Dissecting the role of the HA1-226 leucine residue in the fitness and airborne transmission of an A(H9N2) avian influenza virus

Abstract

A better understanding of viral factors that contribute to influenza A virus (IAV) airborne transmission is crucial for pandemic preparedness. A limited capacity for airborne transmission was recently observed in a human A(H9N2) virus isolate (A/Anhui-Lujiang/39/2018, AL/39) that possesses a leucine (L) residue at position HA1-226 (H3 numbering), indicative of human-like receptor binding potential. To evaluate the roles of the residue at this position in virus fitness and airborne transmission, a wild-type AL/39 (AL/39-wt) and a mutant virus (AL/39-HA1-L226Q) with a single substitution at position HA1-226 from leucine to glutamine (Q), a consensus residue in avian influenza viruses, were rescued and assessed in the ferret model. The AL/39-HA1-L226Q virus lost the ability to transmit by air, although the virus had a comparable capacity for replication, induced similar levels of host innate immune responses, and was detected at comparable levels in the air surrounding the inoculated ferrets relative to AL/39-wt virus. However, ferrets showed a lower susceptibility to AL/39-HA1-L226Q virus infection compared to the AL/39-wt virus. Furthermore, the AL/39-wt and AL/39-HA1-L226Q viruses each gained dominance in different anatomic sites in the respiratory tract in a co-infection competition model in ferrets. Taken together, our findings demonstrate that the increasing dominance of HA1-L226 residue in an avian A(H9N2) virus plays multifaceted roles in virus infection and transmission in the ferret model, including improved virus fitness and infectivity.

Importance: Although the capacity for human-like receptor binding is a key prerequisite for non-human origin influenza A virus (IAV) to become airborne transmissible in mammalian hosts, the underlying molecular basis is not well understood. In this study, we investigated a naturally occurring substitution (leucine to glutamine) at residue 226 in the HA of an avian-origin A(H9N2) virus and assessed the impact on virus replication and airborne transmission in the ferret model. We demonstrate that the enhanced airborne transmission associated with the HA1-L226 virus was mainly due to the increased infectivity of the virus. Interestingly, we found that, unlike most sites in the ferret respiratory tract, ferret ethmoid turbinate lined with olfactory epithelium favors replication of the AL/39-HA1-L226Q virus, suggesting that this site may serve as a unique niche for IAV with avian-like receptor binding specificity to potentially allow the virus to spread to extrapulmonary tissues and to facilitate adaptation of the virus to human hosts.

Keywords: H9N2; airborne transmission; influenza A virus; receptor binding.

Fig 1

Viral replication in a human bronchial epithelial cell line (Calu-3) and in ferrets. (A) Calu-3 cells grown at the air-liquid interface on transwell inserts in triplicate were inoculated with AL/39-wt or AL/39-HA1-L226Q at an MOI of 0.01 EID₅₀. Viral specimens were collected from the apical surface by incubating 200 µL of viral infection medium with the infected cells at 37°C for 15 min at indicated time points p.i. and subsequently titrated in 10-day-old embryonated hens’ eggs. The graph represents mean viral titers with standard deviation from three culture replicates. (B) Groups of three naïve ferrets intranasally inoculated with 10^6.0 EID₅₀ of AL/39-wt or AL/39-HA1-L226Q virus were humanely sacrificed on day 3 p.i. to assess viral replication in tissues, including nasal turbinate (NT), ethmoid turbinate (Eth), trachea (Tra), lung, and soft palate (SP). Bar graph represents the mean viral titers in tissues expressed as log₁₀ EID₅₀/mL (for NT, Eth, SP) or /g (Tra, Lung) from three inoculated ferrets except in lungs, in which only the specimens with viral titers above the limit of detection (1.5 log₁₀ EID₅₀/mL or g, dashed line) were shown (2/3 and 1/3 ferrets in AL/39-wt and AL/39-HA1-L226Q group, respectively). Error bars represent standard deviation (SD).

Fig 2

Respiratory droplet (RD) transmission of AL/39-wt and AL/39-HA1-L226Q virus in ferrets and variants detected from the inoculated and contact ferrets. Groups of three naïve ferrets were intranasally inoculated with 10^6.0 EID₅₀ of AL/39-wt (A) or AL/39-HA1-L226Q virus (B) and were used as donor ferrets in the transmission experiment after placement adjacent to three naïve contact ferrets. Viral titers in NW from the inoculated individual donor ferrets (left sets of bars) and contact ferrets (right sets of bars) are shown for the indicated time points p.i. or p.c. Dashed lines represent the limit of detection, which was 10^1.5 EID₅₀/mL. (C) Areas under the curve of NW titers through days 1–5 and 1–9 for AL/39-wt and AL/39-HA1-L226Q inoculated ferrets were calculated and compared for statistical analysis with unpaired t-test with GraphPad Prism 7.05. **** P < 0.0001, ns P > 0.05. (D) Major variants with mutations in the HA were detected from three inoculated donors (RD-I1, I2, I3), one positive contact (RD-C2), and three inoculated necropsy (NE-I1, I2, I3) ferrets in the RD transmission study. * Indicates the samples with no available next-generation sequencing (NGS) data.

Fig 3

mRNA expression of ferret immune mediator genes in NW specimens. Groups of four ferrets were intranasally inoculated with 10⁶ EID₅₀ of either AL/39-wt or AL/39-HA1-L226Q virus and NW specimens were collected on days 1, 2, and 3 p.i. The fold change of mRNA expression of key immune mediator genes relative to naïve ferret NW baseline (day 0) samples was calculated using relative quantification with kinetic PCR correction. Graphs represent the mean fold changes with standard deviation.

Fig 4

Detection of IAV M gene copy numbers in the aerosol samples collected from virus-inoculated ferrets. Groups of three naïve ferrets were intranasally inoculated with 10^6.0 EID₅₀ of AL/39-wt or AL/39-HA1-L226Q virus and were housed in individual cages. Aerosol samples were collected from the cages using a NIOSH sampler at a flow rate of 3.5 L/min for 2 h and fractionized into three particle sizes (>4 µm, 1–4 µm, <1 µm). IAV M gene copy number in each fraction was determined by qPCR. Dots represent values from individual specimens and short solid lines represent the mean of three specimens. The dashed line represents the limit of detection, which was 0.5 copies per liter of air.

Fig 5

Co-infection of AL/39-wt and AL/39-HA1-L226Q virus in ferrets by intranasal or respiratory inhalation route inoculation. Groups of three naïve ferrets were intranasally inoculated with an inoculum (Ino) of AL/39-wt and AL/39-HA1-L226Q virus mixture at a ratio of 50:50 based on viral EID₅₀ titers (42:58 based on viral RNA copy number) (A, B) or were inoculated by inhalation with a ratio of 24:76 based on viral RNA copy numbers (C.D). Viral titers from individual virus-inoculated ferrets are shown (A, C) with tissues (NT, Eth, Tra, lung, and SP) collected on day 3 (A) or 4 (C) p.i. The proportions of AL/39-wt and AL/39-HA1-L226Q in NW, exhaled air, and tissues (B, D) were determined by NGS analysis.