Characterising viable virus from air exhaled by H1N1 influenza-infected ferrets reveals the importance of haemagglutinin stability for airborne infectivity

Abstract

The transmissibility and pandemic potential of influenza viruses depends on their ability to efficiently replicate and be released from an infected host, retain viability as they pass through the environment, and then initiate infection in the next host. There is a significant gap in knowledge about viral properties that enable survival of influenza viruses between hosts, due to a lack of experimental methods to reliably isolate viable virus from the air. Using a novel technique, we isolate and characterise infectious virus from droplets emitted by 2009 pandemic H1N1-infected ferrets. We demonstrate that infectious virus is predominantly released early after infection. A virus containing a mutation destabilising the haemagglutinin (HA) surface protein displayed reduced survival in air. Infectious virus recovered from droplets exhaled by ferrets inoculated with this virus contained mutations that conferred restabilisation of HA, indicating the importance of influenza HA stability for between-host survival. Using this unique approach can improve knowledge about the determinants and mechanisms of influenza transmissibility and ultimately could be applied to studies of airborne virus exhaled from infected people.

Fig 1. The influenza virus transmission tunnel (IVTT).

(A) Schematic diagram of the IVTT apparatus. Airflow is generated using a bias flow pump, which connects to a 37.5cm (height) x 25cm (diameter) ferret chamber for in vivo experiments, or a 10cm (height) x 9cm (diameter) nebuliser chamber for in vitro experiments with nebulised virus. The IVTT is a half cylindrical clear acrylic 100cm (length) x 18cm (width) x 9cm (height) exposure tunnel containing cell culture plates situated 30cm, 60cm and 90cm from the tunnel opening. Air can be sampled from the end of the IVTT into a SKC Biosampler. (B) Photographs of the IVTT in use for isolating virus from an influenza virus-infected ferret.

Fig 2. Ferrets emit a peak of infectious virus early after infection.

Ferrets were intranasally inoculated with 10⁴ PFU of Eng/09 virus diluted in 0.2mL PBS. On days 1 to 6, air was sampled for 10 minutes using the IVTT and then each ferret was nasal washed while conscious. Virus titre (PFU/mL) detected by plaque assay from nasal wash samples is shown as lines in (A) to (F) on the left y-axis. Total numbers of viral plaques (PFU) detected in the IVTT is shown as grey bars in (A) to (F) on the right y-axis.

Fig 3. Infectious virus emitted from ferrets infected with pH1N1 virus mutants of varying pH stability.

Four ferrets were infected with either Y7H (orange/red) or E21K (green/blue) viruses. In each group donors #1 and #2 were infected with 10⁴ PFU and donors #3 and #4 with 10⁶ PFU. Viral titres in nasal wash samples were quantified by plaque assay for (A) Y7H and (B) E21K infected ferrets. Virus emitted in airborne droplets was collected from (C) Y7H and (D) E21K infected ferrets in the IVTT for 10 minutes on days 1 to 4 and detected on culture plates placed at 30cm, 60cm and 90cm along the tunnel.

Fig 4. Mutations that promote stability of haemagglutinin enhance virus survival in the air.

(A) Upper panel: Virus emitted by Y7H-infected ferret donor #3 on days 1 to 4 was collected as plaques picked from plates 1 (30cm), 2 (60cm) and 3 (90cm) of the IVTT and viral RNA extracted. The haemagglutinin (HA) gene was Sanger sequenced. HA mutations identified in viral plaques are represented by the colours orange H7Y, blue E47K-HA2, green V55I-HA2 and purple V19I. Lower panel: The proportion of corresponding HA mutations at positions 7 and 19 in HA1 and 47 and 55 in HA2 detected on days 1 to 4 in the nasal wash was determined by next-generation sequencing. An additional mutation detected in the nasal wash at >5% frequency but not in any picked plaques is also shown (N31S in red). In each of the bar graphs, the proportion of virus in nasal wash with sequence encoding the amino acid as in the parental virus (Y7H) is shown in grey. (B) HA mutations are modelled on a HA monomer using Pymol molecular visualization tool (PDB: 4jtv). H1 numbering using the mature HA sequence is used throughout[54]. HA1 is shaded light brown, HA2 is teal and the fusion peptide is black. (C) The acid stability of viruses in air emitted from donor #3 was tested by incubating virus propagated from IVTT plaques at low (pH 5.5) and neutral (pH 7) pH, in triplicate. The remaining infectivity detected at pH 5.5 is expressed relative to infectivity detected at pH 7. Each grey bar represents an individual virus propagated from a picked plaque with its HA mutation on the x-axis. Stability of parental virus Y7H is shown on the left of the panel. Error bars represent standard deviation of three independent experiments. One-way ANOVA with Tukey post-test was used to compare each plaque with the Y7H parent virus. *p<0.05, **p<0.01 ***p<0.001 ****p<0.0001.

Fig 5. Virus with increased acid stability has improved survival when nebulised into droplets.

(A) 40%:60% (B) 25%:75% and (C) 10%:90% mixtures of E21K and Y7H based on their infectivity by PFU/mL were nebulised into the IVTT in three independent experiments. Input viral titres were 10⁶ PFU in (A) and 5x10⁴ PFU in (B) and (C). Viral plaques that formed on IVTT culture plates 1 (at 30cm), 2 (at 60cm) and 3 (at 90cm) and a sample of plaques from the inoculum were picked and Sanger sequencing of the haemagglutinin gene carried out. Where the distribution of plaques was such that too few (n = <5) or too many plaques were collected to enable purification and/or analysis is indicated. Fisher’s exact test was used to compare the proportions of E21K/Y7H genotype detected on each plate. *p≤0.05, **p≤0.01, ***p≤0.001.