Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets

Abstract

Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian-origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from a ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.IMPORTANCE Subtype H10 avian influenza A viruses (IAVs) have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.

Keywords: H10N7; aerosol droplet; alpha 2,3-linked sialic acids; alpha 2,6-linked sialic acids; avian influenza virus; glycan receptor binding; gull; influenza A virus; pathogenesis; transmission.

FIG 1

Phylogenetic and antigenic analyses of subtype H10 influenza A viruses (IAVs). (A) Hemagglutinin gene, (B) neuraminidase gene, and (C) polymerase acidic protein. The phylogenetic tree for each gene segment was inferred by using a maximum-likelihood method by running RAxML v8.2.9 and by using a Gamma model of rate heterogeneity and a generalized time-reversible substitution model (72). The bootstrap values were labeled for the selected representative branches with bootstrap values of ≥70. Phylogenetic trees were visualized by using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Scale bars represent nucleotide substitutions per site. Red indicates the H10N7 IAV strain isolated from an Iceland gull in 2015; green indicates the subtype H10N7 avian IAVs isolated from European seals in 2014; and the virus marked in blue indicates the IAVs isolated from Iceland from other studies.

FIG 2

Antigenic map of 32 subtype H10 influenza A viruses (IAVs). The map was constructed using hemagglutination inhibition (HI) data derived from ferret antisera. The open triangles indicate viruses isolated from humans or other mammals; the open circles indicate viruses isolated from domestic poultry; and the black circles indicate viruses isolated from wild birds. IAVs with identical HI titers may have overlapping positions on this antigenic map. The map was constructed using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap) (20, 21). Each gridline (horizontal and vertical) in the map represents one antigenic unit distance corresponding to a 2-fold difference in HI titers. Ig/4266, A/Iceland gull/Iceland/4266/2015(H10N7); Gg/4552, A/glaucous gull/Iceland/4552/2015(H10N7); Gg/4270, A/glaucous gull/Iceland/4270/2015(H10N7); Ig/4402, A/Iceland gull/Iceland/4402/2015(H10N7); rg-A/Jiangxi-Donghu/346/2013[R] (6:2) (H10N8), a reassortant with HA and NA genes from A/Jiangxi-Donghu/346/2013 (H10N8) and six other genes from A/PR/8/1934(H1N1).

FIG 3

Replication kinetics of four subtype H10 influenza A viruses isolates in vitro. Growth curves for influenza A/Iceland gull/Iceland/4266/2015(H10N7) virus (Ig/4266), A/chicken/Jiangxi/34609/2013(H10N8) (Ck/34609) virus, A/mink/Sweden/E12665/84(H10N4) virus (Mink/E12665), and A/seal/Netherlands/P14-221/2014(H10N7) virus (Seal/221) in Madin-Darby canine kidney (MDCK), adenocarcinomic human alveolar basal epithelial (A549), and chicken embryo fibroblast (DF-1) cells. Cells were infected with viruses at a multiplicity of infection of 0.01; dashed lines indicate the limit of virus detection, 1.50 log10 (50% tissue culture infective dose/ml [TCID50/ml]). Virus titers are expressed as mean ± standard deviation of log₁₀ TCID₅₀/ml.

FIG 4

Glycan binding specificity of two subtype H10 influenza A viruses to (A) biotinylated α2,3-linked sialic acid (3ʹSLN) and (B) α2,6-linked sialic acid (6ʹSLN) glycan analogs as determined by biolayer interferometry using an Octet RED instrument (Pall FortéBio, Fremont, CA, USA). Streptavidin-coated biosensors were immobilized with biotinylated glycans at different levels. Sugar-loading-dependent binding signals were captured in the association step and normalized to the same background. Binding curves were fitted by using the saturation binding method in GraphPad Prism 7. Horizontal dashed line indicates half of the fractional saturation (f = 0.5); vertical dashed line indicates relative sugar loading (RSL_0.5) at f = 0.5; the higher the RSL_0.5, the smaller the binding affinity. Ig/4266, A/Iceland gull/Iceland/4266/2015(H10N7); Ck/34609, A/chicken/Jiangxi/34609/2013 (H10N8); H1N1, A/California/04/2009(H1N1); and H5N1, A/duck/Hunan/795/2002(HA, NA) × A/PR/8/34 (H5N1).

FIG 5

Mean titers of influenza viruses recovered from nasal wash fluids of virus-inoculated and contact ferrets in transmission experiments. Ferrets (n = 3 per group/experiment) were inoculated with 10⁶ 50% tissue culture infectious doses/ml (TCID₅₀/ml) of influenza A/Iceland gull/Iceland/4266/2015(H10N7) virus. Twenty-four hours later, naive ferrets (n = 3) were each randomly paired with an inoculated ferret and either housed in same cages or on a different side of a 1-cm-thick, double-layered, steel partition with 5-mm perforations (A, B, and C represent each direct contact transmission group [white bars, inoculated ferrets; gray bars, naive ferrets]; and D, E, and F represent each aerosol transmission group [white bars, inoculated ferrets; black bars, naive ferrets]). Nasal wash fluids were collected on the indicated days after inoculation or exposure for virus quantification using endpoint titration in Madin-Darby canine kidney cells; ending titers were expressed as log₁₀TCID₅₀/ml. Each panel represents a set of paired ferrets. Dashed lines indicate the limit of detection, log₁₀10^1.5 TCID₅₀/ml. Ferrets represented by the individual panels correspond to those listed in Table 1, namely, (A) ferrets in cage 4, (B) ferrets in cage 5, (C) ferrets in cage 6, (D) ferrets in cage 1, (E) ferrets in cage 2, and (F) ferrets in cage 3.

FIG 6

Mean titers of influenza virus recovered from respiratory tract tissues of ferrets nasally inoculated with 10⁶ 50% tissue culture infectious doses (TCID₅₀) of influenza A/Iceland gull/Iceland/4266/2015(H10N7) virus. Two ferrets were euthanized 5 days postinoculation (dpi), and virus titers in the respiratory tissues of each ferret were determined by using endpoint titration in Madin-Darby canine kidney cells. The results shown are log10 TCID₅₀/gram. Results for tissues from the control group were negative (data not shown). Abbreviations: SP, soft palate; TR-U, upper trachea; TR-D, distal trachea; LCR, left cranial lung; LCD, left caudal lung; RCR, right cranial lung; RCD, right caudal; RMD, right middle lung; RA, right accessory lobes. Dashed line indicates the limit of detection, 1.50 log₁₀TCID₅₀/ml.

FIG 7

Pathogenic changes in respiratory tract tissues of ferrets inoculated with influenza A/Iceland gull/Iceland/4266/2015(H10N7) virus at 5 days postinoculation (dpi). Immunohistochemistry staining showed the presence of H10 antigen in respiratory epithelium cells within nasal turbinate (A, brown staining), ciliated epithelial cells within the trachea (B, brown staining), and scattered ciliated epithelial cells in bronchioles tissue were immunoreactive (C, arrows). (D to G) Hematoxylin and eosin-stained ethmoid turbinate sections at 5 dpi in control (D, F) and infected (E, G) ferrets. Ethmoid turbinates from virus-inoculated ferrets had intraluminal aggregates of degenerate neutrophils, macrophages, and cellular debris. In addition, there are scattered neutrophils within the epithelium (G, arrows). Bars = 20 μm/100 μm (in D, E).