10, 52, 53). Recent gain-of-function studies on Eurasian H5N1 viruses also identified a number of substitutions in the HA, including the RBS, that are important for airborne transmission of avian influenza virus A(H5N1) in ferrets (54), the best available animal model for mammalian transmission. Sequence alignment of the H5N8 and H5N6 HAs with those from the HPAI A(H5N1) viruses showed that these H5Nx HAs possess an avian influenza virus-like RBS (Fig. 2) and do not harbor any of the residues known to be critical for the adaptation of avian influenza virus H1, H2, and H3 to pandemic strains, or any residues shown to be important in the transmission of avian H5 influenza viruses among ferrets. However, four amino acid substitutions of A(H5N1) HAs—Ser123Pro, Ser133Ala, Thr156Ala, and Gln192Lys—have been reported to be associated with increased binding of the virus to mammalian receptors (55–58), and all of these were present in the H5Nx HAs (Fig. 1B). Ser133Ala is located on the 130-loop and may affect the binding properties of the RBS, while Gln192Lys is near the 190-helix and also resides in antigenic site Sb in A(H1N1) viruses.

Glycan microarray analysis helps provide a detailed profile of influenza virus receptor specificity (28) and has been used as part of ongoing public health risk assessment activities (26, 59). To gain further insight into species specificity, the interactions of H5Nx viruses with various host receptors were examined using glycan-binding analyses with the various H5Nx RecHAs. Glycan microarray analyses of H5N6, H5N2, and H5N8 RecHAs were compared to that for RecHA from a human influenza virus A(H3N2) that has circulated in people for 40 years (Fig. 3A to D). The data show that all three H5 HAs had an avian influenza virus-like receptor binding preference. The HAs bound to a range of α2-3-linked sialosides (glycans 3 to 26 and 28 to 34), including linear and biantennary sialosides with or without fucosylation/sulfation (Fig. 3A to C; Table 2). These results suggest that the H5 viruses that were first detected in the Northwest U.S. states in late 2014 and early 2015 encode an HA that would not favor human-to-human transmission.

FIG 3.

Glycan array analysis of RecHAs. (A) H5N2 HA; (B) H5N6 HA; (C) H5N8 HA; (D) A(H3N2) HA. Glycans on the microarray are grouped according to sialoside linkage: α2-3 sialosides (blue), α2-6 sialosides (red), α2-6/α2-3 mixed sialosides (purple), N-glycolyl sialosides (green), α2-8 sialosides (brown), β2-6 and 9-O-acetyl sialosides (yellow), and asialoglycans (gray). Error bars reflect the standard error in each signal for six independent replicates on the array. The structure of each of the numbered glycans is found in Table 2. The specific glycan structures that were used in BLI assays are glycans 22 and 56 on the array.

During the following weeks and months, H5Nx viruses continued to spread along the Pacific flyway and across into both the Central and Mississippi flyways. As they moved east, these viruses were detected in wild birds as well as in poultry flocks. Both A(H5N2) and A(H5N8) viruses have been found in recent poultry outbreaks, and all these Eurasian clade 2.3.4.4 HA viruses analyzed to date are highly pathogenic for poultry. Interestingly, while the majority of viruses isolated from wild birds are identical across the HA1 component of the HA protein, differences have been observed in poultry viruses. In particular, a number of turkey A(H5N2) viruses have been found to contain a Ser141Pro substitution relative to the gyrfalcon/Washington/41088-6/2014 A(H5N8) virus. A smaller number of viruses also contain an additional Pro136Ser substitution (60). While both changes were located in/near the known H1 (Ca) and H3 (site A) antigenic sites (Fig. 2), they reside at either end of the 130-loop (Fig. 1B), and the gain/loss of proline could affect the flexibility of this loop. Turkeys and other terrestrial poultry express α2-6-linked sialosides in their respiratory tracts, and the proximity of these proline substitutions to the RBS suggested that they could affect the receptor binding preference. To determine if these natural substitutions impacted receptor utilization, site-directed mutants were generated on both the gyrfalcon/Washington/41088-6/2014 A(H5N8) and northern pintail/Washington/40964/2014 HAs and were subjected to both glycan microarray and biolayer interferometry analyses (Fig. 4 and 5; Table 3). In both analyses, in contrast to wild-type (WT) RecHAs (Fig. 3 and 5; Table 3), HAs with Pro/Ser substitutions at residues 136 and/or 141 bound only avian influenza virus-type receptors. There was no indication of any detectable binding to human influenza virus-type receptors.

FIG 4.

Glycan array analysis of mutant RecHAs. (A) H5N8 Pro136Ser HA; (B) H5N8 Ser141Pro HA; (C) H5N8 Pro136Ser Ser141Pro HA; (D) H5N2 Ser141Pro HA. The glycans on the microarray are described in the legend to Fig. 3. The structure of each of the numbered glycans is found in Table 2. The specific glycan structures that were used in BLI assays are glycans 22 and 56 on the array.

FIG 5.

The binding of WT and mutant H5Nx RecHA proteins to specific biotinylated glycans, 3-SLNLN-b and 6-SLNLN-b, immobilized on streptavidin-coated biosensors was analyzed by BLI. Twofold dilutions of HA were analyzed, and their binding, as measured by the shift in the interference pattern (nm), was plotted. Binding kinetics are shown in Table 2.

TABLE 3.

Kinetic results for glycan binding to WT and mutant H5Nx recHAs^a

RecHA	Glycan	Apparent KD (μM)	kon (ms−1)	kobs ± SE (10−2 s−1)	koff ± SE (10−2 s−1)
Gyr14_WT	3SLNLN-b	1.26	75,286	50.71 ± 2.61	9.52 ± 0.65
	6SLNLN-b	NB	NB	NB	NB
Gyr14_136	3SLNLN-b	1.32	84,740	57.88 ± 3.24	11.18 ± 0.71
	6SLNLN-b	NB	NB	NB	NB
Gyr14_141	3SLNLN-b	1.10	81,281	53.75 ± 2.85	8.97 ± 0.60
	6SLNLN-b	NB	NB	NB	NB
Gyr14_136_141	3SLNLN-b	1.43	86,241	59.88 ± 3.49	12.36 ± 0.79
	6SLNLN-b	NB	NB	NB	NB
pin14_WT	3SLNLN-b	1.88	94,675	69.39 ± 4.16	17.59 ± 0.87
	6SLNLN-b	NB	NB	NB	NB
pin14_141	3SLNLN-b	0.30	85,474	49.34 ± 2.40	2.58 ± 0.21
	6SLNLN-b	NB	NB	NB	NB
Sich14	3SLNLN-b	0.93	81,637	52.55 ± 2.84	7.63 ± 0.51
	6SLNLN-b	NB	NB	NB	NB
SwH3_WT	3SLNLN-b	NB	NB	NB	NB
	6SLNLN-b	141.2	2,040	30.23 ± 1.95	28.81 ± 1.78

^aK_D, equilibrium dissociation constant; k_on, k_obs, and k_off, association, observed, and dissociation rates, respectively; NB, no detectable binding.

Structural analysis of the H5Nx NAs.

Multiple recombinant H5Nx NAs (N1, N2, N6 and N8) were also expressed using a baculovirus expression system and were subjected to structural analyses. Despite variable sequence identities among these different NA subtypes (identities between the H5Nx NAs in this study range from 45% to 56%) (Fig. 6), the overall NA structure is very similar, with the typical “box-shaped” tetrameric association of identical monomers, containing six four-stranded, antiparallel β-sheets that form a propeller-like arrangement (Fig. 7A) (25, 61–68). The nine subtypes of influenza A virus NA found in birds have been classified into two groups according to their sequences (69). Group 1 comprises N1, N4, N5, and N8, and group 2 comprises N2, N3, N6, N7, and N9 (70). Comparison of the four H5Nx NAs shows that the loops are the most variable regions of the NA structures (Fig. 7B). The NA subtypes within the same groups have similar features in the 150-loop (residues 147 to 152), the 270-loop (residues 267 to 276), and the 430-loop (residues 429 to 433). The numbering follows the N2 numbering (for mature protein numbering for each subtype, see the structure-based sequence alignment in Fig. 6). H5N1 NA and H5N8 NA have the 150-cavity, which is a group 1 specific conformation/feature (63). H5N2 NA and H5N6 NA are typical group 2 NAs without the 150-cavity. The N6 NA possesses a unique 340-cavity similar to the previously published N6 and N7 structures (Fig. 8) (25). This small cavity has not been observed in any other NA structures, and given its close proximity to the active site and the conserved Ca²⁺ binding site, it is likely to play a role in NA function.

FIG 6.

Structure-based sequence alignment of H5Nx NA. The secondary structure (ss) is highlighted in cyan for the β-sheet and in magenta for the α-helix. Residues involved in substrate binding are represented by green letters, and surrounding conserved residues by red letters.

FIG 7.

Structure of H5Nx NA. (A) The overall structure of the NA tetramer is shown. One monomer is highlighted in rainbow colors, with the position of the enzyme active site indicated by an arrow. (B) Overlap of the NA active site in N1 NA (green), N8 NA (cyan), N2 NA (magenta), and N6 NA (yellow),\with surrounding variable loops.

FIG 8.

Surface representations of NA monomers.

One calcium ion binding site, which is conserved in all known influenza A and influenza B virus NAs, has a Ca²⁺ ion in each of the four H5Nx NAs. Ca²⁺ is bound through interactions with four backbone carbonyl oxygen atoms from Asn293, Gly297, Gly345, and Tyr347, one of the carboxyl oxygens from Asp324, and a water molecule (Fig. 9A). Calcium ions have been shown to be critical for the thermostability and activity of influenza virus NAs (71, 72), and this conserved metal site has been proposed to be important in stabilizing a reactive conformation of the active site by otherwise flexible loops (73). Interestingly, influenza virus A(H5N1) NA has an extra Ca²⁺ binding site in the 380-loop. Ca²⁺ is bound through interactions with carboxyl oxygens from Asp379, Asn381, Asp387, Ser389, and two water molecules (Fig. 9A). This observation is similar to that for calcium binding site 3 of the 1918 H1N1 virus NA (15). However, the contribution of this binding site to the function of N1 NA is not yet clear.

FIG 9.

(A) Two Ca²⁺ binding sites of N1 NA, with interacting residues shown as sticks. (B) The active site of NA. Highly conserved residues involved in direct substrate binding are shown as green sticks, and surrounding conserved residues are shown as orange sticks.

Among all predicted potential N-linked glycosylation sites, Asn146, which is situated on the membrane-distal surface close to the active site, is the only glycosylation sequon conserved among all other influenza virus A and B NAs (61, 63, 74). At this position, electron density for a glycan was observed only in the N1, N2, and N6 NAs, but only one N-acetylglucosamine could be interpreted in each model. N1 NA has two additional potential glycosylation sites at Asn88 and Asn234, and both had interpretable glycan density in the final model. N2 NA has additional potential glycosylation sites at Asn86, Asn200, Asn234, and Asn402, but interpretable glycan density was observed only at Asn200 and Asn234. Interestingly, density for a biantennary glycan was observed at Asn200 in this model. N6 NA also has putative glycosylation sites at Asn86, Asn200, and Asn402, but only Asn86 had interpretable glycan density for one N-acetylglucosamine. Finally, while N8 NA has additional glycosylation sites at Asn86, Asn295, and Asn400, none of them had interpretable glycan density. There is a suggestion that N-glycosylation might be one of the factors to differentiate individual NAs within each subtype (25); however, more data are needed to confirm this correlation.

Active site of H5Nx NAs and antiviral drug susceptibility to NAIs.

In all the NA subtypes, the enzyme active site includes eight highly conserved residues: Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and Tyr406. These are all charged/polar residues that interact directly with the substrate in the catalytic site. The geometry of the catalytic site is structurally stabilized through a network of hydrogen bonds and salt bridges by a constellation of largely conserved framework residues: Glu119, Arg156, Trp178, Ser179, Asp/Asn198, Ile222, Glu227, His274, Glu277, Asn294, and Glu425 (Fig. 9B) (65).

The NA activities of H5Nx NAs were assessed using the fluorescent reagent 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) as the substrate (33). Despite the structural difference near the active site between group 1 and group 2 NAs, all recombinant H5Nx NAs had similarly high activities compared to human influenza virus NAs (Table 4). In the NI assay, the activities of the recombinant N1, N2, N6, and N8 NAs of various H5Nx viruses were effectively inhibited by the antivirals currently approved by the FDA (oseltamivir carboxylate, zanamivir, peramivir) and an investigational NAI (laninamivir), indicating a neuraminidase inhibitor-susceptible phenotype for these H5Nx viruses (Table 4).

TABLE 4.

Drug susceptibility assessment in the fluorescent neuraminidase inhibition assay

Virus name	NA subtype	Parameter			Mean IC50 ± SD (nM)a
		kcat ± SD (s−1)	Km ± SD (μM)	kcat/Km (M−1 s−1)	Oseltamivir carboxylate	Zanamivir	Peramivir	Laninamivir
A/American green-winged teal/Washington/195750/2014	N1	134,254 ± 2,841	5.08 ± 0.41	3.29 × 1011	3.45 ± 0.35	0.33 ± 0.10	0.10 ± 0.03	0.19 ± 0.02
A/northern pintail/Washington/40964/2014	N2	115,191 ± 2,523	5.48 ± 0.45	2.56 × 1011	0.18 ± 0.06	0.75 ± 0.22	0.20 ± 0.03	1.05 ± 0.10
A/Sichuan/26221/2014	N6	147,235 ± 3,693	7.00 ± 0.63	2.36 × 1011	0.98 ± 0.18	0.98 ± 0.18	0.21 ± 0.03	1.95 ± 0.24
A/gyrfalcon/Washington/41088-6/2014	N8	173,671 ± 3,018	5.98 ± 0.38	4.55 × 1011	0.99 ± 0.20	0.35 ± 0.12	0.06 ± 0.02	0.22 ± 0.01

^aFrom the results of three independent experiments run in triplicate.

Conclusion.

Here we report the molecular characterization of 3 HAs and 4 NAs from the H5Nx subclade 2.3.4.4. Even though H5N6 HA is an isolate from a fatal human case, results for both avian influenza virus H5N8 HA and human influenza virus H5N6 HA show that they possess an avian receptor binding preference, thus reducing their potential to efficiently infect humans. The H5Nx NAs are also sensitive to FDA-approved (zanamivir, oseltamivir, and peramivir) and investigational (laninamivir) neuraminidase inhibitors. It is important to monitor H5Nx viruses in poultry and to continue to assess changes in circulating H5Nx viruses and their pandemic potential. Collectively, the data indicate that additional changes in the H5 HA would be needed for efficient transmission among humans and that currently available antivirals would be an effective tool to combat these viruses.