Airborne transmission of highly pathogenic H7N1 influenza virus in ferrets

Abstract

Avian H7 influenza viruses are recognized as potential pandemic viruses, as personnel often become infected during poultry outbreaks. H7 infections in humans typically cause mild conjunctivitis; however, the H7N9 outbreak in the spring of 2013 has resulted in severe respiratory disease. To date, no H7 viruses have acquired the ability for sustained transmission among humans. Airborne transmission is considered a requirement for the emergence of pandemic influenza, and advanced knowledge of the molecular changes or signature required for transmission would allow early identification of pandemic vaccine seed stocks, screening and stockpiling of antiviral compounds, and eradication efforts focused on flocks harboring threatening viruses. Thus, we sought to determine if a highly pathogenic influenza A H7N1 (A/H7N1) virus with no history of human infection could become capable of airborne transmission among ferrets. We show that after 10 serial passages, A/H7N1 developed the ability to be transmitted to cohoused and airborne contact ferrets. Four amino acid mutations (PB2 T81I, NP V284M, and M1 R95K and Q211K) in the internal genes and a minimal amino acid mutation (K/R313R) in the stalk region of the hemagglutinin protein were associated with airborne transmission. Furthermore, transmission was not associated with loss of virulence. These findings highlight the importance of the internal genes in host adaptation and suggest that natural isolates carrying these mutations be further evaluated. Our results demonstrate that a highly pathogenic avian H7 virus can become capable of airborne transmission in a mammalian host, and they support ongoing surveillance and pandemic H7 vaccine development.

Importance: The major findings of this report are that a highly pathogenic strain of H7N1 avian influenza virus can be adapted to become capable of airborne transmission in mammals without mutations altering receptor specificity. Changes in receptor specificity have been shown to play a role in the ability of avian influenza viruses to cross the species barrier, and these changes are assumed to be essential. The work reported here challenges this paradigm, at least for the influenza viruses of the H7 subtype, which have recently become the focus of major attention, as they have crossed to humans.

FIG 1

Schematic representation of serial-passage experimental design. Experiment 2 in the ferret study section of Materials and Methods is represented. In serial-passage experiments with each line, passage and evaluation of transmission were limited (n = 1).

FIG 2

WT H7N1 shows limited transmission to CH animals in the absence of virus shedding. Four ferrets received 1 × 10⁶ TCID₅₀ of WT H7N1 virus. At 24 hpi, a CH and an AC ferret were introduced to each DI animal. Nasal wash fluid samples were collected daily for virus titration. Viral titers and weight losses of DI (A), CH (B), and AC (C) ferrets. Red (blue, green, or pink) corresponds to one experimental cage setup consisting of three ferrets (one DI, one CH, and one AC ferret) housed in the same isolator. In parentheses is the proportion of ferrets that succumbed to infection or were euthanized. Daggers denote animals that were euthanized or succumbed to infection. N denotes neurological symptoms.

FIG 3

Serial passage yields an H7N1 virus capable of direct and airborne transmission. Two ferrets received 1 × 10⁶ TCID₅₀ of WT H7N1 virus. Serial passage was initiated with nasal wash fluid samples from infected ferrets to inoculate an additional two ferrets. Eight serial passages were performed and maintained as two separate lines, A (red) and B (blue). On the eighth passage, CH animals were introduced and became infected. The nasal wash fluid samples from CH animals were then used to initiate passage 10 (p10). At 24 hpi, a CH and an AC ferret were introduced. Viral titers and weight losses of p10 DI (A), CH (B), and AC (C) animals are shown. In parentheses is the proportion of animals that were euthanized or that succumbed to infection (†).

FIG 4

Ferret-adapted H7N1 is transmitted to CH and AC animals in a larger transmission study. Three ferrets received a pool of nasal wash fluid samples from line A p10 DI ferret (1 × 10⁵ TCID₅₀ of pooled virus). At 24 hpi, a CH and an AC ferret were introduced to the DI animals. Viral titers and percent weight losses of DI (A), CH (B), and AC (C) animals are shown. Red (blue or green) corresponds to one experimental cage setup consisting of three ferrets (one DI, one CH, and one AC) housed in the same isolator. Shown in parentheses is the proportion of ferrets that developed severe disease and were euthanized or succumbed to infection (†). N denotes neurological symptoms.

FIG 5

Titration of brain and lung samples from AC animals shows high virus titers in the brain. In experiment 3, two AC animals became infected and developed neurological disease requiring euthanasia. Both ferrets were subjected to necropsy. Brain and lung samples were collected for titration. Shown above are viral titers in the lungs and brains of both animals. Both animals had replicating virus in the brain, while only one animal had detectable virus in the lungs.

FIG 6

Viral loads in the respiratory tract and nervous system tissues of WT H7N1- and ferret-adapted ACp11-infected ferrets. Six ferrets were inoculated with 1 × 10⁵ TCID₅₀ of either WT H7N1 or ACp11 H7N1 (virus stock generated from p11 AC 3). Tissue samples were collected from the large and small intestines, kidneys, spleen, liver, lungs, trachea, nasal turbinates, brain, and olfactory bulb. Tissue samples were titrated and normalized to the weight of input tissue. No virus was isolated from the intestines, kidneys, spleen, or liver. Each bar represents the viral load of an individual ferret. Viral titers are shown for upper respiratory tract tissue (nasal turbinates and trachea) (A), the lower respiratory tract or lungs (B), and nervous system tissue (olfactory bulb and brain) (C).

FIG 7

Histopathology of lung and brain samples showing differences in inflammation between animals infected with WT H7N1 or ACp11 virus on dpi 5 and 7. Ferrets (n = 6/virus) were infected with either WT H7N1 or ACp11. On dpi 3, 5, and 7, lung, trachea, and brain samples were collected for evaluation of histopathology. All sections were fixed and subjected to H&E staining. Sections were viewed and scored by a veterinary pathologist blinded to the virus strain's identity. Shown are representative images of dpi 5 lung sections from WT H7N1 (A)- and ACp11 (B)-infected ferrets and dpi 7 brain sections from WT H7N1 (C)- and ACp11 (D)-infected ferrets.