Advancing A(H5N1) influenza risk assessment in ferrets through comparative evaluation of airborne virus shedding patterns

Domain: neuroscience
Published: 2026-02-02
Editorial outcome: major_revision

Abstract
Recent A(H5N1) zoonotic cases linked to poultry and cattle in North America highlight the urgent need to assess the pandemic potential of emerging strains. Using male ferrets, we evaluate two B3.13 and two D1.1 genotype A(H5N1) viruses isolated from humans and observe fatal disease and varying capacities for direct contact transmission. To enhance pandemic risk assessment, we conduct aerosol sampling using cyclone BC251 and water condensation capture-based SPOT samplers and perform comparative analyses to include additional A(H5N1), A(H9N2), A(H7N9), and A(H1N1)pdm09 strains with known transmissibility profiles. Although none of the A(H5N1) strains transmit via the air, B3.13 viruses are detected at significantly higher levels compared to D1.1 strains. Here we show strong correlations between viral loads in nasal washes, airborne virus shedding, and transmissibility in ferrets, highlighting the value of these metrics for identifying zoonotic influenza viruses that may be adapting toward increased transmission potential.

Introduction
A(H5N1), a highly pathogenic avian influenza (HPAI) virus, has caused outbreaks in birds and occasional human infections since its emergence in East Asia in 1996. Through ongoing genetic mutation, the virus has diversified into multiple genotypes and clades. Clade 2.3.4.4b A(H5N1) virus was introduced into North America via migratory wild birds on multiple occasions and was first detected in the U.S. during December 20211. By 2024, following reassortment with North American avian influenza viruses, two major genotypes, B3.13 and D1.1, had been identified in the U.S. Genotype B3.13 has been detected in domestic poultry, dairy cattle, cats, and other mammals, and was associated with the first U.S. human case linked to cattle exposure in Texas2,3. Genotype D1.1, initially identified in wild birds and poultry outbreaks, was later detected in Nevada and Arizona dairy herds in 2025, likely introduced through contact with infected wild birds4. Between 2024 and 2025, over 70 confirmed human infections with viruses from both genotypes have been reported in North America, primarily following exposure to infected poultry or cattle. While most cases were mild, characterized by conjunctivitis, cough, sore throat, fever, or fatigue, severe illness occasionally occurred, including a fatal case involving a D1.1 genotype virus in Louisiana, raising concerns about its pandemic potential5. Genomic analyses indicate that neither genotype currently shows evidence of widespread human-adaptive mutations6. Nonetheless, the expanding host range and sporadic detection of mutations associated with reduced antiviral susceptibility highlight the risk that a pandemic-capable strain could emerge. These developments underscore the importance of sustained surveillance, genomic monitoring, and experimental studies to assess the pandemic potential of evolving A(H5N1) viruses7.

Research using the ferret model is essential for evaluating the mammalian transmission potential of A(H5N1) viruses and informing pandemic risk assessments8. Ferrets are considered the most relevant small-animal model for influenza studies due to their respiratory tract anatomy and clinical symptoms that are similar to those of humans9. Historically, airborne transmission of A(H5N1) viruses in ferrets was not observed prior to the emergence of clade 2.3.4.4b viruses10,11, heightening concern about currently circulating strains. Notably, two B3.13 genotype strains, isolated from dairy farm workers in Texas [A/Texas/37/2024 (TX/37)] and Michigan [A/Michigan/90/2024 (MI/90)] early in 2024, were shown to transmit between ferrets via the airborne route12–14. However, D1.1 genotype viruses have not yet been evaluated in the ferret model, limiting our understanding of their transmission potential and the associated public health impact.

Refining our understanding of the ferret transmission model is essential for robust influenza risk assessment. A key gap is how the kinetics and magnitude of viral replication in the ferret respiratory tract affect the levels of virus released into the air, and how this correlates with transmissibility. While higher nasal wash titers often coincide with transmission events, they do not always reliably predict airborne transmissibility across strains15. Detection of infectious virus in the air may be a better predictor of transmission16–18. However, progress in measuring airborne influenza virus has been hampered by the lack of standardized sampling procedures. Currently, no sampler has been validated as a gold standard for influenza that both efficiently captures low levels of virus into a small, concentrated collection volume while preserving viral viability19. Consequently, previous studies in humans and animal models have more frequently focused on quantifying viral genomic RNA in air samples, rather than infectious virus. Nonetheless, with the use of new-generation samplers and improved protocols, growing evidence across multiple influenza strains point to a link between airborne virus levels and transmissibility profiles, supporting routine airborne virus quantification as a complement to existing risk assessment protocols11,14,18,20. Recent B3.13 A(H5N1) isolates from the 2024 Texas and Michigan human cases showed limited but measurable airborne transmission in ferrets. These findings were corroborated by the detection of infectious virus in the air collected from inoculated ferrets12,14. An independent study also demonstrated a direct link between detection of airborne A(H5N1) virus and observed transmissibility in the ferret model11. In contrast, ferrets inoculated with non-transmitting A(H5N1) strains shed little or no infectious virus into the air, suggesting that the lack of transmission reflected insufficient levels of infectious virus in the air. What remains unknown is whether D1.1 genotype viruses are shed into the air and transmit via air, and whether more recent B3.13 isolates are further adapting toward increased airborne shedding and more efficient airborne transmissibility than what was observed with earlier isolates of that genotype.

In this work, we assess the pathogenesis and transmissibility of two D1.1 and two B3.13 A(H5N1) viruses isolated from humans to better understand the evolutionary dynamics of these genotypes and to inform pandemic risk assessments. We show that the A(H5N1) viruses have the capacity to cause fatal disease, exhibit varying capacities for transmission in the presence of direct contact, but do not display an airborne-transmissible phenotype. To further examine the relationship between transmission outcomes and airborne virus shedding in the ferret model, we conduct side-by-side comparisons of a cyclone sampler (BC251) and a water condensation-based sampler (SPOT). While both samplers demonstrate high utility for studying influenza viruses in the air, the data indicate that the BC251 sampler has greater collection efficiency, whereas the SPOT sampler preserves infectious virus more efficiently. We then compare the levels of both airborne genomic RNA and airborne infectious virus shed by the D1.1 and B3.13 A(H5N1) viruses, along with a panel of additional viruses, including contemporary and historical A(H5N1) strains, as well as human seasonal A(H1N1)pdm09 and zoonotic A(H7N9) and A(H9N2) strains, and show that B3.13 viruses are detected in the air at significantly higher levels compared to D1.1 strains. Lastly, we show strong correlations between viral loads in nasal washes, airborne virus shedding, and transmissibility profiles, and employ logistic regression to model the probability of airborne transmission.

Results
Pathogenesis of genotype B3.13 and D1.1 A(H5N1) influenza viruses in the ferret model
Genotype B3.13 A(H5N1) viruses associated with outbreaks in dairy cows have been shown to replicate efficiently in the ferret respiratory tract leading, in most cases, to severe disease followed by systemic spread and death within a week12–14,21. In this study, we evaluated the pathogenesis of two B3.13 genotype viruses and two D1.1 genotype viruses isolated from humans between July and November of 2024. A/Washington/239/2024 (WA/239; genotype D1.1) and A/Colorado/137/2024 (CO/137; genotype B3.13) viruses were isolated from patients exposed to infected poultry, A/British Columbia/PHL-2032/2024 (BC/PHL; genotype D1.1) was isolated from a patient with an unknown exposure source, and A/California/147/2024 (CA/147; genotype B3.13) was isolated from a patient exposed to infected cows (Table 1)5,22.

To evaluate the pathogenesis of these strains, ferrets were inoculated intranasally with 6 log10 PFU of virus and assessed for clinical signs and symptoms of infection. Viral replication kinetics were monitored by collecting nasal wash and rectal swab samples. All the inoculated ferrets displayed rapid weight loss, fever, nasal discharge, and lethargy. Diarrhea was also frequently observed, and infectious virus was detected in the blood of most of the ferrets indicating systemic spread (Table 2, Supplementary Fig. 1a, b). Mean maximum peak titers in nasal washes ranged from 5.3 to 7.6 log10 PFU/mL, with virus detected in rectal swabs from the majority of inoculated ferrets (Fig. 1a–f, Supplementary Fig. 2a-f). All animals developed severe disease and necessitated humane euthanasia between days 2–7 post inoculation (p.i.). To assess systemic spread into tissues, three separate ferrets per group were inoculated with a representative strain of each genotype, CO/137 (B3.13) and WA/239 (D1.1), and euthanized on day 3 p.i. (Fig. 1g). Although no additional animals were included for tissue distribution analysis on day 3 p.i., for CA/147 (B3.13) and BC/PHL (D1.1), tissues were collected from the inoculated ferrets used in the transmission experiments (shown in Fig. 1c, f) at the time they reached humane endpoint criteria (days 4–5 p.i.). Regardless of virus strain or timepoint p.i., high viral titers were consistently detected throughout the respiratory tract. Evidence of systemic spread in all animals was demonstrated by the presence of virus in other tissues including the olfactory bulb, intestine, spleen, and liver (Fig. 1g), which was consistent with previously studied A(H5N1) viruses that were lethal in ferrets23.

To determine whether variants harboring mammalian adaptation markers emerged following infection in ferrets, we performed next-generation deep sequencing analyses of viruses shed in nasal washes and tissues from ferrets inoculated with representative strains of each genotype (B3.13 CO/137 and D1.1 WA/239). No genetic changes at known mammalian adaptation markers were detected in samples collected from inoculated animals, and the identified variants were not consistently enriched across groups (BioProjectID: PRJNA1338708). These findings indicate a lack of strong selective pressure against these viruses, with previous reports showing no apparent trends of mammalian adaptation in ferrets infected with other clade 2.3.4.4b A(H5N1) viruses10,13,14.

Transmission of genotype B3.13 and D1.1 A(H5N1) influenza viruses in the ferret model
Ferret transmissibility studies provide critical experimental evidence on the potential for human-to-human spread of novel influenza viruses, directly informing pandemic risk assessment rubrics24,25. In the direct contact transmission (DCT) model, an inoculated ferret is co-housed with a naïve ferret, allowing transmission via multiple routes: direct contact, indirect contact through fomites, and inhalation of airborne virus26. Previously evaluated genotype B3.13 viruses displayed capacity for transmission in the DCT model12,14,27. To determine whether other clade 2.3.4.4b viruses retain the capacity for transmission in this setting, we tested a representative strain of each genotype CO/137 (B3.13) and WA/239 (D1.1). Similar to what was observed in previously tested genotype B3.13 strains, CO/137 virus efficiently transmitted between each of the three co-housed ferret pairs as evidenced by the detection of virus in nasal wash samples (mean maximum of 5.7 log10 PFU/mL) and rectal swabs (mean maximum of 2.5 log10 PFU/mL) of contact animals (Fig. 1a). All ferrets that became infected with CO/137 virus developed fatal disease with clinical signs including fever, weight loss, lethargy, nasal discharge, and diarrhea. The virus was also detected in blood, lungs, and nasal turbinates (1.9–6.5 log10 PFU/mL or g) in all contact animals. In contrast, direct contact transmission was less apparent with the D1.1 isolate, WA/239. Although virus was not detected in any of the contact nasal wash samples, a rectal swab sample from one animal collected on day 3 post-contact (p.c.) had a titer of 3.4 log10 PFU/mL. This animal displayed weight loss, nasal discharge, diarrhea, and lethargy and reached endpoint criteria on day 3 p.c. (Fig. 1d, Supplementary Fig. 2d). Blood, lung, and trachea samples collected at the time of euthanasia had detectable virus (1.3–2.0 log10 PFU/mL or g), while the nasal turbinate sample did not. The remaining two ferrets did not display signs of infection and did not have detectable virus in nasal wash and rectal swab samples: however, one seroconverted to WA/239 (HAI 160 on day 21 p.c.). These data show a higher transmission capacity of the B3.13 virus as compared to the D1.1 virus in a direct contact setting.

Next, we used the respiratory droplet transmission model (RDT) to determine if recently isolated A(H5N1) viruses (CO/137, CA/147, WA/239, and BC/PHL) were capable of airborne transmission (Fig. 1b, c, e, f). In this set-up, the inoculated and contact ferrets are housed in adjacent cages that permit air exchange but prevent physical contact26. Three ferret pairs were used for each virus group. None of the four tested viruses were capable of airborne transmission as evidenced by the lack of virus detection in nasal wash and rectal swab samples, and the lack of seroconversion to the challenge virus. Notably, earlier B3.13 viruses were capable of limited airborne transmission, suggesting both intra- and inter-genotypic variability in transmission phenotypes12–14.

Comparative evaluation of airborne virus shedding using BC251 and SPOT samplers
To better understand the relationship between airborne virus shedding and transmission in the ferret RDT model, we used a panel of influenza viruses representing a range of airborne transmissibility profiles (Table 1). The viruses were categorized into three groups based on their known transmission frequencies: non-transmissible, low-transmissible, and high-transmissible. In addition to testing clade 2.3.4.4b A(H5N1) strains (B3.13 genotype: CO/137, CA/147 and D.1.1 genotype: WA/239, BC/PHL), we included a non-transmissible predecessor strain, clade 1 A(H5N1) A/Vietnam/1203/2004 (VN/1203) strain, for comparison. Two previously characterized low-transmissible strains were also included: clade 2.3.4.4b A(H5N1) (MI/90; genotype B3.13), which transmitted between 50% of ferret pairs12, and A(H7N9) A/Anhui/1/2013 (Anhui/1), which transmitted in 33% of pairs28. Finally, two high-transmissible viruses were tested: human seasonal A(H1N1)pdm09 A/Nebraska/14/2019 (NE/14) and A(H9N2) A/Anhui-Lujiang/39/2018 (AL/39), which transmitted between 100% and 83% of tested ferret pairs, respectively29,30. Since different air sampling devices have varying collection efficiencies and preserve viral particle infectivity to differing degrees19, we employed two aerosol sampling devices for comparison. The NIOSH BC251 two-stage cyclone sampler collected air at 3.5 L/min and size-fractionated particles into a 15 mL tube (>4 µm), a 1.5 mL tube (1–4 µm), and a 37 mm PTFE filter (<1 µm). Because most viral RNA and infectious particles were recovered in the >4 µm fraction, consistent with a previous study employing diverse influenza virus strains18, we report totals combined across all fractions. The water condensation capture-based SPOT sampler collected particles into liquid media at 1.5 L/min. Air was collected sequentially from inoculated ferrets (number of replicates and collection times are shown in Supplementary Table 1) and the data were normalized to sampled air volume as described in the Methods section to permit direct comparison between samplers and to account for sampling time and flow rate differences. Each of the aerosol sampling devices was capable of capturing both genomic RNA and infectious virus; however, differences in the detection frequencies and viral titers were observed, indicating variability in sampler-specific collection efficiencies as well as virus strain-specific variation in the amount of airborne virus shed by infected animals. In all air samples with detectable virus, genomic RNA copy titers consistently exceeded infectious virus titers (Fig. 2, Supplementary Fig. 3). Out of 184 samples generated using the BC251 sampler throughout all experiments, infectious virus and viral RNA were detected in 68% and 93% of samples, respectively. In comparison, out of 97 samples collected using the SPOT sampler, infectious virus and viral RNA were detected in 52% and 77% of samples, respectively. Fisher’s Exact Test analysis showed that the odds of virus detection with the BC251 sampler were significantly higher than with the SPOT sampler (infectious virus: p≤0.006, genomic RNA: p≤0.0002). Specifically, the BC251 sampler was 2 times more likely to detect infectious virus and 4.2 times more likely to detect viral RNA than the SPOT sampler (Supplementary Table 2). Furthermore, the BC251 sampler collected genomic RNA and infectious virus from transmissible viruses at significantly higher levels compared to the SPOT sampler, supportive of better collection efficiency of the BC251 sampler (Fig. 3a, b; high-transmissible: p≤0.0005, low transmissible: p≤0.0008, non-transmissible: p≥0.07). It is important to note that without normalization to the sampled air volume, the statistical differences between the samplers would have been even greater, as the SPOT sampler drew about 2.33 times less air per minute compared to the BC251 sampler. To assess the differences in sampler-specific retention of virus viability during the collection, we compared the ratios of genomic RNA to PFU titers in samples where both measurements were detected. Lower mean RNA/PFU ratios were observed in samples collected by the SPOT sampler relative to the BC251 sampler, indicative of higher retention of virus viability when using the SPOT sampler. However, this difference reached statistical significance only in samples from ferrets inoculated with the high-transmissible virus group (p≤0.02) (Fig. 3c). Overall, both samplers demonstrated high utility for influenza virus aerosol collection, each offering distinct advantages, suggesting that their parallel use can provide a more comprehensive understanding of airborne virus dynamics across a wider range of viruses.

Correlation between influenza virus loads in ferret nasal washes and air samples
In our previous study, we detected significantly higher levels of genomic RNA in nasal washes and air samples collected from ferrets inoculated with transmissible viruses compared to those inoculated with non-transmissible viruses18. However, since the presence of influenza genomic RNA does not necessarily indicate the presence of viable, infectious virus, in this study, we performed side-by-side comparisons of both viral RNA and infectious virus shed by inoculated ferrets. To measure the correlation between virus exhaled into the air and virus load in nasal washes, the area under the curve (AUC) was calculated for samples collected over days 1, 2, and 3 p.i. (day 5 was omitted due to an incomplete dataset). This approach was selected to allow comparisons between viruses displaying differing peak shedding times. A strong positive correlation using Pearson’s method was observed between AUC values, with R=0.7 for infectious virus and R=0.82 for genomic RNA (Fig. 4a. b), suggesting that higher viral loads in nasal wash specimens during the acute phase of infection are associated with increased virus exhaled into the air (collected by the BC251 samplers). Similar trends were observed for the SPOT sampler, although the correlations were more modest (R=0.61 for infectious virus and R=0.64 for genomic RNA; Supplementary Fig. 4a, b). To better understand the nature of this correlation, it was important to determine whether it was driven by viral loads on specific days or by cumulative viral shedding over multiple days, as represented by the AUC. When viral loads from individual days were compared between nasal wash and air samples collected by BC251 or SPOT samplers using Pearson’s method, positive correlations were still observed, albeit reduced (R=0.5–0.21 for infectious virus and R=0.57–0.33 for genomic RNA; Supplementary Fig. 5). When linear mixed-effects model was utilized with a random intercept for individual ferret, nasal wash to airborne virus collected by BC251 slopes were near zero on day 1 and day 2 and positive on day 3 (infectious virus: 0.369, 95% CI 0.047 to 0.692 and genomic RNA: 0.857, 95% CI 0.156–1.558). Tukey-adjusted pairwise comparisons of slopes showed trends toward stronger day 3 associations versus day 1 (infectious virus: 0.389, p=0.074; genomic RNA: 0.805, p=0.064), with other contrasts not significant. These findings indicate that while viral loads in nasal washes on individual days show some correlation with airborne virus levels, the cumulative viral shedding captured by the AUC provides a more robust and reliable measure when comparing viruses with different shedding kinetics. When comparing across transmissibility groups, AUC means for nasal wash and air samples from animals infected with highly transmissible viruses were significantly higher compared to those from animals infected with non-transmissible viruses. Low-transmissible viruses generally displayed AUCs that were not statistically different from those of either high- or non-transmissible viruses, indicating a more variable shedding phenotype in this intermediate group (Fig. 4, Supplementary Table 3). This trend was consistent across both infectious virus and genomic RNA datasets. Taken together, these findings strengthen the central conclusion that transmissibility is linked to the magnitude of virus shed in nasal washes and the surrounding air.

Probability of airborne transmission of A(H5N1) influenza between ferrets based on virus shedding measurements
To better evaluate the pandemic risk associated with A(H5N1) influenza viruses, we classified the nine tested viruses into two groups, transmissible and non-transmissible, based on their observed transmission outcomes (with viruses associated with airborne transmission between at least one ferret pair classified as transmissible15). We used AUC values, calculated from infectious virus titers and viral RNA copy numbers in BC251 air samples collected on days 1, 2, and 3 post-inoculation to model the probability of airborne transmission using logistic regression with transmissibility as a binary parameter (Fig. 5a, b). We then used the fitted model to calculate the probability of airborne transmission for each virus (Fig. 5c). The non-transmissible D1.1 A(H5N1) viruses, WA/239 and BC/PHL, as well as the clade 1 A(H5N1) VN/1203 virus, had predicted airborne transmission probabilities of ≤16% (Fig. 5c). In contrast, highly transmissible NE/14 and AL/39 viruses showed a high predicted transmission potential of over 80%. Interestingly, viruses that did not transmit between ferret pairs, such as B3.13 CA/147 and CO/137, showed a wide range of predicted transmission probabilities (19–95%) similar to what was observed for MI/90 and Anhui/1 which were previously reported to transmit between 33% and 50% of ferret pairs, highlighting the variability in airborne viral shedding among ferrets inoculated with viruses that are not capable of efficient airborne transmission. These findings show that although both D1.1 (WA/239, BC/PHL) and B3.13 (CA/147, CO/137) viruses did not transmit in the ferret RDT model in this study, B3.13 strains were shed into the air at higher levels, and therefore had higher overall calculated probabilities of airborne transmission potential compared to D1.1 viruses, supporting the utility of assessing airborne virus shedding in the context of pandemic risk assessment.

Methods
Biosafety
The work was conducted to inform public health risk assessments. All animal experiments, including those involving HPAI viruses, were performed under enhanced biosafety level 3 (BSL-3E) containment in a facility under continuous 24/7 monitoring. Facility access was restricted to trained personnel, authorized to enter the laboratory suite as regulated by the Federal Select Agent Program. Enhancements of the BSL-3E suite included HEPA filtration on supply and double HEPA filtration on the exhaust air. All research activities adhered to safety and security protocols established by the U.S. Department of Agriculture and the Select Agent Program, in accordance with the Biosafety in Microbiological and Biomedical Laboratories guidelines54. The work was conducted under strict adherence to administrative controls and the use of personal protective equipment. Biosafety cabinets and secondary containment devices around animal caging were utilized for all work with infectious material. Wild-type viruses isolated and propagated from clinical samples were used for ferret inoculations, and all ferret samples collected in this study were destroyed upon completion of this work.

Virus stocks
Virus stocks of A/Colorado/137/2024 (CO/137), A/Washington/239/2024 (WA/239), A/British Columbia/PHL-2032/2024 (BC/PHL-2032), A/Michigan/90/2024 (MI/90), and A/Nebraska/14/2019 (NE/14) were propagated in MDCK cells at 37°C for 48 h. A/California/147/2024 (CA/147), A/Vietnam/1203/2004 (VN/1203), A/Anhui/1/2013 (Anhui/1), and A/Anhui-Lujiang/39/2018 (AL/39) were grown in the allantoic cavities of 10-day-old embryonated chicken eggs incubated at 37°C for 24–26 h; allantoic fluid from multiple eggs was pooled. Supernatants were clarified by centrifugation, aliquoted, and stored at −80°C55. Viral titers were determined by standard plaque assay, and full-genome sequencing was performed to confirm subtype identity and exclude contamination with other influenza strains.

Ferret experiments
All animal studies were conducted in accordance with protocols approved by the Centers for Disease Control and Prevention (CDC)’s Institutional Animal Care and Use Committee, at an AAALAC International-accredited facility. Fitch ferrets (Mustela putorius furo, Triple F Farms, Sayre, PA), aged 5–10 months, were used, and all animals were male, as sex-related variability has not been shown to affect data reproducibility across influenza risk assessment studies56. The animals were randomly assigned to experimental groups. Smaller animal groups were used for previously studied viruses, while larger groups were used for newly characterized viruses: NE/14 n=2, AL/39 n=3, Anhui/1 n=3, MI/90 n=3, VN/1203 n=3, CA/147 n=6, BC/PHL n=6, WA/239 n=15, and CO/137 n=15. The sample sizes were consistent with previously established protocols and published work57. Using fewer ferrets per virus allowed us to conduct a greater total number of experiments and evaluate multiple viruses within the constraints of ethical animal use. By grouping viruses according to their transmissibility phenotypes, we maintained sufficient sample sizes within each group to ensure robust statistical analyses. Animal studies were not repeated. Prior to study initiation, all ferrets were confirmed seronegative for circulating influenza A and B viruses by hemagglutination inhibition (HAI) assay.

Ferrets were housed individually in stainless steel cages (56×42×42 cm) with soft pellet bedding, placed inside Duo-Flo Bioclean mobile units (Lab Products Inc., Seaford, DE) that provided 150–180 air changes per hour. Ferrets were anesthetized before transponder placement, virus inoculation, nasal and rectal sample collection, and euthanasia. Anesthesia was administered intramuscularly using a combination of ketamine (10–30 mg/kg) and xylazine (1-2 mg/kg). Prior to inoculation, a subcutaneous temperature transponder (IPTT-300, BMDS) was inserted into the dorsal space between the scapulae of each animal. Baseline temperature and weight measurements were obtained one day prior to inoculation. Two to nine ferrets per group were intranasally inoculated with 6 log10 PFU of virus diluted in 1 mL PBS [CO/137 (n=9), CA/147 (n=3), WA/239 (n=9), BC/PHL (n=3), VN/1203 (n=3), MI/90 (n=3), Anhui/1 (n=3), AL/39 (n=3), NE/14 (n=2)]. To assess transmissibility of A(H5N1) viruses, uninfected (naïve) ferrets were introduced 24 hours post-inoculation at a 1:1 ratio. In the DCT model, naïve ferrets were co-housed with inoculated animals (3 pairs). In the RDT model, the ferret pairs were housed in adjacent cages separated by perforated walls that allowed airflow but prevented physical contact (3 pairs)26. Transmission in ferrets was determined by the detection of infectious virus and/or seroconversion. Nasal washes and rectal swabs were collected every 1–2 days from all inoculated and contact animals and stored at −80°C58. All ferrets were observed daily or twice-daily post-inoculation for clinical signs of infection. Animals exhibiting persistent severe illness (e.g., diarrhea, lethargy, labored breathing) were humanely euthanized under anesthesia via intracardiac administration of 1 mL/kg (390 mg pentobarbital sodium and 50 mg phenytoin sodium per mL). Blood samples were collected into BD Vacutainer Plastic Blood Collection Tubes with Lithium Heparin (Becton, Dickinson and Company) on the day of euthanasia to assess viremia stored at −80°C. Tissues were harvested from the euthanized animals to evaluate viral replication and systemic spread of the B3.13 and D1.1 A(H5N1) viruses. Three separate ferrets per group were used to assess systemic dissemination on day 3 p.i. for WA/239 and CO/137, following a timeline consistent with prior studies12,14,23,32. To minimize the number of animals used in this study, the systemic dissemination of CA/147 and BC/PHL was evaluated using euthanized donors from the RDT experiment on the day the animals reached humane endpoint criteria (days 4–5). The tissues were stored at −80°C and subsequently thawed, weighted, and homogenized using Precision Disposable Tissue Grinders (Covidien LLC). The homogenates were clarified by centrifugation at 4°C, and infectious virus titers were determined by standard plaque assay using MDCK cells, along with nasal wash, rectal swab, and whole blood samples.

Air sampling
Air samples were collected from ferrets inoculated with 6 log10 PFU of each virus: NE/14 (n=2), AL/39 (n=3), Anhui/1 (n=3), MI/90 (n=3), CO/137 (n=3), CA/147 (n=3; donors in RDT experiment in this study), WA/239 (n=3, donors in RDT experiment in this study), BC/PHL (n=3; donors in RDT experiment in this study), VN/1203 (n=3). For air collection, awake inoculated ferrets were individually placed in sanitized, ventilated plastic transport containers (23.9 L, solid-walled with a perforated lid)14. Air was sampled for 30–60 minutes using either the BC251 (National Institute for Occupational Safety and Health) or SPOT (Handix Scientific/Aerosol Devices) samplers (sampling durations and replicates are listed in Supplementary Table 1). The BC251 sampler collected air at 3.5 L/min and separated aerosols into >4 µm, 1–4 µm, and <1 µm fractions59. In this study, the >4 µm fraction was resuspended in 600 µL of PBS supplemented with 0.3% bovine serum albumin (BSA) and the 1–4 µm and <1 µm fractions were resuspended in 300 ul of PBS-BSA and pooled for virus quantification. However, most viral RNA and infectious particles were found in the >4 µm fraction, consistent with prior work18, and total combined titers are reported. The SPOT sampler operated at 1.5 L/min and collected air into 450 uL of PBS with 0.3% BSA kept on iced during collection (sampler settings: conditioner 4–5°C, initiator 39–40°C, moderator 21°C, nozzle 28°C). Following collection with either of the samplers, each air sample was split: 140 µL was inactivated in AVL buffer (Qiagen) and stored at −80°C for RNA quantification and the remaining volume was processed immediately by plaque assay to quantify infectious virus18. All titers were normalized to 105 L of air to account for differences in sampling time and flow rates. All sampling equipment and containers were disinfected with 70% ethanol, rinsed with water and air-dried after use.

Virus Quantification
For the detection and quantification of viral RNA copy numbers, total RNA was extracted from the samples using the RNeasy Mini Kit (Qiagen), with 140 µL of sample used for each extraction. Real time RT-PCR was conducted in duplicate using the SuperScript III Platinum One-Step qRT-PCR System (Invitrogen), employing a primer and probe set specific to the influenza A virus M1 gene18. The RNA copy numbers for the influenza virus M gene were determined by extrapolating from a standard curve created with samples of known M gene copy numbers. Infectious virus quantification was conducted using a standard plaque assay in MDCK cells55. After removal of 140 uL of sample for RNA analyses, the entire remaining volume of each air sample was plated onto multiple wells on a 6-well plate within 1–4 h of collection to prevent freezing (samples with suspected higher viral loads were diluted 10-fold). Titers for nasal washes, rectal swabs, homogenized tissues, and whole blood samples were determined using previously frozen samples. The limits of detection were as follows: 2.9 log10 RNA copies/mL of nasal wash, 2.5 log10 RNA copies/105 L of air, 10 PFU/mL or g of nasal wash or tissue, 1 PFU/105 L of air.

Hemagglutination inhibition assay
Blood was collected from surviving contact animals on day 21 post-challenge using a BD Vacutainer Rapid Serum Tube (Becton, Dickinson and Company) to assess seroconversion. Briefly, receptor-destroying enzyme (RDE) (Denka Seiken) treated and 10-fold prediluted sera were 2-fold serially diluted and incubated with 8 HAU of virus for 30 minutes before the addition of 1% (vol/vol) horse red blood cells60. HAI titers were expressed as the reciprocal of the highest dilution of serum that inhibited hemagglutination.

Next generation sequencing
Viral RNA was isolated from nasal wash samples using the QIAamp Viral RNA Mini Kit (Qiagen). Portions of the tissues collected from ferrets inoculated with a representative strain from each genotype, CO/137 (n=3) and WA/239 (n=3), were submerged in RNAprotect Tissue Reagent (Qiagen) and stored at 4°C. The tissues were weighed, placed in buffer RLT Plus (Qiagen), homogenized using an Omni Tissue Homogenizer (Omni International, Inc.), clarified by centrifugation, and viral RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). Whole-influenza-genome amplification was performed using universal Influenza A primers with SuperScript III One-Step RT-PCR with Platinum Taq high-fidelity polymerase kit (Invitrogen) following manufacturer’s instructions at half volume reaction size61. NGS libraries were prepared with Illumina Nextera DNA Flex kits following manufacturers instructions at half volume reaction size. Libraries were sequenced on Illumina iSeq 100 with paired-end 150-bp reads and v2 chemistry reagents. The Fastq files were trimmed using BBDuk and then assembled to the relevant reference genomes with Bowtie262 as implemented in Geneious Prime v2020.2.4, used an CO/137 and WA/239 stock virus sequences in which gene segments were concatenated, separated by a 50 bp “N” spacer. Variants were called with 100x sequence coverage and 5% frequency threshold. Sequencing data have been deposited in the NCBI Sequence Read Archive under BioProjectID: PRJNA1338708.

Statistical analyses and data presentation
All statistical analyses were performed on measurements taken from distinct samples in R v4.4.063. Area under the curve (AUC) values were calculated using infectious virus and viral RNA copy titers on days 1-3 using the DescTools v0.99.57 package64. Statistical assumptions for normality and homogeneity of variances were checked using the Shapiro-Wilk and Levene’s test respectively. Statistical group comparisons were performed using the non-parametric tests of Wilcoxon Rank Sum or Kruskal-Wallis with Dunn’s multiple comparisons (rstatix v0.7.265) using Holm’s method for p-value adjustment, as specified in the results section. Correlations were performed using Pearson’s method with 95% confidence intervals. Pairwise comparisons were conducted using the Kruskal-Wallis test with Dunn’s multiple comparisons post hoc analysis. AUC data for infectious virus and RNA was used separately to perform logistic regression modeling for a binary transmission outcome with 95% confidence intervals. A linear mixed-effects model analyses were performed using data restricted to days 1–3 and structured with one record per ferret per day. Models were fit separately for infectious virus and genomic RNA. Mixed models were fit using lme4 v1.1-35.566 with restricted maximum likelihood (REML), the bobyqa optimizer, and an increased iteration limit. We assessed model stability and diagnostics with lme4::isSingular and performance v0.13.067. Day-specific slopes of NW on air were obtained from emmeans v1.10.468, and differences between day-specific slopes were evaluated with Tukey-adjusted pairwise comparisons. Degrees of freedom and tests for fixed effects and emmeans estimates used Kenward–Roger approximations provided by lmerTest v3.1-369. Figures were created in R using tidyverse v2.0.070, ggplot2 v3.5.171, ggpubr v0.6.072 and patchwork v1.3.073 or GraphPad Prism v10.5.0.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study was conducted by an experienced influenza reference laboratory using the ferret model, the gold standard for risk assessment of emerging B3.13 and D1.1 H5N1 viruses. It provides crucial insights into the potential mammalian transmissibility of these newly emerging H5N1 viruses, particularly the D1.1 variant, which had not previously been assessed in ferrets. Importantly, the authors also employed aerosol sampling using two different air collection platforms and expanded their analysis to include additional H5N1, H9N2, H7N9, and pandemic H1N1 2009 strains with known airborne transmissibility profiles. They provided a detailed analysis of air emission profiles from highly transmissible human seasonal influenza viruses, as well as low and non-transmissible influenza viruses isolated from human cases. The results show that although none of the H5N1 strains transmitted via the air, B3.13 viruses were detected at significantly higher levels compared to D1.1 strains. Strong correlations were observed between viral loads in nasal washes, airborne virus shedding, and transmissibility potential.\nMain Concerns:\n1. In this study, ferrets were inoculated intranasally with a high dose of 10⁶ PFU. While we appreciate that this ensures all ferrets are infected, virus shedding in experimentally infected ferrets may differ from that in real-world settings. For example, in Figure 1, the viral shedding profile in nasal wash samples differed between directly inoculated ferrets and direct-contact ferrets. Since viral replication in directly inoculated ferrets reached peak titers on days 1 and 2, corresponding to higher virus emission in the air, this may not reflect the situation in animals infected with a lower dose. It would be interesting to collect air samples from ferrets infected via direct contact. These ferrets may exhibit different immune response dynamics compared to donor ferrets, which could affect the RNA/PFU ratio, peak viral load, duration, and total amount of virus emitted into the air, as well as onward transmission. If additional animal experiments are not feasible, the authors should discuss these limitations in the Discussion.\n2. In the B.3.13 study, significant amounts of infectious virus and viral RNA were detected in the air from directly inoculated B3.13 ferrets. However, no airborne transmission occurred in respiratory contact ferrets. Compared to highly transmissible influenza viruses, low and non-transmissible influenza viruses may have a much higher ID₅₀, which should be taken into account. While determining the ID₅₀ would require many animals and conflict with the 3Rs principles, this point should be discussed in the Discussion section.\n3. The area under the curve (AUC) of nasal wash and air samples was used to calculate the correlation between influenza viral loads in nasal wash samples and air samples. The authors also compared viral loads from individual days between nasal wash and air samples. It would be helpful to analyse the data using a repeated measures model with data from days 1–3 individually, since the same animals underwent daily nasal wash sampling, which creates multiple repeated measurements within an individual animal.\n4. Did the authors sequence the viruses from the nasal wash samples and those collected by the air samplers? Are there any genetic differences?\nOthers:\nThe comparison between the two air samplers is very useful and should be published as a reference for other studies using air samplers, such as surveillance and animal studies. For the comparison, data were normalized to facilitate comparison between the two samplers. However, if raw data are compared directly, is the NIOSH sampler still better than SPOT?\nLines 313–314: Can the authors add the temperature data? Did virus emission occur before or after fever?", "reviewer_2": "In the proposed manuscript, the authors compared the pathogenesis and airborne virus shedding of the clade 2.3.4.4b H5N1 virus genotypes D3.13 and D1.1 in ferrets. These two virus genotypes have recently been found to cause human infections and are widely circulating in wild birds and non-human mammals, raising major concerns about their pandemic potential. In addition to assessing and comparing the pathogenesis and viral shedding with other less and more transmissible influenza viruses, the authors compared the efficiency of two aerosol collectors. Finally, the authors explored the correlation between airborne virus transmission and infectious virus titers and RNA levels detected in air samples.\nWhile the study is well-designed with a strong selection of controls, the manuscript drastically lacks clarity in both the text and data representation. Notably, important data, collected by the authors (rectal swabs, body weight, clinical scoring (e.g. nasal discharge), temperature) need to be presented, especially given the severity of disease caused by the H5N1 viruses.\nThe Materials and Methods section is lacking key experimental details to be able to reproduce the study by others, for example how viral RNA was quantified, how and when the HI’s were done on ferret sera, how was virus cultured from blood samples (was any pre-treatment of samples needed? Was whole blood or serum/plasma used, and on which days) etc.\nThe results section is difficult to navigate, and some conclusions regarding the overall generalization of D1.1 and D3.13 airborne transmission need to be toned down. Notably, some parts of the results or discussion contradict each other. Line 111/113 (results section):” Notably, earlier B3.13 viruses were capable of limited airborne transmission, suggesting both intra- and inter-genotypic variability in transmission phenotypes”, line 226/227 (discussion section): ”B3.13 viruses are likely not evolving toward a more transmissible phenotype”, line 244/246 (discussion section): ”This suggests a higher estimated likelihood of airborne transmission for B3.13 viruses compared to D1.1 and indicates that their potential for airborne spread should not be dismissed, underscoring the need for continued monitoring of viruses within this genotype”. This final statement is confusing, as no transmission was observed between ferrets with either H5N1 genotype in the presented studies (or in humans). Therefore, it is unclear how the B3.13 viruses can be described as having a higher transmission potential when no transmission was actually detected.\nThe representation of data is complex and, therefore difficult to interpret. For example, Figure 1 does not clearly indicate the relationship between ferret pairs. Please combine donor-recipient pairs in individual panels for each virus. In Figure 1, panel E, it is very difficult to understand which ferrets the data come from. Are these new ferrets from which only tissue samples were collected, without nasal washes, temperature measurements, or weight loss data? The ferret’s group design needs to be better explained and made more easily identifiable in the manuscript. Please also increase the size of the asterisks and ‘d4’ labels.\nThe correlation between influenza virus loads in nasal washes and air samples was only done for the first three days. Why was day 5 not included? The data is available for almost all animals.\nLine 212/213: How is transmission defined? Usually, it is confirmed by the presence of virus replication in the nasal washes, but here it is based on ‘signs of infection’. And what about the other ferret - I don’t think it was the same ferret - that seroconverted?\nOne of the key investigations in the manuscript is the comparison of air sampler efficiency. However, it is surprising that this point is not presented in the title, abstract, or even introduction. We recommend that the authors emphasize their air sampler comparison earlier, rather than only in the results section. Additionally, we advise the authors to present the air sampler comparison in a dedicated paragraph rather than in the ‘Airborne virus shedding by influenza virus-infected ferrets’ section, as this would greatly improve clarity.\nFinally, this study is similar to previous work done by the authors (ref 36). While this manuscript presents novelty in the virus genotypes used and the utilization of the SPOT air sampler in addition to the BC251 air sampler, we advise the authors to clearly place this work in perspective with their previous findings.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "In this manuscript, Pulit-Penaloza et al. used the ferret model to evaluate the pathogenicity, transmission, and aerosol shedding of human-derived A(H5N1) viruses from two genotypes (B3.13 and D1.1). They also applied aersosol sampling and included additional influenza strains (A(H5N1), A(H9N2), A(H1N1)pdm09) with known transmissibility profiles to improve methods for pandemic risk assessment.\nOverall, this is a well-executed study and a clearly written manuscript.\nHowever, a few minor issues need to be addressed that will improve the manuscript.\n1. While the nomenclature is overall correct, the presentation of the four isolates could be made clearer by consistently incorporating their genotype information throughout the text, which would help readers follow the experimental comparisons more easily.\n2. Separating Figure 1A–D to align more closely with the narrative in the Results section would make the text easier to follow, as the current layout is somewhat difficult to track while reading.\n3. Providing the abbreviations ‘DCT’ and ‘RDT’ in the Results section corresponding to Figure 1A–D would improve clarity for the reader.\n4. Consider using the same visualization style as in Figure 3, where virus isolates are stratified by transmissibility, to make Figure 2 easier to interpret.\n5. It seems like that Figure 5 is mislabeled. The authors refer to Figure 5C, which does not exist."}, "author_response": "Thank you for your valuable critiques and input; the authors sincerely appreciate the feedback. We are pleased to receive many positive comments, particularly regarding the significance of this work and the interest in our air sampling methods and findings. We agree that all the points raised are important considerations to better understand the multifactorial nature of transmission. We have responded to each comment point by point and made extensive additions to the manuscript as detailed in the responses below. The line numbers refer to specific locations in the clean copy of the resubmitted manuscript. A copy of the manuscript containing tracked changes is also available for review. We believe these changes have significantly enhanced the quality and readability of the manuscript. We hope these improvements meet the reviewers' expectations and that the manuscript is now suitable for publication in Nature Communications. Thank you once again for your time and expert input.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis study was conducted by an experienced influenza reference laboratory using the ferret model, the gold standard for risk assessment of emerging B3.13 and D1.1 H5N1 viruses. It provides crucial insights into the potential mammalian transmissibility of these newly emerging H5N1 viruses, particularly the D1.1 variant, which had not previously been assessed in ferrets. Importantly, the authors also employed aerosol sampling using two different air collection platforms and expanded their analysis to include additional H5N1, H9N2, H7N9, and pandemic H1N1 2009 strains with known airborne transmissibility profiles. They provided a detailed analysis of air emission profiles from highly transmissible human seasonal influenza viruses, as well as low and non-transmissible influenza viruses isolated from human cases. The results show that although none of the H5N1 strains transmitted via the air, B3.13 viruses were detected at significantly higher levels compared to D1.1 strains. Strong correlations were observed between viral loads in nasal washes, airborne virus shedding, and transmissibility potential.\nMain Concerns:\n1.In this study, ferrets were inoculated intranasally with a high dose of 10⁶ PFU. While we appreciate that this ensures all ferrets are infected, virus shedding in experimentally infected ferrets may differ from that in real-world settings. For example, in Figure 1, the viral shedding profile in nasal wash samples differed between directly inoculated ferrets and direct-contact ferrets. Since viral replication in directly inoculated ferrets reached peak titers on days 1 and 2, corresponding to higher virus emission in the air, this may not reflect the situation in animals infected with a lower dose. It would be interesting to collect air samples from ferrets infected via direct contact. These ferrets may exhibit different immune response dynamics compared to donor ferrets, which could affect the RNA/PFU ratio, peak viral load, duration, and total amount of virus emitted into the air, as well as onward transmission. If additional animal experiments are not feasible, the authors should discuss these limitations in the Discussion.\nResponse: One of the rationales for this study was to characterize four new H5N1 viruses isolated from humans and to generate data for risk assessment (IRAT) using protocols that enable comparisons with previous studies. To achieve this, the inoculation dose was set at 10^6 PFU to align with standard protocols. However, we acknowledge the value of conducting further studies with lower, more physiologically relevant doses and/or sampling from contact ferrets to better understand the kinetics and magnitude of strain-specific shedding under varying conditions. Currently, no alternative dose has been recognized as a standard that is considered most physiologically relevant. Ideally, to ensure that all the ferrets are infected following inoculation with a lower dose, the ferret infectious dose 50 would need to be established for all tested viruses. As the reviewer noted, this would require a substantial number of animals and resources, making it difficult to test a large panel of viruses. To acknowledge these considerations, we have added a paragraph to the discussion (lines 364-370). We also reference studies that enhance our understanding of how dose-dependent ferret inoculations can influence the kinetics and magnitude of shedding. We believe that our comparative analyses using a larger panel of viruses to identify trends in shedding in both nasal washes and air at a fixed dose (ensuring robust infection across strains) provide a solid framework for follow-up studies exploring the feasibility of testing airborne virus shedding following lower inoculation doses.\n2. In the B.3.13 study, significant amounts of infectious virus and viral RNA were detected in the air from directly inoculated B3.13 ferrets. However, no airborne transmission occurred in respiratory contact ferrets. Compared to highly transmissible influenza viruses, low and non-transmissible influenza viruses may have a much higher ID₅₀, which should be taken into account. While determining the ID₅₀ would require many animals and conflict with the 3Rs principles, this point should be discussed in the Discussion section.\nResponse: Thank you for your valuable feedback. We agree that this is an important consideration, closely related to the concerns raised in comment 1. We recognize that conducting these experiments would indeed require a large number of animals, which is a significant reason why this area remains understudied in the field. While we were unable to include these additional ferret groups in our study due to the required resources, we referenced studies that support this line of inquiry and highlighted the need for future investigations into the relationship between airborne influenza shedding and the airborne infectious dose 50 in ferrets. We also note that by evaluating ID50 (lines 310-313), this research may provide further insights into the airborne transmissibility of the newly emerging A(H5N1) viruses, particularly regarding the differences between transmissible and non-transmissible B3.13 genotype viruses. We believe that these additions strengthen the manuscript by more clearly discussing the findings and acknowledging the remaining gaps in knowledge.\n3. The area under the curve (AUC) of nasal wash and air samples was used to calculate the correlation between influenza viral loads in nasal wash samples and air samples. The authors also compared viral loads from individual days between nasal wash and air samples. It would be helpful to analyse the data using a repeated measures model with data from days 1–3 individually, since the same animals underwent daily nasal wash sampling, which creates multiple repeated measurements within an individual animal.\nResponse: We acknowledge that there are various methods to analyze this data set. As requested, we utilized linear mixed-effects analyses, which revealed a positive association on day 3 but not on days 1 and 2. Corresponding updates have been made in the Results section (lines 220–225) and the Methods section (lines 488-492). The R code has also been updated on GitHub. Given that this comparative study involves distinct influenza strains and subtypes with significant heterogeneity in host adaptation, frequently reflected in virus shedding kinetics, we consider per-day comparisons to be a weak primary measure. We believe that AUC analysis provides a more robust and reliable assessment of the relationship between nasal wash viral load and airborne virus shedding across diverse viruses.\n4. Did the authors sequence the viruses from the nasal wash samples and those collected by the air samplers? Are there any genetic differences?\nResponse: We performed additional experiments to sequence nasal wash and tissue samples from ferrets infected with representative strains of the B3.13 and D1.1 genotypes (CO/137 and WA/239), to assess potential viral evolution. The data have been deposited in the NCBI Sequence Read Archive (BioProject ID: PRJNA1338708), and relevant text has been added (lines 116-123). No genetic changes at known mammalian adaptation markers or consistent variant patterns were detected. These results align with previous reports indicating no emergence of mammalian-adaptive variants in ferrets infected with the A/TX/37/2024 2.3.4.4b H5N1 virus. Air samples could not be sequenced due to low viral loads in most samples and workflow limitations. All air samples (the entire volume) were used for plaque assays, leaving no material for further RNA extraction and troubleshooting of low-titer samples by NGS. We have revised the Discussion to acknowledge this limitation and emphasize the need for studies using improved methods to facilitate sequencing of air samples (lines 327-334).\nOthers:\nThe comparison between the two air samplers is very useful and should be published as a reference for other studies using air samplers, such as surveillance and animal studies. For the comparison, data were normalized to facilitate comparison between the two samples. However, if raw data are compared directly, is the NIOSH sampler still better than SPOT?\nResponse: We have expanded the Methods section to provide a clearer description of the procedures used. We carefully considered how to compare the samplers, given that they draw air at different rates. The SPOT sampler draws 1.5 L/min, versus 3.5 L/min for the NIOSH sampler (2.33-fold difference). While normalized data could be expressed as a value per minute or hour, our normalized data are presented as a value per liter of air. To normalize the data, the SPOT sampler values were multiplied by 2.33 to align with those of the NIOSH sampler. Without this normalization, the SPOT sampler would have values 2.33-times lower than those presented here. Consequently, the SPOT sampler collects fewer particles than the NIOSH sampler and has a lower collection efficiency. We have added additional text to clarify this point (lines 191-193).\nLines 313–314: Can the authors add the temperature data? Did virus emission occur before or after fever?\nResponse: The peak emission occurred concurrently with or after the peak in temperature. We have added Supplementary Figure 1A and 1B to show the weight and temperature data. Additionally, the mean peak temperature values and the median day of peak occurrence are now presented in Supplementary Table 5.\nReviewer #2 (Remarks to the Author):\nIn the proposed manuscript, the authors compared the pathogenesis and airborne virus shedding of the clade 2.3.4.4b H5N1 virus genotypes D3.13 and D1.1 in ferrets. These two virus genotypes have recently been found to cause human infections and are widely circulating in wild birds and non-human mammals, raising major concerns about their pandemic potential. In addition to assessing and comparing the pathogenesis and viral shedding with other less and more transmissible influenza viruses, the authors compared the efficiency of two aerosol collectors. Finally, the authors explored the correlation between airborne virus transmission and infectious virus titers and RNA levels detected in air samples.\nWhile the study is well-designed with a strong selection of controls, the manuscript drastically lacks clarity in both the text and data representation.\nNotably, important data, collected by the authors (rectal swabs, body weight, clinical scoring (e.g. nasal discharge), temperature) need to be presented, especially given the severity of disease caused by the H5N1 viruses.\nResponse: Thank you for this comment. The clinical signs, including mean maximum temperature (including the median peak day), weight change from the baseline, nasal discharge, diarrhea, and viremia are presented in Table 2. We have added Supplementary Fig. 1A and B to show weight and temperature change kinetics, and we also added Supplementary Fig. 2 A-F to show the time course for rectal swab titers.\nThe Materials and Methods section is lacking key experimental details to be able to reproduce the study by others, for example how viral RNA was quantified, how and when the HI’s were done on ferret sera, how was virus cultured from blood samples (was any pre-treatment of samples needed Was whole blood or serum/plasma used, and on which days) etc.\nResponse: We have made extensive revisions to the Methods section (with tracked changes) to include more detailed information, and we now provide all the requested information. We are happy to hear that our methods could serve as a valuable resource for others.\nThe results section is difficult to navigate, and some conclusions regarding the overall generalization of D1.1 and D3.13 airborne transmission need to be toned down. Notably, some parts of the results or discussion contradict each other. Line 111/113 (results section):” Notably, earlier B3.13 viruses were capable of limited airborne transmission, suggesting both intra- and inter-genotypic variability in transmission phenotypes”, line 226/227 (discussion section): ”B3.13 viruses are likely not evolving toward a more transmissible phenotype”, line 244/246 (discussion section): ”This suggests a higher estimated likelihood of airborne transmission for B3.13 viruses compared to D1.1 and indicates that their potential for airborne spread should not be dismissed, underscoring the need for continued monitoring of viruses within this genotype”. This final statement is confusing, as no transmission was observed between ferrets with either H5N1 genotype in the presented studies (or in humans). Therefore, it is unclear how the B3.13 viruses can be described as having a higher transmission potential when no transmission was detected.\nResponse: Thank you for pointing out these inconsistencies, your feedback is very helpful. We have removed lines 226-227 and 244-246 to avoid confusion and to prevent generalizations about virus evolution, especially given the mixed phenotypes observed. We also toned down our conclusions throughout the manuscript.\nThe representation of data is complex and, therefore difficult to interpret. For example, Figure 1 does not clearly indicate the relationship between ferret pairs. Please combine donor-recipient pairs in individual panels for each virus. In Figure 1, panel E, it is very difficult to understand which ferrets the data come from. Are these new ferrets from which only tissue samples were collected, without nasal washes, temperature measurements, or weight loss data? The ferret’s group design needs to be better explained and made more easily identifiable in the manuscript. Please also increase the size of the asterisks and ‘d4’ labels.\nResponse: As requested, we have combined donor-recipient pairs in individual panels for each virus and increased the size of the asterisks and d4 labels in Figure 1. The ferrets in Figure 1 G (previously 1E) represent 6 new ferrets inoculated with CO/137 and WA/239 (for the sole purpose of examining day 3 tissue titers, as performed in previous publications on other H5N1 strains), while the ferrets inoculated with CA/147 and BC/PHL are the same ferrets as the donors in Fig 1C and F (from which tissues were collected when these ferrets reached humane endpoint criteria). We revised the Figure 1 caption for better readability and updated the color scheme.\nThe correlation between influenza virus loads in nasal washes and air samples was only done for the first three days. Why was day 5 not included? The data is available for almost all animals.\nResponse: We limited our analyses to days 1-3 because some virus groups lacked day 5 data, either due to animals meeting humane endpoint criteria or because the data were collected on day 4 instead of day 5. Including incomplete day 5 data could skew the analyses and lead to inaccurate conclusions. We have added text to acknowledge this experimental choice in lines 209, 359-364.\nLine 212/213: How is transmission defined? Usually, it is confirmed by the presence of virus replication in the nasal washes, but here it is based on ‘signs of infection’. And what about the other ferret - I don’t think it was the same ferret - that seroconverted?\nResponse: Thank you for your feedback. We have clarified the definition of transmission throughout the study, emphasizing that it is not based on signs of infection. In this study, transmission is defined by virus detection in any specimen collected and/or seroconversion. We updated the Methods section (lines 413-314) and the caption of Table 1 for clarification, and we further refined the Discussion to better describe which ferrets seroconverted (lines 275-279). One WA/239 ferret had virus detected in a rectal swab sample and, due to severe illness, required euthanasia; we subsequently collected tissues and detected infectious virus in the lower respiratory tract. The second ferret seroconverted without viral detection and survived, so we were unable to test its tissues.\nOne of the key investigations in the manuscript is the comparison of air sampler efficiency. However, it is surprising that this point is not presented in the title, abstract, or even introduction. We recommend that the authors emphasize their air sampler comparison earlier, rather than only in the results section. Additionally, we advise the authors to present the air sampler comparison in a dedicated paragraph rather than in the ‘Airborne virus shedding by influenza virus-infected ferrets’ section, as this would greatly improve clarity.\nNeeds revision of text and maybe supp fig.\nResponse: Thank you for your valuable suggestion. We agree that it is important to present the sampler comparison more effectively. Due to word limit, we were unable to include additional detail in the title, and for the same reason, we could only make minor adjustments in the abstract (we now specify the types of samplers in the abstract). We have significantly expanded the introduction (lines 56-77) and revised the section titled “Airborne Virus Shedding by Influenza Virus-Infected Ferrets” to “Comparative Evaluation of Airborne Virus Shedding Using BC251 and SPOT Samplers,” dedicating that section to a detailed comparison of the samplers. We have two figures and one table focused on sampler comparisons: Figure 3, Supplementary Figure 3, Supplementary Table 2.\nFinally, this study is similar to previous work done by the authors (ref 36). While this manuscript presents novelty in the virus genotypes used and the utilization of the SPOT air sampler in addition to the BC251 air sampler, we advise the authors to clearly place this work in perspective with their previous findings.\nResponse: We have added an additional paragraph to the Introduction to clarify how this current research builds on previous findings (lines 56-77, 202-207) and to provide more context regarding the importance and challenges of aerosol sampling, as requested in comment 7.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nIn this manuscript, Pulit-Penaloza et al. used the ferret model to evaluate the pathogenicity, transmission, and aerosol shedding of human-derived A(H5N1) viruses from two genotypes (B3.13 and D1.1). They also applied aersosol sampling and included additional influenza strains (A(H5N1), A(H9N2), A(H1N1)pdm09) with known transmissibility profiles to improve methods for pandemic risk assessment.\nOverall, this is a well-executed study and a clearly written manuscript.\nHowever, a few minor issues need to be addressed that will improve the manuscript.\n1. While the nomenclature is overall correct, the presentation of the four isolates could be made clearer by consistently incorporating their genotype information throughout the text, which would help readers follow the experimental comparisons more easily.\nResponse: We made additions throughout the text to more consistently incorporate their genotype information.\n2. Separating Figure 1A–D to align more closely with the narrative in the Results section would make the text easier to follow, as the current layout is somewhat difficult to track while reading. ?\nResponse: We have edited the figure to separate the data on direct contact and respiratory transmission for improved clarity. Additionally, we updated the color scheme to facilitate tracking of individual ferrets. We also made edits to the results text and the figure caption for better readability.\n3. Providing the abbreviations ‘DCT’ and ‘RDT’ in the Results section corresponding to Figure 1A–D would improve clarity for the reader.\nResponse: We clarified the abbreviations and provided additional background information to enhance readability. Lines 126-128, 147-150.\n4. Consider using the same visualization style as in Figure 3, where virus isolates are stratified by transmissibility, to make Figure 2 easier to interpret.\nResponse: We have made changes to Figure 2 as suggested. The viruses are now ordered and grouped by their transmissibility profiles to ensure that the figure formatting aligns exactly with that of Figure 3.\n5. It seems like that Figure 5 is mislabeled. The authors refer to Figure 5C, which does not exist.\nResponse: Thank you for bringing this error to our attention. We have corrected the figure labels."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all issues satisfactorily", "reviewer_2": "I’m very pleased with the authors’ responses to the comments and think that the manuscript has significantly improved compared to the initial submission. I only have some minor issues:\nLine 422: remove space after viruses.\nTable 1: The superscript associated with column 4 (named ‘Exposure source') should be a not b.\nThe superscript associated with columns 5 and 6 (named ‘DCT’ and ‘RDT’) should be b not c.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed my earlier concerns, and I find their revisions and responses satisfactory."}, "author_response": "Thank you for re-reviewing the manuscript. We are pleased that the changes we made are satisfactory.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all issues satisfactorily\nResponse: Thank you.\nReviewer #2 (Remarks to the Author):\nI’m very pleased with the authors’ responses to the comments and think that the manuscript has significantly\nimproved compared to the initial submission. I only have some minor issues:\nLine 422: remove space after viruses.\nTable 1: The superscript associated with column 4 (named ‘Exposure source') should be a not b.\nThe superscript associated with columns 5 and 6 (named ‘DCT’ and ‘RDT’) should be b not c.\nResponse: All typos were corrected as requested. Thank you.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my earlier concerns, and I find their revisions and responses satisfactory.\nResponse: Thank you."}]

Decision letter