Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal

Abstract

The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

Figure 1. Genetic relationship amongst H3 hemagglutinin genes

(a) Phylogenetic tree for H3 HA gene. The genetic relationship of the H3N8 HA genes was determined by aligning nucleotide sequences, and the tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. (b) Amino acid changes associated with viruses in the harbour seal clade. Four amino acid changes were observed between harbour seal associated viruses and viruses in the second North American avian subclade. All four amino acids were located in the head domain of the HA protein.

Figure 2. Sialic acid binding affinities of the different viruses

Viruses associated with the harbour seal clade had increased binding to α2,6 sialic acids suggesting increased mammalian binding efficiency. Data are representative of two experiments with three replicates per virus. Error bars represent the s.e. of the mean.

Figure 3. Transmission of H3N8 viruses

(a–g) Ferrets (n = 3 per group/experiment, n = 2 for control avian virus) were inoculated with 10⁶ units of H3N8 influenza virus (black lines). Twenty-four hours later, naïve ferrets (n = 2–3) were either placed in direct contact with the infected group (blue lines) or housed in separate cages (respiratory transmission, red lines) and nasal washes were collected on the indicated d.p.i. for virus quantification. Lines represent individual animals. Data are representative of three separate experiments for seal H3N8 and two separate experiments for other viruses.

Figure 4. Replicative capacity of H3N8 viruses in vitro and in vivo

To evaluate the replication of the H3N8 viruses in vitro, MDCK (a) human lung A549 (b) or primary NHBE cells grown in an air–liquid interface (c) were infected at an multiplicity of infection of 0.01–0.03 (NHBE) and cell culture supernatants collected at 6, 24, 48 and 72 h.p.i. Viral titres were determined by TCID₅₀ analysis in triplicate. Data are representative of at least two experiments. Error bars represent the s.e. of the mean. To evaluate the pathogenicity and transmissibility of avian and mammalian H3N8 viruses 6–8-week-old BALB/c mice (n = 10/group/experiment) were i.n. infected with 10⁵ TCID₅₀ units of the indicated viruses and weight loss (d) was monitored for 12 d.p.i. (e) At 3 and 6 d.p.i., lungs were collected from three mice per group and viral titres were determined in homogenates by TCID₅₀ analysis. Error bars represent the s.e. of the mean. Data are representative of two experiments.