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.

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.

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.

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.

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.

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.

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.

Efficient human-to-human transmission represents a necessary adaptation for a zoonotic influenza A virus (IAV) to cause a pandemic. As such, many emerging IAVs are characterized for transmissibility phenotypes in mammalian models, with an emphasis on elucidating viral determinants of transmission and the role host immune responses contribute to mammalian adaptation. Investigations of virus infectivity and stability in aerosols concurrent with transmission assessments have increased in recent years, enhancing our understanding of this dynamic process. Here, we employ a diverse panel of 17 human and zoonotic IAVs, inclusive of seasonally circulating H1N1 and H3N2 viruses, and avian and swine viruses associated with human infection, to evaluate differences in spray factor (a value that assesses efficiency of the aerosolization process), stability, and infectivity following aerosolization. While most seasonal influenza viruses did not exhibit substantial variability within these parameters, there was more heterogeneity among zoonotic influenza viruses, which possess a diverse range of transmission phenotypes. Aging of aerosols at different relative humidities identified strain-specific levels of stability with different profiles identified between zoonotic H3, H5, and H7 subtype viruses associated with human infection. As studies continue to elucidate the complex components governing virus transmissibility, notably aerosol matrices and environmental parameters, considering the relative role of subtype- and strain-specific factors to modulate these parameters will improve our understanding of the pandemic potential of zoonotic influenza A viruses. Importance Transmission of respiratory pathogens through the air can facilitate the rapid and expansive spread of infection and disease through a susceptible population. While seasonal influenza viruses are quite capable of airborne spread, there is a lack of knowledge regarding how well influenza viruses remain viable after aerosolization, and if influenza viruses capable of jumping species barriers to cause human infection differ in this property from seasonal strains. We evaluated a diverse panel of influenza viruses associated with human infection (originating from human, avian, and swine reservoirs) for their ability to remain viable after aerosolization in the laboratory under a range of conditions. We found greater diversity among avian and swine-origin viruses compared with seasonal influenza viruses; strain-specific stability was also noted. Although influenza virus stability in aerosols is an underreported property, if molecular markers associated with enhanced stability are identified, we will be able to quickly recognize emerging strains of influenza that present the greatest pandemic threat.

Highly pathogenic avian influenza (HPAI) H5 viruses, of the A/goose/Guangdong/1/1996 lineage, have exhibited substantial geographic spread worldwide since the first detection of H5N1 virus in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health and can cause lethal human disease following virus exposure. Here, HPAI H5N6 and H5N2 viruses (isolated between 2014 and 2017) of the 2.3.4.4 clade were assessed for their capacity to replicate in human respiratory tract cells, and to cause disease and transmit in the ferret model. All H5N6 viruses possessed increased virulence in ferrets compared to the H5N2 virus; however, pathogenicity profiles varied among the H5N6 viruses tested, from mild infection with sporadic virus dissemination beyond the respiratory tract, to severe disease with fatal outcome. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not with the H5N2 virus. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the identification of mammalian adaptation markers in key genes associated with pathogenesis supports these findings.

Low pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Co-circulation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world, and for over twenty years, have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported and was found to exhibit properties of adaptation to humans and, importantly, show similarities to strains isolated from the live bird markets of Vietnam.

ABSTRACTLow pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naive ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

As influenza A viruses continue to jump species barriers, data generated in the ferret model to assess influenza virus pathogenicity, transmissibility, and tropism of these novel strains continues to inform an increasing scope of public health-based applications. This review presents the suitability of ferrets as a small mammalian model for influenza viruses and describes the breadth of pathogenicity and transmissibility profiles possible in this species following inoculation with a diverse range of viruses. Adaptation of aerobiology-based techniques and analyses have furthered our understanding of data obtained from this model and provide insight into the capacity of novel and emerging influenza viruses to cause human infection and disease.

Swine-origin (variant) H1 influenza A viruses associated with numerous human infections in North America in recent years have been extensively studied in vitro and in mammalian models to determine their pandemic potential. However, limited information is available on Eurasian avian-like lineage variant H1 influenza viruses. In 2015, A/Hunan/42443/2015 virus was isolated from a child in China with a severe infection. Molecular analysis revealed that this virus possessed several key virulence and human adaptation markers. Similar to what was previously observed in C57BL/6J mice, we report here that in the BALB/c mouse model, A/Hunan/42443/2015 virus caused more severe morbidity and higher mortality than did North American variant H1 virus isolates. Furthermore, the virus efficiently replicated throughout the respiratory tract of ferrets and exhibited a capacity for transmission in this model, underscoring the need to monitor zoonotic viruses that cross the species barrier as they continue to pose a pandemic threat.

We previously demonstrated that despite no airborne transmissibility increase compared to low pathogenic avian influenza viruses, select human isolates of highly pathogenic avian influenza A(H7N9) virus exhibit greater virulence in animal models and a lower threshold pH for fusion. In the current study, we utilized both in vitro and in vivo approaches to identify key residues responsible for hemagglutinin (HA) intracellular cleavage, acid stability, and virulence in mice. We found that the four amino acid insertion (-KRTA-) at the HA cleavage site of A/Taiwan/1/2017 virus is essential for HA intracellular cleavage and contributes to disease in mice. Furthermore, a lysine to glutamic acid mutation at position HA2-64 increased the threshold pH for HA activation, reduced virus stability, and replication in mice. Identification of a key residue responsible for enhanced acid stability of A(H7N9) viruses is of great significance for future surveillance activities and improvements in vaccine stability.

The relative importance of influenza virus transmission via aerosols is not fully understood, but experimental data suggests that aerosol transmission may represent a critical mode of influenza virus spread among humans. Decades ago, prototypical laboratory strains of influenza were shown to persist in aerosols; however, there is a paucity of data available covering currently circulating influenza viruses, which differ significantly from their predecessors. In this study, we evaluated the longevity of influenza viruses in aerosols generated in the laboratory. We selected a panel of H1 viruses that exhibit diverse transmission profiles in the ferret model, including four human isolates of swine-origin (referred to as variant) and a seasonal strain. By measuring the ratio of viral RNA to infectious virus maintained in aerosols over time, we show that influenza viruses known to transmit efficiently through the air, display enhanced stability in an aerosol state for prolonged periods compared with those viruses that do not transmit as efficiently. We then assessed whether H1 influenza virus was still capable of infecting and causing disease in ferrets after being aged in suspended aerosols. Ferrets exposed to very low levels of influenza virus (</=17 PFU) in aerosols aged for 15 or 30 min, became infected, with 5/6 ferrets shedding virus in nasal washes at titers on par with ferrets who inhaled higher doses of unaged influenza virus. We describe here, an underreported characteristic of influenza viruses, stability in aerosols, and make a direct connection to the role it plays in influenza transmission.Importance Each time a swine influenza virus transmits to a human, it provides an opportunity for the virus to acquire adaptations needed for sustained human-to-human transmission. Here, we use aerobiology techniques to test the stability of swine-origin H1 subtype viruses in aerosols and evaluate their infectivity in ferrets. Our results show that highly transmissible influenza viruses display enhanced stability in an aerosol state compared with viruses that do not transmit as efficiently. Similar to human-adapted strains, swine-origin influenza viruses are infectious in ferrets at low doses even after prolonged suspension in the air. These data underscore the risk of airborne swine-origin influenza viruses, and support the need for continued surveillance and refinement of innovative laboratory methods to investigate mammalian exposure to inhaled pathogens. Determination of molecular markers that affect longevity of airborne influenza viruses will improve our ability to quickly identify emerging strains that present the greatest threat to public health.

Emergence of genetically and antigenically diverse strains of influenza to which the human population has no or limited immunity necessitates continuous risk assessments to determine the likelihood of these viruses acquiring adaptations that facilitate sustained human-to-human transmission. As the North American swine H1 virus population has diversified over the last century by means of both antigenic drift and shift, in vivo assessments to study multifactorial traits like mammalian pathogenicity and transmissibility of these emerging influenza viruses are critical. In this review, we examine genetic, molecular, and pathogenicity and transmissibility data from a panel of contemporary North American H1 subtype swine-origin viruses isolated from humans, as compared to H1N1 seasonal and pandemic viruses, including the reconstructed 1918 virus. We present side-by-side analyses of experiments performed in the mouse and ferret models using consistent experimental protocols to facilitate enhanced interpretation of in vivo data. Contextualizing these analyses in a broader context permits a greater appreciation of the role that in vivo risk assessment experiments play in pandemic preparedness. Collectively, we find that despite strain-specific heterogeneity among swine-origin H1 viruses, contemporary swine viruses isolated from humans possess many attributes shared by prior pandemic strains, warranting heightened surveillance and evaluation of these zoonotic viruses.

The fifth-wave of the H7N9 influenza epidemic in China was distinguished by a sudden increase in human infections, an extended geographic distribution, and the emergence of highly pathogenic avian influenza (HPAI) viruses. Genetically, some H7N9 viruses from the fifth-wave have acquired novel amino acid changes at positions involved in mammalian adaptation, antigenicity, and HA cleavability. Here, several low pathogenic avian influenza (LPAI) and HPAI H7N9 human isolates from the fifth epidemic wave were assessed for their pathogenicity and transmissibility in mammalian models, as well as their ability to replicate in human airway epithelial cells. We found that a LPAI virus exhibited a similar capacity to replicate and cause disease in two animal species as viruses from previous waves. In contrast, HPAI H7N9 viruses possessed enhanced virulence, causing greater lethargy and mortality, with an extended tropism for brain tissues in both ferret and mouse models. These HPAI viruses also showed signs of adaptation to mammalian hosts by acquiring the ability to fuse at a lower pH threshold compared with other H7N9 viruses. All of the fifth-wave H7N9 viruses were able to transmit among cohoused ferrets, but exhibited a limited capacity to transmit by respiratory droplets and deep sequencing analysis revealed that the H7N9 viruses sampled after transmission showed a reduced amount of minor variants. Taken together, we conclude that the fifth-wave HPAI H7N9 viruses have gained the ability to cause enhanced disease in mammalian models, and with further adaptation may acquire the ability to cause an H7N9 pandemic.ImportanceThe potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increased number of human infections and the emergence of antigenically distinct LPAI and HPAI H7N9 viruses. In this study, a group of fifth-wave HPAI and LPAI viruses were evaluated for their ability to infect, cause disease, and transmit in small animal models. The ability of HPAI H7N9 viruses to cause more severe disease and to replicate in brain tissues in animal models as well as their ability to fuse at a lower pH threshold compared to LPAI H7N9 viruses suggest that the fifth-wave H7N9 viruses have evolved to acquire novel traits with the potential to pose a higher risk to humans. Although the fifth-wave H7N9 viruses have not yet gained the ability to transmit efficiently by air, continuous surveillance and risk assessment remain essential parts of our pandemic preparedness efforts.

Influenza A virus pandemics are rare events caused by novel viruses which have the ability to spread in susceptible human populations. With respect to H1 subtype viruses, swine H1N1 and H1N2 viruses occasionally cross the species barrier to cause human infection. Recently isolated from humans (termed variants), swine viruses were shown to display great genetic and antigenic diversity, hence posing considerable public health risk. Here, we utilized in vitro and in vivo approaches to provide characterization of H1 subtype variant viruses isolated since the 2009 pandemic and discuss the findings in context with previously studied H1 subtype human isolates. The variant viruses were well adapted to replicate in human respiratory cell line, Calu-3, and the respiratory tracts of mice and ferrets. However, with respect to HA activation pH, the variant viruses had fusion pH thresholds closer to that of most classical swine and triple reassortant H1 isolates rather than viruses that had adapted to humans. Consistent with previous observations for swine isolates, the tested variant viruses were capable of efficient transmission between co-housed ferrets but could transmit via respiratory droplets to differing degrees. Overall, this investigation demonstrates that swine H1 viruses that infected humans possess adaptations required for robust replication and, in some cases, efficient respiratory droplet transmission in a mammalian model, and therefore, need to be closely monitored for additional molecular changes that could facilitate transmission among humans. This work highlights the need for risk assessments of emerging H1 viruses as they continue to evolve and cause human infections.IMPORTANCE Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infections. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no pre-existing immunity is an ongoing threat to public health. Recently isolated variant H1 subtype viruses were shown to display extensive genetic diversity and in many instances were antigenically distinct from seasonal vaccine strains. In this study, we provide characterization of representative H1N1v and H1N2v viruses isolated since the 2009 pandemic. Our results show that, although recent variant H1 viruses possess some adaptation markers of concern, these viruses have not fully adapted to humans and require further adaptation to present a pandemic threat. This investigation highlights the need for close monitoring of emerging variant influenza viruses for molecular changes that could facilitate efficient transmission among humans.

While several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011-2016 were evaluated for their replicative ability in both in vitro and in vivo mammalian models, as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacity in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.Importance: Swine-origin influenza viruses of the H3N2 subtype, with the HA and NA derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the U.S between 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the ability of these viruses to replicate, caused disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlights their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

Influenza A(H1) viruses circulating in swine represent an emerging virus threat as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus (A/Iowa/39/2015), as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Pre-existing immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a non-fatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with pre-existing immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015 demonstrating decreased protection. This data illustrates the potential for currently circulating swine influenza viruses to infect and cause illness in humans with pre-existing immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Infections with low pathogenicity and highly pathogenic avian influenza A(H7N9) viruses affected poultry in 4 states in the southeastern United States in 2017. We evaluated pathogenicity and transmission of representative viruses in mouse and ferret models and examined replication kinetics in human respiratory tract cells. These viruses can cause respiratory infections in mammalian models.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.