Assessment of transmission, pathogenesis and adaptation of H2 subtype influenza viruses in ferrets

Abstract

After their disappearance from the human population in 1968, influenza H2 viruses have continued to circulate in the natural avian reservoir. The isolation of this virus subtype from multiple bird species as well as swine highlights the need to better understand the potential of these viruses to spread and cause disease in humans. Here we analyzed the virulence, transmissibility and receptor-binding preference of two avian influenza H2 viruses (H2N2 and H2N3) and compared them to a swine H2N3 (A/swine/Missouri/2124514/2006 [swMO]), and a human H2N2 (A/England/10/1967 [Eng/67]) virus using the ferret model as a mammalian host. Both avian H2 viruses possessed the capacity to spread efficiently between cohoused ferrets, and the swine (swMO) and human (Eng/67) viruses transmitted to naïve ferrets by respiratory droplets. Further characterization of the swMO hemagglutinin (HA) by x-ray crystallography and glycan microarray array identified receptor-specific adaptive mutations. As influenza virus quasispecies dynamics during transmission have not been well characterized, we sequenced nasal washes collected during transmission studies to better understand experimental adaptation of H2 HA. The avian H2 viruses isolated from ferret nasal washes contained mutations in the HA1, including a Gln226Leu substitution, which is a mutation associated with α2,6 sialic acid (human-like) binding preference. These results suggest that the molecular structure of HA in viruses of the H2 subtype continue to have the potential to adapt to a mammalian host and become transmissible, after acquiring additional genetic markers.

Keywords: Ferret; Hemagglutinin; Influenza; Transmission.

Fig. 1

Plaque morphology of MDCK cells (London) infected with H2 viruses of human, avian and swine origin. For every virus, the same dilution was used to inoculate a confluent cell monolayer and was incubated at either 37 °C or 33 °C for 65 h.

Fig. 2

Respiratory droplet transmission experiments using H2 viruses of human, avian and swine origin. Each experiment was done in triplicate; ferrets were i.n. inoculated with 10⁶ EID₅₀ of virus and placed in individual cages. Twenty four hours later a naïve ferret was housed in adjacent cage to allow exchange of respiratory droplets through perforations on the adjoining walls. Nasal wash samples were collected from inoculated and indirect ferrets on alternating days, starting on day 1 post inoculation/post indirect contact (p.i./p.i.c.). (A) Ferrets inoculated with human virus Eng67; (B) swMO; (C) mallMD. Bars with the same pattern represent nasal wash titers of inoculated/indirect contact ferret pair, the bars on the left side correspond to inoculated ferret and on the right side to the indirect contact ferret.

Fig. 3

Influenza virus titers recovered from respiratory tract ferret tissues. Three ferrets were i.n. inoculated with 10⁶ EID₅₀ of Eng67, swMO, mallMD and ckPA, and tissues were collected on day 3 p.i. Tissues homogenates were titrated in 10-day embryonated eggs. The limit of detection is 1.5 Log₁₀ EID₅₀/g or mL. Each bar represents the average titer for three ferrets, plus standard deviation. Significance was determined by student t-test and compared against swMO values: *p<0.05, **p<0.1, ***p<0.01.

Fig. 4

Glycan microarray analysis of swMO recombinant HA. Colored bars highlight glycans that contain α2–3 SA (blue) and α2–6 SA (red), α2–6/α2–3-mixed SA (purple), N-glycolyl SA (green), α2–8 SA (brown), β2–6 and 9-O-acetyl SA, and non-SA (gray). Error bars reflect the standard deviation in the signal for six independent replicates on the array. Structures of each of the numbered glycans are found in Supplementary Table 2.

Fig. 5

Structural overview of swMO HA. (A) One swMO monomer is shown with the HA1 chain colored in green and the HA2 chain in cyan. The locations of the glycosylation sites are shown in red sticks. (B) Expanded view of swMO HA RBS with its three structural elements, the 130-loop, 190-helix, and 220-loop, colored in purple, red and yellow, respectively. Mutations found in the nasal washes of ferrets inoculated with ckPA (colored in orange and red sticks) and mallMD (colored in orange and blue sticks). (C) Amino acid differences in HA region that contains receptor binding amino acids (HA1 positions 130 to 230). All structural Figs were generated with MacPyMol (DeLano, 2002).