Influenza A(H5N1) viruses have been detected in US dairy cow herds since 2024. We assessed the pathogenesis, transmission, and airborne release of A/Michigan/90/2024, an H5N1 isolate from a dairy farm worker in Michigan, in the ferret model. Results show this virus caused airborne transmission with moderate pathogenicity, including limited extrapulmonary spread, without lethality.

Reports of human infections with influenza A(H5N1) clade 2.3.4.4b viruses associated with outbreaks in dairy cows in the United States underscore the need to assess the potential cross-protection conferred by existing influenza immunity. We serologically evaluated ferrets previously infected with an influenza A(H1N1)pdm09 virus for cross-reactive antibodies and then challenged 3 months later with either highly pathogenic H5N1 clade 2.3.4.4b or low pathogenicity H7N9 virus. Our results showed that prior influenza A(H1N1)pdm09 virus infection more effectively reduced the replication and transmission of the H5N1 virus than did the H7N9 virus, a finding supported by the presence of group 1 hemagglutinin stalk and N1 neuraminidase antibodies in preimmune ferrets. Our findings suggest that prior influenza A(H1N1)pdm09 virus infection may confer some level of protection against influenza A(H5N1) clade 2.3.4.4.b virus.

BACKGROUND: The human eye represents a potential site of influenza A virus (IAV) replication, and an entry point for the virus to reach the respiratory tract. The frequent detection of conjunctivitis among farm workers with confirmed infection with clade 2.3.4.4b A(H5N1) IAV from this ongoing outbreak represents an atypical disease presentation for this virus subtype. We aimed to investigate whether the occurrence of ocular complications reported following clade 2.3.4.4b A(H5N1) virus infection was associated with an enhanced capacity of this virus to replicate in mammalian ocular tissue and cause infection following ocular exposure. METHODS: Primary human nasal and corneal tissue constructs were infected with A(H5N1) A/Texas/37/2024 (Texas/37), A(H1N1)pdm09 A/Nebraska/14/2019 (Neb/14), and A(H7N7) A/Netherlands/219/2003 (NL/219) viruses (multiplicity of infection [MOI] of 0·01-0·02, 33°C). Corneal tissue constructs were also infected with an expanded panel of IAVs (Texas/37, A[H5N1] A/Michigan/90/2024 [MI/90], A[H5N1] A/Chile/25945/2023 [Chile/25945], NL/219, A/Netherlands/230/2003 [NL/230], and Neb/14; MOI of 0·01, 37°C). In-vitro infections of tissue constructs were used to assess replication kinetics by infectious virus titration. Induction of innate host antiviral responses in infected corneal tissue constructs was assessed by PCR array (MOI of 2·00, 37°C). Ferrets (serologically naive or pre-immune to A[H1N1]pdm09 virus) were inoculated by the ocular route with Texas/37 A(H5N1) virus-using a liquid inoculum (10⁶ plaque forming units [PFU]), aerosol inhalation (15-16 PFU), or ocular-only aerosol exposure (18-132 PFU)-to assess pathogenicity and tropism of the virus following different exposure routes. Transmissibility was assessed by placing serologically naive or pre-immune ferrets inoculated by ocular-only aerosol exposure in direct contact with serologically naive ferrets, monitoring pathogenicity in contact animals, and measuring viral titres in nasal washes of both inoculated and contact ferrets. FINDINGS: Nasal and corneal tissue constructs supported replication of all IAVs tested. In corneal tissue constructs, A(H7N7) and A(H1N1)pdm09 viruses reached 10-fold higher overall titres than A(H5N1) isolates. Relatively few genes (n=13) related to antiviral responses were significantly differentially expressed in corneal tissue constructs infected with IAV, with no consistent differential expression among clade 2.3.4.4b A(H5N1) viruses associated with either conjunctivitis or severe respiratory disease, although strain-specific differences were observed. Serologically naive ferrets inoculated by liquid ocular, aerosol inhalation, or aerosol-only ocular routes with Texas/37 virus exhibited a systemic and fatal infection in all animals, transmitting the virus to naive cagemates. By contrast, reduced disease severity following ocular-only aerosol inoculation was observed in animals with pre-existing heterosubtypic immunity. No serologically naive ferrets placed in direct contact with pre-immune ferrets inoculated with Texas/37 virus by the ocular-only aerosol route became infected. INTERPRETATION: A clade 2.3.4.4b A(H5N1) virus from the dairy cattle outbreak in the USA that was first detected in March, 2024, does not appear to possess features indicative of an ocular tropism. However, this virus can maintain a virulent and transmissible phenotype in ferrets following ocular exposure, highlighting the importance of ocular protection. FUNDING: US Centers for Disease Control and Prevention.

Although assessments of influenza A virus transmissibility in the ferret model play a critical role in pandemic risk evaluations, few studies have investigated which virological data collected from virus-inoculated animals are most predictive of subsequent virus transmission to naïve contacts. We compiled viral titer data from >475 ferrets inoculated with 97 contemporary IAV (including high- and low-pathogenicity avian, swine-origin, and human viruses of multiple HA subtypes) that served as donors for assessments of virus transmission in the presence of direct contact (DCT) or via respiratory droplets (RDT). A diversity of molecular determinants, clinical parameters, and infectious titer measurements and derived quantities were examined to identify which metrics were most statistically supported with transmission outcome. Higher viral loads in nasal wash (NW) specimens were strongly associated with higher transmission frequencies in DCT, but not RDT models. However, viruses that reached peak titers in NW specimens early (day 1 p.i.) were strongly associated with higher transmission in both models. Interestingly, viruses with 'intermediate' transmission outcomes (33-66%) had NW titers and derived quantities more similar to non-transmissible viruses (<33%) in a DCT setting, but with efficiently transmissible viruses (>67%) in a RDT setting. Machine learning was employed to further assess the predictive role of summary measures and varied interpretation of intermediate transmission outcomes in both DCT and RDT models, with models employing these different thresholds yielding high performance metrics against both internal and external datasets. Collectively, these findings suggest that higher viral load in inoculated animals can be predictive of DCT outcomes, whereas the timing of when peak titers are detected in inoculated animals can inform RDT outcomes. Identification that intermediate transmission outcomes should be contextualized relative to the transmission mode assessed provides needed refinement towards improving interpretation of ferret transmission studies in the context of pandemic risk assessment.

A better understanding of viral factors that contribute to influenza A virus (IAV) airborne transmission is crucial for pandemic preparedness. A limited capacity for airborne transmission was recently observed in a human A(H9N2) virus isolate (A/Anhui-Lujiang/39/2018, AL/39) that possesses a leucine (L) residue at position HA1-226 (H3 numbering), indicative of human-like receptor binding potential. To evaluate the roles of the residue at this position in virus fitness and airborne transmission, a wild-type AL/39 (AL/39-wt) and a mutant virus (AL/39-HA1-L226Q) with a single substitution at position HA1-226 from leucine to glutamine (Q), a consensus residue in avian influenza viruses, were rescued and assessed in the ferret model. The AL/39-HA1-L226Q virus lost the ability to transmit by air, although the virus had a comparable capacity for replication, induced similar levels of host innate immune responses, and was detected at comparable levels in the air surrounding the inoculated ferrets relative to AL/39-wt virus. However, ferrets showed a lower susceptibility to AL/39-HA1-L226Q virus infection compared to the AL/39-wt virus. Furthermore, the AL/39-wt and AL/39-HA1-L226Q viruses each gained dominance in different anatomic sites in the respiratory tract in a co-infection competition model in ferrets. Taken together, our findings demonstrate that the increasing dominance of HA1-L226 residue in an avian A(H9N2) virus plays multifaceted roles in virus infection and transmission in the ferret model, including improved virus fitness and infectivity. IMPORTANCE: Although the capacity for human-like receptor binding is a key prerequisite for non-human origin influenza A virus (IAV) to become airborne transmissible in mammalian hosts, the underlying molecular basis is not well understood. In this study, we investigated a naturally occurring substitution (leucine to glutamine) at residue 226 in the HA of an avian-origin A(H9N2) virus and assessed the impact on virus replication and airborne transmission in the ferret model. We demonstrate that the enhanced airborne transmission associated with the HA1-L226 virus was mainly due to the increased infectivity of the virus. Interestingly, we found that, unlike most sites in the ferret respiratory tract, ferret ethmoid turbinate lined with olfactory epithelium favors replication of the AL/39-HA1-L226Q virus, suggesting that this site may serve as a unique niche for IAV with avian-like receptor binding specificity to potentially allow the virus to spread to extrapulmonary tissues and to facilitate adaptation of the virus to human hosts.

Since 2020, there has been unprecedented global spread of highly pathogenic avian influenza A(H5N1) in wild bird populations with spillover into a variety of mammalian species and sporadically humans(1). In March 2024, clade 2.3.4.4b A(H5N1) virus was first detected in dairy cattle in the U.S., with subsequent detection in numerous states(2), leading to over a dozen confirmed human cases(3,4). In this study, we employed the ferret model, a well-characterized species that permits concurrent investigation of viral pathogenicity and transmissibility(5) in the evaluation of A/Texas/37/2024 (TX/37) A(H5N1) virus isolated from a dairy farm worker in Texas(6). Here, we show that the virus has a remarkable ability for robust systemic infection in ferrets, leading to high levels of virus shedding and spread to naïve contacts. Ferrets inoculated with TX/37 rapidly exhibited a severe and fatal infection, characterized by viremia and extrapulmonary spread. The virus efficiently transmitted in a direct contact setting and was capable of indirect transmission via fomites. Airborne transmission was corroborated by the detection of infectious virus shed into the air by infected animals, albeit at lower levels compared to the highly transmissible human seasonal and swine-origin H1 subtype strains. Our results show that despite maintaining an avian-like receptor binding specificity, TX/37 displays heightened virulence, transmissibility, and airborne shedding relative to other clade 2.3.4.4b virus isolated prior to the 2024 cattle outbreaks(7), underscoring the need for continued public health vigilance.

The sporadic occurrence of human infections with swine-origin influenza A(H3N2) viruses and the continual emergence of novel A(H3N2) viruses in swine herds underscore the necessity for ongoing assessment of the pandemic risk posed by these viruses. Here, we selected 3 recent novel swine-origin A(H3N2) viruses isolated between 2017 to 2020, bearing hemagglutinins from the 1990.1, 2010.1, or 2010.2 clades, and evaluated their ability to cause disease and transmit in a ferret model. We conclude that despite considerable genetic variances, all 3 contemporary swine-origin A(H3N2) viruses displayed a capacity for robust replication in the ferret respiratory tract and were also capable of limited airborne transmission. These findings highlight the continued public health risk of swine-origin A(H3N2) strains, especially in human populations with low cross-reactive immunity.

In vivo assessments of influenza A virus (IAV) pathogenicity and transmissibility in ferrets represent a crucial component of many pandemic risk assessment rubrics, but few systematic efforts to identify which data from in vivo experimentation are most useful for predicting pathogenesis and transmission outcomes have been conducted. To this aim, we aggregated viral and molecular data from 125 contemporary IAV (H1, H2, H3, H5, H7, and H9 subtypes) evaluated in ferrets under a consistent protocol. Three overarching predictive classification outcomes (lethality, morbidity, transmissibility) were constructed using machine learning (ML) techniques, employing datasets emphasizing virological and clinical parameters from inoculated ferrets, limited to viral sequence-based information, or combining both data types. Among 11 different ML algorithms tested and assessed, gradient boosting machines and random forest algorithms yielded the highest performance, with models for lethality and transmission consistently better performing than models predicting morbidity. Comparisons of feature selection among models was performed, and highest performing models were validated with results from external risk assessment studies. Our findings show that ML algorithms can be used to summarize complex in vivo experimental work into succinct summaries that inform and enhance risk assessment criteria for pandemic preparedness that take in vivo data into account.

Ocular inoculation of a clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) virus caused severe and fatal infection in ferrets. Virus was transmitted to ferrets in direct contact. The results highlight the potential capacity of these viruses to cause human disease after either respiratory or ocular exposure.

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 728 ferrets inoculated with 126 unique IAV, conducted by a single research group under a uniform experimental protocol. This collection of morbidity, mortality, and viral titer data represents the largest publicly available dataset to date of in vivo-generated IAV infection outcomes on a per-ferret level.

Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses have caused large outbreaks within avian populations on five continents, with concurrent spillover into a variety of mammalian species. Mutations associated with mammalian adaptation have been sporadically identified in avian isolates, and more frequently among mammalian isolates following infection. Reports of human infection with A(H5N1) viruses following contact with infected wildlife have been reported on multiple continents, highlighting the need for pandemic risk assessment of these viruses. In this study, the pathogenicity and transmissibility of A/Chile/25945/2023 HPAI A(H5N1) virus, a novel reassortment with four gene segments (PB1, PB2, NP, MP) from North America lineage, isolated from a severe human case in Chile, was evaluated in vitro and using the ferret model. This virus possessed a high capacity to cause fatal disease, characterized by high morbidity and extrapulmonary spread in virus-inoculated ferrets. The virus was capable of transmission to naïve contacts in a direct contact setting, with contact animals similarly exhibiting severe disease, but did not exhibit productive transmission in respiratory droplet or fomite transmission models. Our results indicate that the virus would need to acquire an airborne transmissible phenotype in mammals to potentially cause a pandemic. Nonetheless, this work warrants continuous monitoring of mammalian adaptations in avian viruses, especially in strains isolated from humans, to aid pandemic preparedness efforts.

Viral pathogenesis and therapeutic screening studies that utilize small mammalian models rely on the accurate quantification and interpretation of morbidity measurements, such as weight and body temperature, which can vary depending on the model, agent and/or experimental design used. As a result, morbidity-related data are frequently normalized within and across screening studies to aid with their interpretation. However, such data normalization can be performed in a variety of ways, leading to differences in conclusions drawn and making comparisons between studies challenging. Here, we discuss variability in the normalization, interpretation, and presentation of morbidity measurements for four model species frequently used to study a diverse range of human viral pathogens - mice, hamsters, guinea pigs and ferrets. We also analyze findings aggregated from influenza A virus-infected ferrets to contextualize this discussion. We focus on serially collected weight and temperature data to illustrate how the conclusions drawn from this information can vary depending on how raw data are collected, normalized and measured. Taken together, this work supports continued efforts in understanding how normalization affects the interpretation of morbidity data and highlights best practices to improve the interpretation and utility of these findings for extrapolation to public health contexts.

As use of the ferret model to study influenza A virus (IAV) pathogenicity increases, periodic assessment of data generated in this model is warranted, to identify features associated with virus replication throughout the respiratory tract and to refine future analyses. However, protocol-specific differences present between independent laboratories limit easy aggregation of virological data. We compiled viral titer and clinical data from >1,000 ferrets inoculated with 125 contemporary IAV under a consistent experimental protocol (including high- and low-pathogenicity avian, swine-origin, and human viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes) and examined which meaningful and statistically supported associations were present among numerous quantitative measurements. Viral titers correlated positively between ferret nasal turbinate tissue, lung tissue, and nasal wash specimens, though the strength of the associations varied, notably regarding the particular nasal wash summary measure employed and properties of the virus itself. Use of correlation coefficients and mediation analyses further supported the interconnectedness of viral titer measurements taken at different sites throughout the respiratory tract. IAV possessing mammalian host adaptation markers in the HA and PB2 exhibited more rapid growth in the ferret upper respiratory tract early after infection, supported by quantities derived from infectious titer data to capture infection progression, compared with viruses bearing hallmarks of avian IAV. Collectively, this work identifies summary metrics most closely linked with virological and phenotypic outcomes in ferrets, supporting continued refinement of data analyzed from in vivo experimentation, notably from studies conducted to evaluate the public health risk posed by novel and emerging IAV.IMPORTANCEFerrets are frequently employed to study the pandemic potential of novel and emerging influenza A viruses. However, systematic retrospective analyses of data generated from these experiments are rarely performed, limiting our ability to identify trends in this data and explore how analyses can be refined. Using logarithmic viral titer and clinical data aggregated from one research group over 20 years, we assessed which meaningful and statistically supported associations were present among numerous quantitative measurements obtained from influenza A virus (IAV)-infected ferrets, including those capturing viral titers, infection progression, and disease severity. We identified numerous linear correlations between parameters assessing virus replication at discrete sites in vivo, including parameters capturing infection progression not frequently employed in the field, and sought to investigate the interconnected nature of these associations. This work supports continued refinement of data analyzed from in vivo experimentation, notably from studies which evaluate the public health risk posed by IAV.

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.

Environmental enrichment is a necessary component of all research vivarium settings. However, appropriate enrichmentdecisions vary greatly depending on the species involved and the research use of the animals. The increasing use of ferrets inresearch settings-notably for modeling the pathogenicity and transmissibility of viral pathogens that require containmentin ABSL-2 to -4 environments-presents a particular challenge for veterinary and research staff to ensure that enrichmentneeds for these animals are met consistently. Here, we discuss the species-specific enrichment needs of ferrets, enrichmentconsiderations for ferrets housed in research settings, and the challenges and importance of providing appropriate enrichmentduring experimentation, including when ferrets are housed in high-containment facilities. This article is organizedto support the easy availability of information that will facilitate the design and implementation of optimal environmentalenrichment for ferrets used in diverse research efforts in vivarium settings.

Ferrets represent an invaluable model for the study of influenza virus pathogenicity and transmissibility due to the ability of this species to recapitulate clinical symptoms of influenza infection present in humans. Ferrets are also employed for the study of bacterial pathogens that naturally infect humans at different anatomical sites, including the respiratory and gastrointestinal tracts. While viral and bacterial infection studies in isolation using animal models are important for furthering our understanding of pathogen biology and for the development of improved therapeutics, it is also critical to extend our knowledge to pathogen coinfections in vivo, to more closely examine interkingdom dynamics that may contribute to overall disease outcomes. Despite the increasing use of ferrets for studies with influenza virus, few reports have investigated influenza and bacterial coinfection challenges in ferrets. In this review, we discuss how ferrets have been employed to study a diverse range of both influenza viruses and bacterial species and summarize key studies that have utilized the ferret model for primary influenza virus challenge followed by secondary bacterial infection. These co-pathogenesis studies have provided critical insight into the dynamic interplay between these pathogens, underscoring the utility of ferrets as a model system for investigating influenza virus-bacteria interactions.

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.Competing Interest StatementThe authors have declared no competing interest.

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.

Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.2) variant displaced the prototype Washington/1/2020 (WA/1) strain. While WA/1 virus exhibited a moderately increased proportion compared to that in the inoculum following co-infection in human respiratory cells, Delta variant possessed a substantial in vivo fitness advantage as this virus becoming predominant in both inoculated and contact animals. This work identifies critical traits of the Delta variant that likely played a role in it becoming a dominant variant and highlights the necessities of employing multiple model systems to assess the fitness of newly emerged SARS-CoV-2 variants.

Airborne transmission of viral pathogens is dependent on the generation, exhalation, and deposition of virus-laden aerosols from infected to susceptible hosts. Rigorous evaluations of virus transmissibility in laboratory settings have provided critical insight into viral and host features that contribute to this property, but historically have not included concurrent evaluations of viral load in the air. Similarly, investigation of respiratory pathogens in an aerosol state has been understudied relative to other areas focused on transmission dynamics between mammalian hosts. What makes collection and quantification of virus-laden aerosols in the laboratory so challenging? Here, we discuss practical obstacles and limitations on performing this work in laboratory environments, the additional challenges posed by conducting these experiments concurrent with in vivo experimentation, and how continued investment in this work will provide greater understanding of the role aerosols play in viral transmission.

The ferret transmission model is routinely used to evaluate the pandemic potential of newly emerging influenza A viruses. However, concurrent measurement of viral load in the air is typically not a component of such studies. To address this knowledge gap, we measured the levels of virus in ferret nasal washes as well as viral RNA emitted into the air for 14 diverse influenza viruses, encompassing human-, swine-, and avian-origin strains. Here we show that transmissible viruses display robust replication and fast release into the air. In contrast, poorly- and non-transmissible viruses show significantly reduced or delayed replication along with lower detection of airborne viral RNA at early time points post inoculation. These findings indicate that efficient ferret-to-ferret transmission via the air is directly associated with fast emission of virus-laden particles; as such, quantification of viral RNA in the air represents a useful addition to established assessments of new influenza virus strains.

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

Despite reports of confirmed human infection following ocular exposure with both influenza A virus (IAV) and SARS-CoV-2, the dynamics of virus spread throughout oculonasal tissues and the relative capacity of virus transmission following ocular inoculation remain poorly understood. Furthermore, the impact of exposure route on subsequent release of airborne viral particles into the air has not been examined previously. To assess this, ferrets were inoculated by the ocular route with A(H1N1)pdm09 and A(H7N9) IAVs and two SARS-CoV-2 (early pandemic Washington/1 and Delta variant) viruses. Virus replication was assessed in both respiratory and ocular specimens, and transmission was evaluated in direct contact or respiratory droplet settings. Viral RNA in aerosols shed by inoculated ferrets was quantified with a two-stage cyclone aerosol sampler (National Institute for Occupational Safety and Health [NIOSH]). All IAV and SARS-CoV-2 viruses mounted a productive and transmissible infection in ferrets following ocular inoculation, with peak viral titers and release of virus-laden aerosols from ferrets indistinguishable from those from ferrets inoculated by previously characterized intranasal inoculation methods. Viral RNA was detected in ferret conjunctival washes from all viruses examined, though infectious virus in this specimen was recovered only following IAV inoculation. Low-dose ocular-only aerosol exposure or inhalation aerosol exposure of ferrets to IAV similarly led to productive infection of ferrets and shedding of aerosolized virus. Viral evolution during infection was comparable between all inoculation routes examined. These data support that both IAV and SARS-CoV-2 can establish a high-titer mammalian infection following ocular exposure that is associated with rapid detection of virus-laden aerosols shed by inoculated animals. IMPORTANCE Documented human infection with influenza viruses and SARS-CoV-2 has been reported among individuals wearing respiratory protection in the absence of eye protection, highlighting the capacity of these respiratory tract-tropic viruses to exploit nonrespiratory routes of exposure to initiate productive infection. However, comprehensive evaluations of how ocular exposure may modulate virus pathogenicity and transmissibility in mammals relative to respiratory exposure are limited and have not investigated multiple virus families side by side. Using the ferret model, we show that ocular exposure with multiple strains of either coronaviruses or influenza A viruses leads to an infection that results in shedding of detectable aerosolized virus from inoculated animals, contributing toward onward transmission of both viruses to susceptible contacts. Collectively, these studies support that the ocular surface represents a susceptible mucosal surface that, if exposed to a sufficient quantity of either virus, permits establishment of an infection which is similarly transmissible as that following respiratory exposure.

The continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans necessitates evaluation of variants for enhanced virulence and transmission. We used the ferret model to perform a comparative analysis of four SARS-CoV-2 strains, including an early pandemic isolate from the United States (WA1), and representatives of the Alpha, Beta, and Delta lineages. While Beta virus was not capable of pronounced replication in ferrets, WA1, Alpha, and Delta viruses productively replicated in the ferret upper respiratory tract, despite causing only mild disease with no overt histopathological changes. Strain-specific transmissibility was observed; WA1 and Delta viruses transmitted in a direct contact setting, whereas Delta virus was also capable of limited airborne transmission. Viral RNA was shed in exhaled air particles from all inoculated animals but was highest for Delta virus. Prior infection with SARS-CoV-2 offered varied protection against reinfection with either homologous or heterologous variants. Notable genomic variants in the spike protein were most frequently detected following WA1 and Delta virus infection. IMPORTANCE Continued surveillance and risk assessment of emerging SARS-CoV-2 variants are critical for pandemic response and preparedness. As such, in vivo evaluations are indispensable for early detection of variants with enhanced virulence and transmission. Here, we used the ferret model to compare the pathogenicity and transmissibility of an original SARS-CoV-2 isolate (USA-WA1/2020 [WA1]) to those of a panel of Alpha, Beta, and Delta variants, as well as to evaluate protection from homologous and heterologous reinfection. We observed strain-specific differences in replication kinetics in the ferret respiratory tract and virus load emitted into the air, revealing enhanced transmissibility of the Delta virus relative to previously detected strains. Prior infection with SARS-CoV-2 provided varied levels of protection from reinfection, with the Beta strain eliciting the lowest level of protection. Overall, we found that ferrets represent a useful model for comparative assessments of SARS-CoV-2 infection, transmission, and reinfection.

Highly pathogenic avian influenza A(H5N1) viruses have spread rapidly throughout North American flyways in recent months, affecting wild birds in over 40 states. We evaluated the pathogenicity and transmissibility of a representative virus using a ferret model and examined replication kinetics of this virus in human respiratory tract cells.

Past pandemic influenza viruses with sustained human-to-human transmissibility have emerged from animal influenza viruses. Employment of experimental models to assess the pandemic risk of emerging zoonotic influenza viruses provides critical information supporting public health efforts. Ferret transmission experiments have been utilized to predict the human-to-human transmission potential of novel influenza viruses. However, small sample sizes and a lack of standardized protocols can introduce interlaboratory variability, complicating interpretation of transmission experimental data. To assess the range of variation in ferret transmission experiments, a global exercise was conducted by 11 laboratories using two common stock H1N1 influenza viruses with different transmission characteristics in ferrets. Parameters known to affect transmission were standardized, including the inoculation route, dose, and volume, as well as a strict 1:1 donor/contact ratio for respiratory droplet transmission. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored and analyzed. The overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. Among environmental parameters that varied across laboratories, donor-to-contact airflow directionality was associated with increased transmissibility. To attain high confidence in identifying viruses with moderate to high transmissibility or low transmissibility under a smaller number of participating laboratories, our analyses support the notion that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment. IMPORTANCE Following detection of a novel virus, rapid characterization efforts (both in vitro and in vivo) are undertaken at numerous laboratories worldwide to evaluate the relative risk posed to human health. Aggregation of these data are critical, but the use of nonstandardized protocols can make interpretation of divergent results a challenge. For evaluation of virus transmissibility, a multifactorial trait which can only be evaluated in vivo, identifying intrinsic levels of variability between groups can improve the utility of these data, as well as ensure that experiments are performed with sufficient replication to ensure high confidence in compiled results. Using the ferret transmission model and two influenza A viruses, we conducted a multicenter standardization exercise to improve the interpretation of transmission data generated during risk assessment activities; this exercise serves as a model for future efforts employing both in vitro and in vivo models against possible pandemic pathogens.

Influenza A viruses (IAV) cause mammalian infections following several transmission routes. Considering the anatomic proximity and connection between the nasopharynx and periocular tissues, there is a need to understand the dynamics of virus spread between these sites following both respiratory and nonrespiratory viral transmission. We examined virus distribution and associated inflammation within nasal and periocular tissues during the acute phase of H1N1 IAV infection in ferrets following intranasal or ocular inoculation. Ocular and intranasal inoculations with IAV caused comparable viral antigen distribution and inflammation in the nasal passages, though infection kinetics and magnitude differed by inoculation route. Ocular inoculation was associated with inflammation in the conjunctiva and lacrimal glands. Although intranasal inoculation was also associated with periocular inflammation, the onset was delayed relative to ocular inoculation. This work underscores the importance of investigating extrapulmonary tissues following mammalian infection with respiratory pathogens, even after intranasal inoculation.

Influenza A viruses (IAVs) in the swine reservoir constantly evolve, resulting in expanding genetic and antigenic diversity of strains that occasionally cause infections in humans and pose threat of emerging as a strain capable of human-to-human transmission. For these reasons, there is an ongoing need for surveillance and characterization of newly emerging strains to aid pandemic preparedness efforts, particularly for the selection of candidate vaccine viruses and conducting risk assessments. Here, we performed a parallel comparison of the pathogenesis and transmission of genetically and antigenically diverse swine-origin A(H1N1) variant (v) and A(H1N2)v, and human seasonal A(H1N1)pdm09 IAVs using the ferret model. Both groups of viruses were capable of replication in the ferret upper respiratory tract; however, variant viruses were more frequently isolated from the lower respiratory tract as compared to the human-adapted viruses. Regardless of virus origin, observed clinical signs of infection differed greatly between strains, with some viruses causing nasal discharge, sneezing and, in some instances, diarrhea in ferrets. The most striking difference between the viruses was the ability to transmit through the air. Human-adapted viruses were capable of airborne transmission between all ferret pairs. In contrast, only one out of the four tested variant viruses was able to transmit via the air as efficiently as the human-adapted viruses. Overall, this work highlights the need for sustained monitoring of emerging swine IAVs to identify strains of concern such as those that are antigenically different from vaccine strains and that possess adaptations required for efficient respiratory droplet transmission in mammals.