Airborne transmissibility is a prerequisite for a pandemic influenza A virus (IAV), and a better understanding of how zoonotic IAV evolves to acquire a transmissible phenotype is essential for pandemic preparedness. Select contemporary influenza A(H9N2) viruses such as A/Anhui-Lujiang/39/2018 (AL/39) have exhibited a limited transmission capability by the airborne route in the ferret model; therefore, it is of great importance to identify viral factors that contribute to enhanced transmission. To investigate the role of virus acid stability in virus airborne transmission, we rescued a pair of isogenic A(H9N2) viruses, including the wild-type (wt) AL/39 and the mutant virus bearing a naturally occurring substitution HA1-Y17H, with a resulting difference in virus pH thresholds for hemagglutinin activation. We next assessed virus replication, airborne transmission, and fitness in a co-infection competition model in ferrets. We found that the HA1-Y17H mutant virus yielded only non-productive airborne transmission despite possessing a comparative replication as the wt virus in the ferret upper respiratory tract. Furthermore, ferrets inoculated with the wt virus emitted more virus-laden particles into the air than the HA1-Y17H mutant virus-inoculated animals. During ferret co-infection experiments, the wt virus was the dominant species in multiple types of specimens following different inoculation routes. Taken together, our study demonstrates that an acid-stable IAV had a greater capacity to establish a productive infection in the ferret upper respiratory tract and was emitted in greater quantities from infected animals, features that may contribute to virus airborne transmission in a synergistic manner in mammalian hosts.


Despite the accumulation of evidence showing that airborne transmissible influenza A virus (IAV) typically has a lower pH threshold for hemagglutinin (HA) fusion activation, the underlying mechanism for such a link remains unclear. In our study, by using a pair of isogenic recombinant A(H9N2) viruses with a phenotypical difference in virus airborne transmission in a ferret model due to an acid-destabilizing mutation (HA1-Y17H) in the HA, we demonstrate that an acid-stable A(H9N2) virus possesses a multitude of advantages over its less stable counterpart, including better fitness in the ferret respiratory tract, more effective aerosol emission from infected animals, and improved host susceptibility. Our study provides supporting evidence for the requirement of acid stability in efficient airborne transmission of IAV and sheds light on fundamental mechanisms for virus airborne transmission.


Influenza A viruses (IAVs) residing in avian hosts pose a significant risk to public health. The majority of documented human infections with avian IAV have been caused by the H5N1, H7N9, and H9N2 subtypes with over 2,000 confirmed cases as of April 2023 (1). While H5, H7, and H9 avian IAV have not yet caused sustained human-to-human transmission, such viruses have the potential to cause a pandemic in an immunologically naive population should the viruses acquire an airborne transmissible phenotype. There is an urgent need to better understand how IAVs evolve to cross species barriers and the molecular drivers associated with their ability to transmit between humans.
Two seminal studies on transmissible H5N1 mutant viruses identified pathways for an avian IAV to become airborne transmissible through only a handful of mutations (2, 3). In addition to key substitutions in the hemagglutinin (HA) receptor binding site for a switch from an avian-like receptor (α2,3-linked sialic acid moieties) to a human-like receptor (α2,6-linked sialic acid moieties) binding preference and mutations in polymerase genes for efficient replication in mammalian hosts, both studies identified that an additional mutation (HA-H110Y or T318I, H3 numbering throughout) resulting in virus enhanced thermostability or acid stability was also essential for an airborne transmissible H5N1 mutant virus in the ferret model (2, 3). IAV stability is mainly regulated by key residues in the HA protein, and the exact pH value for HA activation varies for different IAVs. Generally, most avian and swine IAVs have a higher threshold pH for HA activation compared to human isolates within the same subtype (4, 5), indicating that an acid-stable IAV may have a fitness advantage in infecting and transmitting among humans. A link between virus acid stability and airborne transmission has also been observed for 2009 pandemic H1N1 (2009pdmH1N1), swine-origin A(H1N1), and human A(H3N2) IAVs (68). Currently circulating 2009pdmH1N1 viruses share HA activation values ranging from 5.2 to 5.4, which are lower compared with early pandemic strains in humans (pH 5.5) and precursor H1N1 viruses in swine (pH 5.5–6.0) (9). However, despite the large body of evidence supporting the requirement for an acid-stable HA for efficient airborne transmission, the underlying mechanism is still poorly understood, and it remains unclear whether non-H5 subtype zoonotic viruses such as H7 and H9 would employ similar pathways to acquire an airborne transmissible phenotype.
IAV has been studied extensively in animal models, with ferrets being regarded as the best small animal model to capture both disease symptoms and transmission (10). Avian IAVs, including highly pathogenic avian influenza H5N1 viruses associated with human infection, typically exhibit no or limited transmission between ferrets in the presence of direct contact (11, 12). However, select contemporary avian influenza A(H7N9) and A(H9N2) viruses have exhibited limited transmission by the airborne route in addition to efficient transmission in the presence of direct contact in the ferret model (1316). Although results from IAV transmission studies in ferrets cannot be fully extrapolated to humans, the heightened transmissibility associated with H7 and H9 viruses nevertheless indicates that such viruses may be more primed to cause a pandemic compared to H5 viruses, warranting in-depth assessment of H7 and H9 subtype IAV transmission dynamics and stability in an aerosol state.
In this study, we focused on a low pathogenic avian influenza A(H9N2) virus associated with human infection and identified a key residue in the HA responsible for virus acid stability. Using reverse genetics (rg), we introduced a single amino acid mutation in the HA of the wild-type (wt) A(H9N2) virus to modulate virus acid stability and compared virus replication both in vitro and in vivo, the capacity for airborne transmission between ferrets, and viral fitness in co-infection competition studies. We show that isogenic A(H9N2) viruses with differing acid stability exhibited no substantial difference in viral replication in either epithelial cell culture models or the ferret respiratory tract but displayed modest differences in viral airborne transmission. We further demonstrate that the improvement in viral transmission associated with the acid-stable virus may partially result from improved fitness in the ferret upper respiratory tract, more effective virus emission from infected donors, and greater host susceptibility.


Recombinant virus rescue and propagation

Generation of recombinant viruses by rg was approved by the Centers for Disease Control and Prevention Institutional Biosafety Committee. All experiments with recombinant A(H9N2) viruses were performed in a biosafety level 3 containment facility with enhancements, as required by the U.S. Department of Agriculture (17). The full-length sequences of all gene segments from A/Anhui-Lujiang/39/2018 (AL/39) A(H9N2) virus were downloaded from the Global Initiative on Sharing All Influenza Data (GISAID) database (isolate identification number: EPI_ISL_330737) and were subsequently synthesized and cloned into pUC57 or pCDNA3.1 vector by Genscript (Piscataway, NJ, USA) for rescuing recombinant virus by rg (18). A single amino acid mutation to the AL/39 HA at the residue HA1-Y17H was introduced by site-directed mutagenesis to the pUC57-HA plasmid to rescue the mutant virus. Stock viruses were prepared and titered in both 10-day-old embryonated hens’ eggs and MDCK cells. Viral titers in eggs were, in general, up to 10-fold higher than in MDCK cells; therefore, subsequent specimens collected in vitro and in vivo experiments were titrated in eggs for higher sensitivity. The sequences of the stock viruses were confirmed by deep sequencing on an Illumina MiSeq Next Generation Sequencer.

pH thresholds for HA fusion activation

The pH thresholds of HA fusion activation of the A(H9N2) viruses were evaluated by a syncytium formation assay as described previously (19). In brief, Vero cells grown on a 24-well plate were infected with 1–50 μL of rg AL/39-wt or AL/39-HA1-Y17H viruses for 18 h before syncytia formation was induced by incubating with low-pH fusion buffers (pH values ranging 5.2–6.0) following treating infected cells with 5  µg/mL of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-trypsin at 37°C for 15  min. The fusion pH threshold was defined as the highest pH value at which 50% or more syncytia could be achieved among infected cells. Average pH thresholds for HA fusion activation from three independent experiments are reported.

Virus growth kinetics in vitro

The human bronchial epithelial cell line, Calu-3, was cultured at air-liquid interface conditions as described previously (20). Ferret primary nasal cells cultured at an air-liquid interface and tracheal cells cultured at liquid submerged conditions were prepared as described (21). Cells were cultured at 37°C and infected in triplicate at an MOI of 0.01 EID50 according to previously described methods (22). Cell supernatants collected from the apical side of insert cultures at 0–2, 24, 48, and 72-h post-inoculation (p.i.) were stored at −80°C until titration in 10-day-old embryonated hens’ eggs. The limit of detection (LOD) was 101.5 EID50/mL.

Virus replication and transmission in ferrets

Replication and transmission of the rg AL/39 virus pair in a respiratory droplet (RD) transmission model were assessed as described previously (23). Briefly, groups of six ferrets (Triple F Farms, Sayre, PA, USA, or Marshall Farms, North Rose, NY, USA), 8–10 months of age and serologically negative by hemagglutination inhibition (HI) assay for currently circulating influenza viruses, were intranasally (i.n.) inoculated with 106.0 EID50 of each virus diluted in 1 mL of phosphate-buffered saline (PBS). Three inoculated ferrets were humanely euthanized on day 3 p.i. to collect tissues [nasal turbinate, trachea (middle section), and lung (pooled from each lobe)] for virus titration. Three ferrets were monitored daily for clinical signs of disease and served as donors in RD transmission studies, in which three naive ferrets were placed adjacent to donor ferrets in individual cages with perforated side walls on day 1 p.i. Nasal wash (NW) specimens were collected from all ferrets on alternating days 1–11 p.i. or post-contact (p.c.) and stored at −80°C until subsequent virus titration in eggs. A successful RD transmission event was determined by the presence of infectious virus in NW specimens from contact ferrets and detectable seroconversion to homologous virus by day 21 p.c. (determined by HI). A non-productive transmission event was defined as a contact ferret with detectable levels of infectious virus in at least one nasal wash specimen in the absence of seroconversion, or seroconversion in the absence of detecting infectious virus in nasal wash specimens. A contact ferret that did not shed detectable infectious virus and did not seroconvert was considered as an unsuccessful transmission event. Fifty percentage of ferret infectious doses (FID50) was determined as previously described (24).

Exhaled aerosol collection from inoculated ferrets

Aerosol samples were collected using a two-stage cyclone aerosol sampler (BC 251, NIOSH) as described previously (25). Briefly, air was collected from cages housing individual inoculated ferrets on days 1–3 p.i. at 3.5 L/min for 2 h. Aerosols were fractioned by size (>4, 1–4, and <1 µm) and were inactivated in AVL buffer (Qiagen) for subsequent viral RNA extraction and quantification.

Ferret inoculation by inhalation

Calculation of spray factor (SF) is required to determine the aerosolized dose of virus presented to ferrets during inhalation inoculation (24) and was determined using a previously described procedure (26). For ferret inoculations with a mixture of the AL/39-wt and AL/39-HA1-Y17H mutant virus at a target ratio of 1:4 by the respiratory inhalation route, both viruses were prediluted based on the calculated SF and then mixed before being placed into a Collison nebulizer for a 15 min continuous spray. The actual viral ratio presented to the exposed animals was confirmed by next-generation sequencing of the virus collected in an SKC BioSampler.

Viral RNA detection by digital droplet RT-PCR

Collected aerosol samples were inactivated using AVL buffer followed by viral RNA extraction using a QIAamp Viral RNA Mini Kit (Qiagen). IAV M gene copy numbers in aerosol samples were detected with One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad) and M gene-specific primers and probes with sequences described previously (27) in duplicate reactions using a Bio-Rad QX200 ddPCR system. The absolute M gene copy number was calculated with Quantasoft (Bio-Rad) software and was expressed as the total copy number of the M gene for each sample.

Real-time quantitative measurement of ferret host immune responses in the nasal wash and tissue specimens

Ferret NW specimens were collected from naive ferrets (12–24 months of age) (n = 4 per group) inoculated with AL/39-wt and AL/39-HA1-Y17H virus on days 1–3 p.i. for RNA extraction. Real-time quantitative PCR reactions were conducted to quantify the mRNA expression of key genes involved in host immune responses using the primer and probe sets described previously (28, 29).

Next-generation sequencing (NGS)

Viral RNAs were extracted from virus inoculum and various ferret specimens (tissues, NW, and aerosol samples) using the QIAamp Viral RNA Mini Kits (Qiagen). One-step RT-PCR using influenza virus universal primers was performed to amplify all eight segments of viral genes as described previously (30). The amplified viral gene segments were processed for next generation sequencing (NGS) library preparation using the Illumina Nextera DNA Flex library prep kit and then sequenced paired end on iSeq100 with v2 300 cycle reagents (Illumina), and variants above 5% frequency with a minimum sequencing depth threshold of 100× coverage were identified.


Residue HA1-Y17 is responsible for a lower threshold pH for HA activation of A(H9N2) virus

We previously reported that contemporary A(H9N2) viruses display a wide pH spectrum for HA fusion activation, independent of host origin (31). To identify the key residue(s) responsible for the diverse pH thresholds for HA activation among A(H9N2) viruses, we performed a sequence alignment for the HAs of a panel of contemporary A(H9N2) viruses isolated from humans or chickens (Table S1). Among the residues shared by the viruses with a pH threshold for fusion ≤5.5, we focused on the residue HA1-17 since variance of tyrosine (Y) or histidine (H) at this position has been previously shown to modulate the pH of HA fusion activation for H1, H3, and H5 subtype IAV (3235); additional residues identified in the alignment were not located in regions predicted to affect virus stability based on H9 HA structural modeling (data not shown). To test whether HA1-17Y residue was responsible for the lowered pH threshold for HA activation of A(H9N2) viruses, we rescued rg wild-type AL/39 virus (AL/39-wt), which possesses a tyrosine residue at HA1-17 or an isogenic mutant virus (AL/39-HA1-Y17H) with a histidine at this position and compared the pH thresholds for fusion activation of these two viruses. As shown by syncytium formation assay in Vero cells (Fig. 1), a single HA1-Y17H mutation in the rg AL/39 virus increased the pH threshold for fusion activation from 5.4 (referred to as acid stable) to 5.8 (referred to as less acid stable), confirming that the residue at the HA1-17 position plays an important role in modulating pH thresholds for fusion activation of A(H9N2) viruses. Interestingly, we noticed that viral infectivity of the AL/39-wt virus in Vero cells was lower than that of the AL/39-HA1-Y17H mutant virus, a trend that has also been previously observed for other pairs of isogenic IAV with differing threshold pHs for HA activation and was believed to be associated with the elevated endosomal pH in Vero cells (36, 37).
Fig 1
Fig 1 Syncytium formation of isogenic rg A(H9N2) viruses in Vero cells. Vero cells grown on a 24-well plate were infected with 1–50 µL of the AL/39-wt or AL/39-HA1-Y17H mutant virus, and the cells were induced for syncytium formation by incubating with fusion buffers of pH values ranging from 5.2 to 6.0 for 5 min following treating infected cells with (TPCK)-trypsin at 37°C for 15 min. Cells were then fixed and processed for immunofluorescence microscopy with staining for viral NP protein (green) and nuclei (blue). Representative images at pH 5.4 and 5.8 are shown; the pH threshold was defined as the highest average pH value at which 50% of NP-positive cells formed syncytia from three independent experiments.

In vitro comparative replication of isogenic rg A(H9N2) viruses with differing pH thresholds for fusion activation

Upon rescuing a pair of isogenic AL/39 viruses with a confirmed phenotypical difference in virus acid stability due to a single mutation in the HA (HA1-Y17H), we next investigated whether virus acid stability affected virus fitness in vitro by assessing the replicative capacity of the rg viruses in either the immortalized human lung epithelial cell line (Calu-3) or ferret primary nasal and tracheal epithelial cells. Both Calu-3 and ferret primary nasal epithelial cells were grown on an air-liquid interface in Transwell inserts, facilitating cell differentiation that closely emulates respiratory epithelium (21). As shown in Fig. 2, the rg AL/39 virus pair exhibited similar replication kinetics and reached comparable titers at all time points examined in both human and ferret cell types. These results indicate that virus acid stability conferred by the residue of HA1-Y17 in an A(H9N2) virus resulted in no substantial advantage in viral replication capacity in vitro at cell culture conditions best mimicking the sites for virus replication in mammalian hosts.
Fig 2
Fig 2 Replication kinetics of isogenic rg A(H9N2) viruses in Calu-3, primary ferret nasal or tracheal cells. Calu-3 and ferret nasal cells were grown on Transwell inserts. All cultures were inoculated with AL/39-wt or AL/39-HA1-Y17H virus at an MOI of 0.01 EID50, and cell culture supernatants were collected at the indicated times p.i. from the apical side and subsequently titrated in eggs for viral titer determination. Dashed lines represent the LOD, which was 1.5 log10 EID50/mL. Graphs represent viral mean titers with standard deviation from three culture replicates. Two-way ANOVA was performed and AL/39-wt and AL/39-HA1-Y17H mutant virus showed no significant difference in viral titers at the time points examined in the study.

Lack of productive airborne transmission of AL/39-HA1-Y17H mutant virus despite efficient viral replication in the ferret upper respiratory tract

To further understand the effect of acid stability on virus fitness in mammalian hosts, we next compared the replication and transmission of the AL/39-wt and AL/39-HA1-Y17H mutant viruses in the ferret model. The AL/39-wt virus caused only mild clinical symptoms (transient fever and sneezing) and moderate weight loss (9.5% mean maximum), in agreement with its biological wt virus (31). AL/39-HA1-Y17H mutant virus caused similar clinical symptoms and weight loss (5.8% mean maximum). Virus shedding based on mean viral titers in NW specimens between this pair of rg AL/39 viruses was not significantly different on days 1 and 3 p.i. but was statistically higher for AL/39-wt virus on day 5 p.i. (Fig. 3A, left panel). Furthermore, both AL/39-wt and AL/39-HA1-Y17H mutant viruses exhibited comparable viral detection and replication in the nasal turbinate and trachea (Fig. 3B). Infectious virus was detected in the lung of two of three (AL/39-wt) or one of three (AL/39-HA1-Y17H) ferrets (Fig. 3B). Taken together, we conclude that the acid-destabilizing mutation HA1-Y17H in an A(H9N2) virus had no substantial effect on virus replication and tissue distribution early post-infection.
Fig 3
Fig 3 Replication and transmission of the rg AL/39 virus pair in ferrets. Groups of six naive ferrets (8–10 months of age) were intranasally inoculated with 106.0 EID50 of rg AL/39-wt or AL/39-HA1-Y17H mutant viruses either for assessing viral replication in tissues from three ferrets on day 3 p.i. or for using as donors (n = 3 per group) in the RD transmission study. (A) Viral titers in NW from the individual inoculated donor ferrets (left sets of bars) and RD contact ferrets (right sets of bars) are shown for the indicated times p.i. or p.c. (B) Individual viral titers in ferret nasal turbinate (NT), trachea (Tra), and lung were expressed in log10 EID50/mL (for NT) or g (for Tra, lung) and are shown in the bar graph. The LOD was 1.5 log10 EID50/mL or g as indicated by the dashed lines.
Wild-type AL/39 H9N2 virus can transmit not only in the presence of direct contact but also by respiratory droplets, albeit at a reduced capacity (31). We next compared the transmissibility of rg AL/39-wt and AL/39-HA1-Y17H mutant viruses by respiratory droplets. The AL/39-wt virus exhibited a limited capacity for airborne transmission as one out of three contact ferrets shed high titers of infectious virus on days 3–11 p.c. with peak titers comparable to inoculated ferrets and seroconverting to the homologous virus (Fig. 3A, left panel). In contrast, two contact ferrets housed adjacent to AL/39-HA1-Y17H virus-inoculated ferrets had low titers of infectious virus detected in nasal wash specimens (102.25-2.75 EID50/mL) on days 7 and 9, respectively, but did not seroconvert to homologous virus; the remaining contact ferret neither shed virus nor seroconverted (Fig. 3A, right panel), suggesting that AL/39-HA1-Y17H virus was only capable of non-productive airborne transmission, in contrast to the limited productive airborne transmission observed for the AL/39-wt virus.
Finally, we used NGS to determine whether the Y or H residue at the HA1-17 position was retained during virus infection in ferrets. For this purpose, viral RNA from ferret NW (days 1, 3, 5, and 7 p.i.) was processed for NGS to identify variants with a particular focus on the mutations in the HA gene. We found that AL/39-wt virus retained the HA1-Y17 residue in NW samples throughout day 7 p.i.; interestingly, a major HA1-Y232C variant emerged starting on day 5 p.i. ultimately reaching 45%–68% frequency in all inoculated ferrets. In the AL/39-wt contact ferret, the major HA1-L222R variant was detected at 50% frequency on day 7 p.c. In ferrets inoculated with AL/39-HA1-Y17H virus, the HA1-Y17H mutation was relatively stable and retained at above 99% frequency in NW samples collected through day 7 p.i. in all inoculated ferrets; additional major variants included HA1-T137K/I/R (7.7%–48%) and HA1-L222R (14%–44.4%), which were also detected in the AL/39-wt virus-inoculated animals (Fig. S1). The residues HA1-137, 222, and 232 are located in or adjacent to the HA receptor binding site, and the emerging variants at these positions may help the virus adapt to the ferret host (Fig. S1).
Collectively, we demonstrate that the HA-destabilizing mutation HA1-Y17H abolished the ability of an A(H9N2) virus to transmit productively by respiratory droplets, despite having only a marginal effect on virus replication in the ferret respiratory tract early post-infection. Furthermore, no substantial reversion was observed in AL/39-HA1-Y17H virus-inoculated animals, indicating that A(H9N2) virus can tolerate both Y and H residues at position HA1-17.

Higher levels of acid stable AL/39-wt virus emitted into the air by inoculated ferrets

It has been recently shown that ferrets inoculated with airborne transmissible IAVs generally shed higher levels of virus into their proximal environment than do ferrets inoculated with viruses that do not transmit by the air (25), with IAV in relatively large-sized aerosol particles playing a major role in virus airborne transmission (24, 38). As both rg AL/39 virus pairs replicated with comparable efficiency in the ferret upper respiratory tract early after infection but differed in transmissibility by respiratory droplets, we investigated whether inoculated ferrets shed comparable amounts of viruses into the air. We employed cyclone-based air samplers to collect aerosol samples fractionated by size (>4, 1–4,and <1 µm) from cages holding inoculated ferrets on days 1–3 p.i. and assessed the viral RNA load in each fraction by quantifying viral M gene copy numbers. Elevated viral RNA loads in collected aerosols were detected by day 2 p.i., with levels in the >4 µm size fraction from AL/39-wt virus-infected ferrets 40-fold higher (day 2 p.i.) and 70-fold higher (day 3 p.i.) compared with AL/39-HA1-Y17H virus (Fig. 4).
Fig 4
Fig 4 Detection of viruses emitted from ferrets inoculated with the rg AL/39 virus pair. Air from the cage housing individual ferrets inoculated intranasally with 106.0 EID50 of rg AL/39-wt or AL/39- HA1-Y17H mutant virus was collected on days 1, 2, and 3 p.i. by a two-stage cyclone aerosol sampler at a flow rate of 3.5 L/min for 2 h and fractionized into three particle sizes (>4, 1–4, and <1 µm). Viral M gene copy numbers in each fraction during 2 h aerosol collection were determined by digital droplet PCR. Dots represent the values from individual ferret cages; dashed line represents the mean from all collected samples. ***P < 0.0001.
To investigate if these differences were driven at the level of innate immune responses, NW specimens from ferrets inoculated with either rg AL/39-wt or AL/39-HA1-Y17H viruses were collected for the analysis of mRNA expression of key immune mediators. Comparable viral titers in NW specimens (Fig. 5A) and levels of mRNA expression of multiple cytokines and chemokines (Fig. 5B) were detected in all animals. In NW specimens, reduced expression of IFNβ, IL-12, IL-6, and TNFα in AL/39-HA1-Y17H mutant virus-inoculated ferrets compared to AL/39-wt day 2 p.i. did not reach statistical significance (Fig. 5B). Taken together, we conclude that ferrets inoculated with the acid-stable AL/39-wt virus shed more virus into the air compared to the animals inoculated with less acid-stable AL/39-HA1-Y17H mutant virus, which may partially account for the heightened airborne transmission associated with acid-stable IAVs in this model, despite comparable levels of localized upper respiratory tract innate immune responses based on the expression of key immune mediators during acute infection.
Fig 5
Fig 5 Viral titers and immune mediator gene mRNA expression in nasal wash specimens following the rg AL/39 virus pair infection in ferrets. Groups of four ferrets (12–24 months of age) were inoculated with 106 EID50 of either rg AL/39-wt or AL/39-HA1-Y17H mutant virus. (A) Mean viral titers in the nasal washes on days 1, 2, and 3 p.i. (B) The fold change of mRNA expression of key immune mediator genes relative to the naive ferret samples was calculated using relative quantification with kinetic PCR correction. Day 0 represents a pool of naive ferret nasal wash samples.

Fitness advantage of acid-stable AL/39-wt virus in ferrets during co-infection by i.n. route

To investigate whether virus acid stability provides a fitness advantage in a competing virus infection model, ferrets were inoculated intranasally with 106.0 EID50 of a mixture of both rg AL/39-wt and AL/39-HA1-Y17H mutant viruses at a 1:1 ratio (based on viral EID50 titers) and various specimens, including NW, tissues, and aerosol samples through day 3 p.i., were collected for viral titration and NGS analysis to assess the ratio of each virus species in the specimens. Consistent with efficient viral replication observed in ferrets inoculated with single species of virus, high viral titers were detected in NW samples and tissues throughout the respiratory tract following inoculation with the rg AL/39 virus mixture (Fig. 6A). When NGS was performed to analyze the proportion of each virus species, we found that the AL39-wt virus became dominant (≥92%) in the day 1 NW specimens and persisted through day 3 p.i. (≥89%) in NW samples and upper respiratory tract tissues (nasal turbinate and ethmoid turbinate). AL/39-wt virus exhibited even higher dominance in exhaled air, soft palate, trachea, and lung tissues on day 3 p.i., achieving close to 100% in at least two out of three ferrets (Fig. 6B). To further confirm the fitness advantage associated with AL/39-wt virus, the experiment was repeated at a target ratio of 10:90 (AL/39-wt:AL/39-HA1-Y17H). Despite being at a substantially lower frequency in the inoculum, AL/39-wt virus reached above 65% dominance in NW samples on day 1 p.i. and completely replaced the mutant virus by day 7 p.i. (Fig. S2). In agreement, the 50% ferret infectious dose (FID50) following i.n. inoculation for AL/39-wt virus was 102.25 EID50, 18-fold lower than the FID50 for AL/39-HA1-Y17H virus (103.5 EID50), further supporting that ferrets were less susceptible to the less acid-stable virus relative to AL/39-wt virus. Collectively, these results suggest that the acid-stable A(H9N2) virus has a strong fitness advantage in replicating in the ferret respiratory tract and being exhaled into the air and is positively selected in a ferret co-infection model.
Fig 6
Fig 6 Co-infection of the rg AL/39 virus pair in ferrets inoculated by the i.n. route. Three naive ferrets (8–10 months of age) were i.n. inoculated with a 1:1 mixture (based on EID50 titers) of rg AL/39-wt and AL/39-HA1-Y17H mutant viruses. (A) Viral titers in NW and tissues including nasal turbinate (NT), ethmoid turbinate (Eth), trachea (Tra), lung, and soft palate (SP) on day 3 p.i. Dashed line represents the LOD, which was 1.5 log10 EID50/mL. (B) Percentages of AL/39-wt and AL/39-HA1-Y17H viruses in the inoculum (Ino), nasal wash, tissues, and exhaled air collected from inoculated ferrets were determined by NGS analysis. One sample with low viral gene copy numbers was excluded from NGS and is marked with “NA” in the graph.

Fitness advantage of acid-stable AL/39-wt virus in ferrets during co-infection by respiratory inhalation

To better emulate the natural route of viral infection and compare viral fitness by different inoculation routes, ferrets were inoculated by the aerosol inhalation route with a mixture of AL/39-wt and AL/39-HA1-Y17H viruses at a target ratio of 25:75 (based on EID50 titers). The presented dose was approximately 105.8 EID50 per ferret, comparable to the dose used for standard liquid intranasal inoculation; the actual viral RNA ratio in the aerosol (based on virus collected in the sampler during animal aerosol exposure) was determined to be 18.6:81.6 by NGS. By this inoculation route, all ferrets were successively infected based on the detection of high-titer infectious virus in NW samples and high M gene copy numbers in various tissues. However, compared to intranasal inoculation, viral replication in the upper respiratory tract was delayed as evidenced by delayed peak NW viral titers (Fig. 7A), in agreement with prior studies employing an aerosol-based inoculum (24). In contrast with viral co-infection studies employing the standard i.n. inoculation route, ferrets inoculated with a mixture of the AL/39-wt and AL/39-HA1-Y17H mutant virus by the aerosol inhalation route exhibited greater variances in virus ratios among individual ferrets, with a general trend of the AL/39-wt virus steadily gaining dominance over time in NW samples. Virus ratios in exhaled air were only analyzed by NGS for aerosol samples collected on day 4 p.i. due to low viral copy numbers in specimens collected prior to this timepoint; AL/39-wt virus was at approximately 68% in this sample type. In tissues collected during necropsy on day 4 p.i., the ratio of the AL/39-wt virus ranged from 40% to 100% (Fig. 7B). Interestingly, the ethmoid turbinate and soft palate harbored AL/39-wt virus at the highest ratios compared to other sample types, with 100% of this virus detected from two of three ferrets and 95% and 68% in ethmoid turbinate and soft palate, respectively, from the third ferret. These results further confirm that the AL/39-wt virus possesses a capacity to steadily gain dominance over the less acid-stable AL/39-HA1-Y17H mutant virus during multiple inoculation routes and that certain tissues, such as ethmoid turbinate and soft palate, may exert greater selection pressure for the acid-stable virus during virus replication in the ferret respiratory tract.
Fig 7
Fig 7 Co-infection of the rg AL/39 virus pair in ferrets inoculated by the respiratory inhalation route. Three naive ferrets (8–10 months of age) were intranasally inoculated with a mixture of AL/39-wt and AL/39-HA1-Y17H mutant viruses at a ratio of 25:75 (based on EID50 titers) at a calculated presented dose of 105.8 EID50 per ferret during a 15-min aerosol exposure. (A) Viral titers in NW (left) or M gene copy numbers in tissues including nasal turbinate (NT), ethmoid turbinate (Eth), trachea (Tra), lung, and soft palate (SP) (right) on day 4 p.i. Dashed lines represent the LOD, which was 1.5 log10 EID50/mL or 1,000 RNA copies/mL. (B) Percentages of the AL/39-wt and AL/39-HA1-Y17H virus in the inoculum (Ino), nasal wash, tissues as well as in the exhaled air (day 4 p.i.) collected from inoculated ferrets were determined by NGS analysis. Samples with low viral gene copy numbers were excluded from NGS and are marked with “NA” in the graph.


A comprehensive understanding of underlying mechanisms governing IAV cross-species infection and airborne transmission is essential for pandemic preparedness. Some emerging zoonotic influenza A(H9N2) isolates already possess certain mutations in the HA for human-like receptor binding and polymerase proteins for efficient viral replication in mammalian cells (39); whether such viruses can further evolve and gain the capacity for sustained human-to-human transmission remains uncertain. In our study, we identified a naturally occurring HA-acid-stabilizing Y residue in the HA1-17 position of a zoonotic A(H9N2) virus that contributed to virus airborne transmission in a ferret model and showed that this residue improved virus infectivity and fitness in the respiratory tract and facilitated virus exhalation from the ferret respiratory tract.
Many residues in the HA have been identified to modulate virus acid stability (4, 33, 40). However, most of these substitutions have been derived from in vitro selection in cells cultured at elevated endosomal pH values or by passaging in a new host (4, 33, 40). Among over 2,000 A(H9N2) isolate sequences deposited in GISAID, approximately 77% of them possess an HA1-Y17 with the rest bearing an HA1-H17 residue. In contrast, human seasonal (H1 and H3) and avian-origin (H5 and H7) IAV subtypes exhibit an HA group-specific preference at the HA1-17 position with H1 and H5 (Group 1) and H3 and H7 (Group 2) subtypes possessing exclusively a Y or H residue at this position, respectively. The substitution of an H and Y for H1 and H5, and H3 HA at position 17, respectively, modulates the pH threshold for HA activation, virus infectivity, pathogenicity, and transmission in both avian and mammalian hosts (9, 32, 34, 35, 4143). A 2009pdmH1N1 virus (A/Tennessee/1–560/2009) bearing a HA1-Y17H mutation (elevating pH for activation from 5.5 to 6.0) displayed reduced viral replication in mice, pigs, and ferrets; moreover, this mutant virus either reverted back to the wt or acquired additional stabilizing mutations during virus transmission between pigs, ferrets, or from pigs to ferrets, supporting that efficient viral replication and transmission in mammalian hosts requires an acid-stable HA (9, 44). In our study, the HA1-Y17H substitution in an A(H9N2) virus backbone showed no substantial effect on virus replication and pathogenicity in ferrets early post-infection but abolished productive airborne transmission relative to the wild-type virus, further confirming the functional significance of the HA1-17 residue for two distinct HA groups. Interestingly, the AL/39-HA1-Y17H mutant virus did not exhibit significant reversion or acquire compensatory mutations during viral infection in ferrets as was previously observed with the 2009pdmH1N1 HA1-Y17 mutant virus (9, 44, 45). Although the low number of inoculated and respiratory droplet contact animals included in our study may limit the detection of reversion or compensatory mutations, the natural divergence at position HA1-Y17 among A(H9N2) isolates suggests that both residues can be tolerated in avian and mammalian hosts. However, some A(H9N2) viruses with lowered pH thresholds for HA activation described by Peacock et al. do not share a Y residue at this position (46). Considering the differences among HA structures of different HA subtypes, and the extent of structural changes that occur during fusion, it is likely that numerous sites throughout the HA will lead to different stability phenotypes; only a limited subset of these have been identified to date. In the future, identification of virus subtype-specific residues in natural isolates that are involved in IAV stability and investigation of whether such residues share a similar effect on virus adaptation and transmission would help us better understand the link between virus stability and virus transmission in mammalian hosts and develop risk assessment strategies based on viral sequences and in vitro phenotypes.
In our study, despite both AL/39-wt and AL/39-HA1-Y17H viruses exhibiting comparable viral replication in the ferret upper respiratory tract early after infection, AL/39-HA1-Y17H virus inoculated ferrets shed fewer virus-laden airborne particles into the surrounding environment relative to AL/39-wt virus. Furthermore, the AL/39-HA1-Y17H virus had a higher FID50 value compared to the AL/39-wt virus (FID50 values of 103.5 and 102.25 EID50 for AL/39-HA1-Y17H and AL/39-wt viruses, respectively). A requirement for higher viral loads to establish infection for a less acid-stable virus has also been observed for a 2009pdmH1N1 mutant virus with a HA2-K47E-destabilizing mutation (47). Lowered virus shedding into the air from donor animals, coupled with increased virus threshold requirements for initiating a productive infection, may collectively contribute to the non-productive airborne transmission associated with AL/39-HA1-Y17H mutant virus shown here; further investigation of the relative role that non-productive airborne transmission events may contribute toward the acquisition of a productive transmissible phenotype among IAVs is warranted. It should be noted the NIOSH BC 251 samplers employed here are not designed to maintain virus viability. A pair of rg 2009pdmH1N1 viruses with differing acid stability due to an HA1-Y17H (destabilizing) or E31K (stabilizing) mutation were evaluated in ferrets and, employing a different experimental protocol that permits quantification of viable virus, found higher levels of viable virus from animals inoculated with the HA1-E31K mutant virus compared with the HA1-Y17H mutant virus. However, the ratios of live to total viral particles and how these proportions changed throughout infection remain unknown since total viral RNA in the air could not be quantified in their experimental setting (45). In addition to reduced virus shedding into the air from infected donor animals, less acid-stable virus may be more likely to be inactivated in an aerosol state compared to acid-stable virus, which may further lower the detectable infectious virus level in emitted air. In the future, concurrent measurement of both viral RNA and viable viruses from aerosols shed by inoculated animals and a comparison of the stability of viruses in exhaled breath will help us understand the scope of multifactorial benefits conferred by the acid stability of IAV.
Elevated induction of host immune responses in IAV-inoculated ferrets early after infection has been found to correlate with the frequency of transmission and clinical signs (sneezing and rhinorrhea) in a comparative ferret study with both seasonal A(H3N2) virus, pandemic A(H1N1), and highly pathogenic A(H5N1) viruses (48). In a murine infection model, a 2009pdmH1N1 virus (A/Tennessee/1–560/2009) carrying the destabilizing mutation HA1-Y17H induced the upregulation of more host genes and increased type I IFN responses and cytokine expression in murine lungs and bone marrow-derived dendritic cells than its wt virus (42). However, we did not detect significant differences in host innate immune responses in ferrets based on cells collected from ferret NW specimens at early times post-infection between AL/39-wt and AL/39-HA1-Y17H virus groups. This may be due to the limited tissue types or collection time points included in our study. Future comprehensive assessments of host immune responses including the utilization of single-cell RNA-Seq platforms may help us better understand the potential link between virus acid stability and virus emission from infected donors.
To compare virus relative fitness in the ferret model, we employed multiple co-infection approaches using varying mixtures of AL/39-wt and AL/39-HA1-Y17H mutant viruses delivered either by the intranasal route or respiratory inhalation route. Both routes supported that the AL/39-wt virus gained predominance throughout the respiratory tract post-inoculation even when present in the inoculum at lower proportions than the AL/39-HA1-Y17H mutant virus. Different virus inoculation routes affect the initial anatomical sites for viral replication as aerosol delivery may allow the virus to initiate infection in the oropharyngeal cavity, whereas intranasal inoculation deposits the virus directly into the ferret nasal cavity, trachea, and lung (24). The possible difference in cell tropism in the upper and lower respiratory tract or receptor binding affinity between the AL/39-wt and AL/39-HA1-Y17H mutant virus may contribute toward variation in viral replication dynamics during co-infection. Interestingly, we observed from both routes of inoculation that nasal ethmoid turbinate possessed the highest proportion of the AL/39-wt virus, followed by the soft palate. The ethmoid turbinate is lined with olfactory epithelium, and the enrichment of acid-stable virus in the ethmoid turbinate and soft palate during this competition study suggests that these two sites may have a strong selection preference for acid-stable IAV. Influenza virus tropism in olfactory epithelium has not yet been studied extensively compared to that in nasal respiratory epithelium. It was recently reported that aerosol transmissible IAV including H1, H3, and recombinant A(H5N1) mutant viruses have a higher infection rate in nasal respiratory epithelium compared to less transmissible virus during intranasal infection (49). Further investigation of cell tropism and infection dynamics of acid-stable and less-stable viruses in the olfactory epithelium during intranasal or aerosol inhalation inoculation is warranted.
IAV airborne transmission is a complex process and both viral and host factors contribute to this dynamic process. In our study, using a pair of isogenic viruses with only one amino acid difference in the HA, which regulates virus pH for HA activation, we revealed a possible mechanism underlying the requirement for an acid-stable HA in IAV airborne transmission and concluded that having a stable HA offers advantages in outcompeting viruses with lower stability, facilitating virus expulsion from the donor animals, and effectively initiating infection in recipient animals. Importantly, this study shows the utility of incorporating aerobiology-based techniques in the evaluations of molecular determinants governing virulence and transmissibility of IAV. Our study lays the foundation for more in-depth mechanistic studies of virus airborne transmission in the future, including but not limited to IAV.


This article is a direct contribution from Terrence M. Tumpey, a Fellow of the American Academy of Microbiology, who arranged for and secured reviews by Charles Russell, St. Jude Children's Research Hospital, and David Steinhauer, Emory University School of Medicine.


Fig S1 - mbio.02957-23-s0001.tiff
Major variants detected in virus-inoculated or RD contact ferrets.
Fig S2 - mbio.02957-23-s0002.tif
Co-infection of the rg AL/39 virus pair in ferrets inoculated by the i.n. route.
Legends - mbio.02957-23-s0003.docx
Supplemental figure legends.
Table S1 - mbio.02957-23-s0004.docx
Alignment of HAs of select A(H9N2) viruses with different pH thresholds for HA activation.
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Information & Contributors


Published In

cover image mBio
Volume 15Number 116 January 2024
eLocator: e02957-23
Editor: Malik Peiris, University of Hong Kong, Pokfulam, Hong Kong Nil, Hong Kong
PubMed: 38112470


Received: 7 November 2023
Accepted: 14 November 2023
Published online: 19 December 2023


  1. influenza A virus
  2. H9N2
  3. HA fusion activation
  4. airborne transmission
  5. acid stability


The findings and conclusions are those of the authors and do not necessarily reflect the views of ASTDR/the Centers for Disease Control and Prevention (CDC). The authors declare no competing interests.



Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, and Writing – review and editing.
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Formal analysis, Methodology, and Writing – review and editing.
Joanna A. Pulit-Penaloza
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Data curation, Formal analysis, Investigation, Methodology, and Writing – review and editing.
Nicole Brock
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Data curation, Formal analysis, Investigation, Methodology, and Writing – review and editing.
Troy J. Kieran
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Data curation, Formal analysis, Methodology, Software, and Writing – review and editing.
Hui Zeng
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Data curation, Formal analysis, Methodology, and Writing – review and editing.
Claudia Pappas
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Data curation, Formal analysis, Methodology, and Writing – review and editing.
Terrence M. Tumpey
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Formal analysis, Resources, Supervision, and Writing – original draft.
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Author Contributions: Conceptualization, Project administration, Supervision, and Writing – review and editing.


Malik Peiris
University of Hong Kong, Pokfulam, Hong Kong Nil, Hong Kong


The authors declare no conflict of interest.

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