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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.

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.

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.

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 a 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.

Repeated influenza vaccination and natural infections generate complex immune profiles in humans that require antibody landscape analysis to assess immunity and evaluate vaccines. However, antibody landscape analyses are difficult to perform using traditional assays., To better understand the antibody landscape changes following influenza virus natural infection and vaccination, we developed a high-throughput multiplex influenza antibody detection assay (MIADA) containing 42 recombinant hemagglutinins (rHAs) (ectodomain and/or globular head domain) from pre-2009 A(H1N1), A(H1N1)pdm09, A(H2N2), A(H3N2), A(H5N1), A(H7N7), A(H7N9), A(H7N2), A(H9N2), A(H13N9), and influenza B viruses. Panels of ferret antisera, 227 paired human sera from vaccinees (children and adults) in 5 influenza seasons (2010 to 2018), and 17 paired human sera collected from real-time reverse transcription-PCR (rRT-PCR)-confirmed influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B virus-infected adults were analyzed by the MIADA. Ferret antisera demonstrated clear strain-specific antibody responses to exposed subtype HA. Adults (19 to 49 years old) had broader antibody landscapes than young children (

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. Cocirculation 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 20 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 it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

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 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 naïve 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.

H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses.

Recently, novel highly pathogenic avian influenza H5Nx viruses (clade 2.3.4.4) caused outbreaks in US poultry. We evaluated the potential of a stockpiled A(H5N1) A/Anhui/1/2005 (clade 2.3.4) vaccine to elicit cross-reactive antibody responses to these emerging viruses. Sera from subjects who received 2 doses of MF59-adjuvanted A/Anhui/1/2005, or 1 dose of MF59-adjuvanted A/Anhui/1/2005 following priming with a clade 1 vaccine were characterized by microneutralization assays and modified hemagglutination inhibition (HI) assays. Only heterologous prime-boost vaccination induced modest cross-reactive HI antibody responses to H5Nx viruses. Heterologous prime-boost may provide a more effective vaccination strategy to broaden the antibody responses to emerging viruses.

Infection of adherent cell monolayers using a liquid inoculum represents an established method to reliably and quantitatively study virus infection, but poorly recapitulates the exposure and infection of cells in the respiratory tract that occurs during infection with aerosolized pathogens. To better simulate natural infection in vitro, we adapted a system that generates viral aerosols similar to those exhaled by infected humans to the inoculation of epithelial cell monolayers. Procedures for cellular infection and calculation of exposure dose were developed and tested using viruses characterized by distinct transmission and pathogenicity phenotypes: an HPAI H5N1, an LPAI H7N9, and a seasonal H3N2 virus. While all three aerosolized viruses were highly infectious in a human bronchial epithelial cell line (Calu-3) cultured submerged in media, differences between the viruses were observed in primary human alveolar epithelial cells and in Calu-3 cells cultured at air-liquid interface. This system provides a novel enhancement to traditional in vitro experiments, particularly those focused on the early stages of infection.

Avian influenza A H7 viruses have caused multiple outbreaks in domestic poultry throughout North America, resulting in occasional infections of humans in close contact with affected birds. In early 2016, the presence of H7N8 highly pathogenic avian influenza (HPAI) viruses and closely related H7N8 low-pathogenic avian influenza (LPAI) viruses was confirmed in commercial turkey farms in Indiana. These H7N8 viruses represent the first isolation of this subtype in domestic poultry in North America, and their virulence in mammalian hosts and the potential risk for human infection are largely unknown. In this study, we assessed the ability of H7N8 HPAI and LPAI viruses to replicate in vitro in human airway cells and in vivo in mouse and ferret models. Both H7N8 viruses replicated efficiently in vitro and in vivo , but they exhibited substantial differences in disease severity in mammals. In mice, while the H7N8 LPAI virus largely remained avirulent, the H7N8 HPAI virus exhibited greater infectivity, virulence, and lethality. Both H7N8 viruses replicated similarly in ferrets, but only the H7N8 HPAI virus caused moderate weight loss, lethargy, and mortality. The H7N8 LPAI virus displayed limited transmissibility in ferrets placed in direct contact with an inoculated animal, while no transmission of H7N8 HPAI virus was detected. Our results indicate that the H7N8 avian influenza viruses from Indiana are able to replicate in mammals and cause severe disease but with limited transmission. The recent appearance of H7N8 viruses in domestic poultry highlights the need for continued influenza surveillance in wild birds and close monitoring of the potential risk to human health. IMPORTANCE H7 influenza viruses circulate in wild birds in the United States, but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) virus and an H7N8 low-pathogenic avian influenza (LPAI) virus were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed their pathogenesis and transmission in vitro and in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence, and although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals.

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

Avian-origin influenza represents a global public health concern. In 2013, the H10N8 virus caused documented human infections for the first time. Currently, there is no approved vaccine against H10 influenza. Recombinant virus-like particles (VLPs) represent a promising vaccine approach. In this study, we evaluated H10 VLPs containing hemagglutinin from H10N8 virus as an experimental vaccine in a ferret challenge model. In addition, we evaluated quadri-subtype VLPs co-localizing H5, H7, H9 and H10 subtypes. Both vaccines elicited serum antibody that reacted with the homologous H10 derived from H10N8 virus and cross-reacted with the heterologous H10N1 virus. Quadri-subtype vaccine also elicited serum antibody to the homologous H5, H7, and H9 antigens and cross-reacted with multiple clades of H5N1 virus. After heterologous challenge with the H10N1 virus, all vaccinated ferrets showed significantly reduced titers of replicating virus in the respiratory tract indicating protective effect of vaccination with either H10 VLPs or with quadri-subtype VLPs.

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation.

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 the human respiratory cell line Calu-3 and the respiratory tracts of mice and ferrets. However, with respect to hemagglutinin (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 cohoused 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 preexisting 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.

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 hemagglutinin cleavability. Here, several human low-pathogenic avian influenza (LPAI) and HPAI H7N9 virus 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 an 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 than 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.IMPORTANCE The potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increase in the 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 was evaluated for its 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 than LPAI H7N9 viruses suggests 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(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 the 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. Preexisting 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 nonfatal 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 preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

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, the recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011 to 2016 were evaluated for their replicative ability in both in vitro and in vivo in mammalian models as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacities 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 hemagglutinin (HA) and neuraminidase (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 United States during the 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the abilities of these viruses to replicate, cause 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 highlight their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.

A novel highly pathogenic avian influenza (HPAI) H5N8 virus, first detected in January 2014 in poultry and wild birds in South Korea, has spread throughout Asia and Europe and caused outbreaks in Canada and the United States by the end of the year. The spread of H5N8 and the novel reassortant viruses, H5N2 and H5N1 (H5Nx), in domestic poultry across multiple states in the United States pose a potential public health risk. To evaluate the potential of cross-species infection, we determined the pathogenicity and transmissibility of two Asian-origin H5Nx viruses in mammalian animal models. The newly isolated H5N2 and H5N8 viruses were able to cause severe disease in mice only at high doses. Both viruses replicated efficiently in the upper and lower respiratory tracts of ferrets; however, the clinical symptoms were generally mild, and there was no evidence of systemic dissemination of virus to multiple organs. Moreover, these influenza H5Nx viruses lacked the ability to transmit between ferrets in a direct contact setting. We further assessed viral replication kinetics of the novel H5Nx viruses in a human bronchial epithelium cell line, Calu-3. Both H5Nx viruses replicated to a level comparable to a human seasonal H1N1 virus, but significantly lower than a virulent Asian-lineage H5N1 HPAI virus. Although the recently isolated H5N2 and H5N8 viruses displayed moderate pathogenicity in mammalian models, their ability to rapidly spread among avian species, reassort, and generate novel strains underscores the need for continued risk assessment in mammals. IMPORTANCE In 2015, highly pathogenic avian influenza (HPAI) H5 viruses have caused outbreaks in domestic poultry in multiple U.S. states. The economic losses incurred with H5N8 and H5N2 subtype virus infection have raised serious concerns for the poultry industry and the general public due to the potential risk of human infection. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals, which is an essential component of pandemic risk assessment. This study demonstrates that the newly isolated H5N2 and H5N8 viruses lacked the ability to transmit between ferrets and exhibited low to moderate virulence in mammals. In human bronchial epithelial (Calu-3) cells, both H5N8 and H5N2 viruses replicated to a level comparable to a human seasonal virus, but significantly lower than a virulent Asian-lineage H5N1 (A/Thailand/16/2004) virus. The results of this study are important for the evaluation of public health risk.

In March 2013, diagnosis of the first reported case of human infection with a novel avian-origin influenza A(H7N9) virus occurred in eastern China. Most human cases have resulted in severe respiratory illness and, in some instances, death. Currently there are no licensed vaccines against H7N9 virus, which continues to cause sporadic human infections. Recombinant virus-like particles (VLPs) have been previously shown to be safe and effective vaccines for influenza. In this study, we evaluated the immunogenicity and protective efficacy of a H7N9 VLP vaccine in the ferret challenge model. Purified recombinant H7N9 VLPs morphologically resembled influenza virions and elicited high-titer serum hemagglutination inhibition (HI) and neutralizing antibodies specific for A/Anhui/1/2013 (H7N9) virus. H7N9 VLP-immunized ferrets subsequently challenged with homologous virus displayed reductions in fever, weight loss, and virus shedding compared to these parameters in unimmunized control ferrets. H7N9 VLP was also effective in protecting against lung and tracheal infection. The addition of either ISCOMATRIX or Matrix-M1 adjuvant improved immunogenicity and protection of the VLP vaccine against H7N9 virus. These results provide support for the development of a safe and effective human VLP vaccine with potent adjuvants against avian influenza H7N9 virus with pandemic potential.

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film. IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract.

In December 2016, a low-pathogenic avian influenza (LPAI) A(H7N2) virus was identified to be the causative source of an outbreak in a cat shelter in New York City, which subsequently spread to multiple shelters in the states of New York and Pennsylvania. One person with occupational exposure to infected cats became infected with the virus, representing the first LPAI H7N2 virus infection in a human in North America since 2003. Considering the close contact that frequently occurs between companion animals and humans, it was critical to assess the relative risk of this novel virus to public health. The virus isolated from the human case, A/New York/108/2016 (NY/108), caused mild and transient illness in ferrets and mice but did not transmit to naive cohoused ferrets following traditional or aerosol-based inoculation methods. The environmental persistence of NY/108 virus was generally comparable to that of other LPAI H7N2 viruses. However, NY/108 virus replicated in human bronchial epithelial cells with an increased efficiency compared with that of previously isolated H7N2 viruses. Furthermore, the novel H7N2 virus was found to utilize a relatively lower pH for hemagglutinin activation, similar to human influenza viruses. Our data suggest that the LPAI H7N2 virus requires further adaptation before representing a substantial threat to public health. However, the reemergence of an LPAI H7N2 virus in the northeastern United States underscores the need for continuous surveillance of emerging zoonotic influenza viruses inclusive of mammalian species, such as domestic felines, that are not commonly considered intermediate hosts for avian influenza viruses. IMPORTANCE Avian influenza viruses are capable of crossing the species barrier to infect mammals, an event of public health concern due to the potential acquisition of a pandemic phenotype. In December 2016, an H7N2 virus caused an outbreak in cats in multiple animal shelters in New York State. This was the first detection of this virus in the northeastern United States in over a decade and the first documented infection of a felid with an H7N2 virus. A veterinarian became infected following occupational exposure to H7N2 virus-infected cats, necessitating the evaluation of this virus for its capacity to cause disease in mammals. While the H7N2 virus was associated with mild illness in mice and ferrets and did not spread well between ferrets, it nonetheless possessed several markers of virulence for mammals. These data highlight the promiscuity of influenza viruses and the need for diligent surveillance across multiple species to quickly identify an emerging strain with pandemic potential.

Respiratory pathogens have traditionally been studied by examining the exposure and infection of respiratory tract tissues. However, these studies typically overlook the role of ocular surfaces, which represent both a potential site of virus replication and a portal of entry for the establishment of a respiratory infection. To model transocular virus entry in a mammalian species, we established a novel inoculation method that delivers an aerosol inoculum exclusively to the ferret ocular surface. Using influenza virus as a representative respiratory pathogen, we found that both human and avian viruses mounted productive respiratory infections in ferrets following ocular-only aerosol inoculation, and we demonstrated that H5N1 virus can result in a fatal infection at doses below 10 PFU or with exposure times as short as 2 min. Ferrets inoculated by the ocular aerosol route with an avian (H7N7, H7N9) or human (H1N1, H3N2v) virus were capable of transmitting the virus to naïve animals in direct-contact or respiratory-droplet models, respectively. Our results reveal that ocular-only exposure to virus-containing aerosols constitutes a valid exposure route for a potentially fatal respiratory infection, even for viruses that do not demonstrate an ocular tropism, underscoring the public health implications of ocular exposure in clinical or occupational settings. IMPORTANCE In the absence of eye protection, the human ocular surface remains vulnerable to infection with aerosolized respiratory viruses. In this study, we present a way to inoculate laboratory mammals that excludes respiratory exposure, infecting ferrets only by ocular exposure to influenza virus-containing aerosols. This study demonstrates that the use of respiratory protection alone does not fully protect against influenza virus exposure, infection, and severe disease.

Avian influenza H5, H7 and H9 viruses top the World Health Organization's (WHO) list of subtypes with the greatest pandemic potential. Here we describe a recombinant virus-like particle (VLP) that co-localizes hemagglutinin (HA) proteins derived from H5N1, H7N2, and H9N2 viruses as an experimental vaccine against these viruses. A baculovirus vector was configured to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as neuraminidase (NA) and matrix (M1) genes from A/Puerto Rico/8/1934 (H1N1) virus. Co-expression of these genes in Sf9 cells resulted in production of triple-subtype VLPs containing HA molecules derived from the three influenza viruses. The triple-subtype VLPs exhibited hemagglutination and neuraminidase activities and morphologically resembled influenza virions. Intranasal vaccination of ferrets with the VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with H5N1, H7N2, and H9N2 viruses.

Coronavirus disease-2019 (COVID-19), caused by a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has been linked to ocular signs and symptoms in several case reports. Research has demonstrated that SARS-CoV-2 is spread primarily through close contact via respiratory droplets, but there is the possibility for ocular transmission, with the conjunctiva as a conduit as well as a source of infection.Ocular manifestations of SARS-CoV-2 include follicular conjunctivitis, and have been repeatedly noted as an initial or subsequent symptom of COVID-19-positive patients. Particularly in patients with ocular manifestations, there is evidence that the virus may present in tears, based on the detection of SARS-CoV-2 in conjunctival swab samples via reverse transcription polymerase chain reaction. The virus may therefore be transmittable from the ocular surface to a new host via contact with the ocular mucosa, tears, or subsequent fomites.All health care professionals should ask patients about ocular symptoms consistent with SARS-CoV-2, and use eye protection such as goggles or face shields as part of the standard personal protective equipment for high-risk patients in addition to wearing of masks by both the patient and provider, and should consider tears to be potentially infectious.

H7 subtype influenza viruses have demonstrated an ocular tropism in humans, causing conjunctivitis and not respiratory symptoms in many infected individuals. However, the molecular determinants which confer ocular tropism are still poorly understood. Here, we used a murine model of ocular inoculation to demonstrate that H7 influenza viruses are more likely to cause infection following ocular exposure than are non-H7 subtype viruses. We included investigation regarding the potential role of several properties of influenza viruses with murine infectivity following ocular inoculation, including virus lineage, pathogenicity, and HA cleavage site composition. Furthermore, we examined the potential contribution of internal proteins to murine ocular infectivity. These studies establish a link between H7 subtype viruses and the risk of heightened infectivity in a mammalian species following ocular exposure, and support the development of non-traditional inoculation methods and models to best understand the human risk posed by influenza viruses of all subtypes.

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.

SUMMARY Ferrets are widely employed to study the pathogenicity, transmissibility, and tropism of influenza viruses. However, inherent variations in inoculation methods, sampling schemes, and experimental designs are often overlooked when contextualizing or aggregating data between laboratories, leading to potential confusion or misinterpretation of results. Here, we provide a comprehensive overview of parameters to consider when planning an experiment using ferrets, collecting data from the experiment, and placing results in context with previously performed studies. This review offers information that is of particular importance for researchers in the field who rely on ferret data but do not perform the experiments themselves. Furthermore, this review highlights the breadth of experimental designs and techniques currently available to study influenza viruses in this model, underscoring the wide heterogeneity of protocols currently used for ferret studies while demonstrating the wealth of information which can benefit risk assessments of emerging influenza viruses.

Three waves of human infection with H7N9 influenza viruses have concluded to date, but only viruses within the first wave (isolated between March and September 2013) have been extensively studied in mammalian models. While second- and third-wave viruses remain closely linked phylogenetically and antigenically, even subtle molecular changes can impart critical shifts in mammalian virulence. To determine if H7N9 viruses isolated from humans during 2013 to 2015 have maintained the phenotype first identified among 2013 isolates, we assessed the ability of first-, second-, and third-wave H7N9 viruses isolated from humans to cause disease in mice and ferrets and to transmit among ferrets. Similar to first-wave viruses, H7N9 viruses from 2013 to 2015 were highly infectious in mice, with lethality comparable to that of the well-studied A/Anhui/1/2013 virus. Second- and third-wave viruses caused moderate disease in ferrets, transmitted efficiently to cohoused, naive contact animals, and demonstrated limited transmissibility by respiratory droplets. All H7N9 viruses replicated efficiently in human bronchial epithelial cells, with subtle changes in pH fusion threshold identified between H7N9 viruses examined. Our results indicate that despite increased genetic diversity and geographical distribution since their initial detection in 2013, H7N9 viruses have maintained a pathogenic phenotype in mammals and continue to represent an immediate threat to public health. IMPORTANCE H7N9 influenza viruses, first isolated in 2013, continue to cause human infection and represent an ongoing public health threat. Now entering the fourth wave of human infection, H7N9 viruses continue to exhibit genetic diversity in avian hosts, necessitating continuous efforts to monitor their pandemic potential. However, viruses isolated post-2013 have not been extensively studied, limiting our understanding of potential changes in virus-host adaptation. In order to ensure that current research with first-wave H7N9 viruses still pertains to more recently isolated strains, we compared the relative virulence and transmissibility of H7N9 viruses isolated during the second and third waves, through 2015, in the mouse and ferret models. Our finding that second- and third-wave viruses generally exhibit disease in mammals comparable to that of first-wave viruses strengthens our ability to extrapolate research from the 2013 viruses to current public health efforts. These data further contribute to our understanding of molecular determinants of pathogenicity, transmissibility, and tropism.

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

Human infections with highly pathogenic H5N1 avian influenza viruses continue to occur in many parts of the world and pose a considerable public health threat. With the use of animal models, the identification of virulence determinants has been instrumental in improving our understanding of how these viruses cause severe disease in humans. Two genetically similar H5N1 viruses (A/Thailand/16/2004 and A/Thailand/SP83/2004) exhibit high or low virulence phenotypes, respectively, in multiple animal models. Reassortant viruses were generated from this virus pair and evaluated in ferrets. Each of the polymerase genes of A/Thailand/16/2004 virus individually conferred increased virulence to A/Thailand/SP83/2004 virus while the neuraminidase of the low virulence virus reduced virulence and replication efficiency of the virulent virus in ferrets unless the homologous HA was present. Our results demonstrate that H5N1 virus virulence determinants are polygenic and that there is an important correlation between polymerase adaptation, efficient replication in the host, and virulence.

Effects of the commercial drug zanamivir (Relenza™) covalently attached to poly-l-glutamine on the infectivity of influenza A viruses are examined using the plaque reduction assay and binding affinity to viral neuraminidase (NA). These multivalent drug conjugates exhibit (i) up to a 20,000-fold improvement in anti-influenza potency compared with the zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants, and (ii) superior neuraminidase (NA) inhibition constants, especially for the mutants. These findings provide a basis for exploring polymer-attached inhibitors as more efficacious therapeutics, particularly against drug-resistant influenza strains., National Institutes of Health (U.S.) (Grant Number U01-AI074443), Fundación Ramón Areces. Postdoctoral Fellowship

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.

The pandemic H1N1 virus of 2009 (2009 H1N1) produced a spectrum of disease ranging from mild illness to severe illness and death. Respiratory symptoms were frequently associated with virus infection, with relatively high rate of gastrointestinal symptoms reported. To better understand 2009 H1N1 virus pathogenesis in humans, we studied virus and host responses following infection of two cell types: polarized bronchial and pharyngeal epithelial cells, which exhibit many features of the human airway epithelium, and colon epithelial cells to serve as a human intestinal cell model. Selected 2009 H1N1 viruses were compared to both seasonal H1N1 and triple-reassortant swine H1N1 influenza viruses that have circulated among North American pigs since before the 2009 pandemic. All H1N1 viruses replicated productively in airway cells; however, in contrast to seasonal H1N1 virus infection, infection with the 2009 H1N1 and triple-reassortant swine H1N1 viruses resulted in an attenuated inflammatory response, a weaker interferon response, and reduced cell death. Additionally, the H1N1 viruses of swine origin replicated less efficiently at the temperature of the human proximal airways (33°C). We also observed that the 2009 H1N1 viruses replicated to significantly higher titers than seasonal H1N1 virus in polarized colon epithelial cells. These studies reveal that in comparison to seasonal influenza virus, H1N1 viruses of swine origin poorly activate multiple aspects of the human innate response, which may contribute to the virulence of these viruses. In addition, their less efficient replication at human upper airway temperatures has implications for the understanding of pandemic H1N1 virus adaptation to humans.

The influenza pandemic of 1918 to 1919 was one of the worst global pandemics in recent history. The highly pathogenic nature of the 1918 virus is thought to be mediated in part by a dysregulation of the host response, including an exacerbated proinflammatory cytokine response. In the present study, we compared the host transcriptional response to infection with the reconstructed 1918 virus in wild-type, tumor necrosis factor (TNF) receptor-1 knockout (TNFRKO), and interleukin-1 (IL-1) receptor-1 knockout (IL1RKO) mice as a means of further understanding the role of proinflammatory cytokine signaling during the acute response to infection. Despite reported redundancy in the functions of IL-1β and TNF-α, we observed that reducing the signaling capacity of each of these molecules by genetic disruption of their key receptor genes had very different effects on the host response to infection. In TNFRKO mice, we found delayed or decreased expression of genes associated with antiviral and innate immune signaling, complement, coagulation, and negative acute-phase response. In contrast, in IL1RKO mice numerous genes were differentially expressed at 1 day postinoculation, including an increase in the expression of genes that contribute to dendritic and natural killer cell processes and cellular movement, and gene expression profiles remained relatively constant at later time points. We also observed a compensatory increase in TNF-α expression in virus-infected IL1RKO mice. Our data suggest that signaling through the IL-1 receptor is protective, whereas signaling through the TNF-α receptor increases the severity of 1918 virus infection. These findings suggest that manipulation of these pathways may have therapeutic benefit.

Toward a General Flu Vaccination Current seasonal influenza virus vaccines are targeted against specific viral strains and do not provide broad, durable protection. Seasonal influenza vaccines induce protective antibody responses against regions of viral hemagglutinin (HA) that rapidly mutate so that very soon, the virus becomes resistant to vaccination. Conserved regions of HA also exist, and a major goal of influenza vaccine development is to design a vaccine that elicits antibodies against the conserved regions so that protection against a wide range of viral strains is achieved. Wei et al. (p. 1060 , published online 15 July; see the Perspective by Doms ) show that a combined HA DNA prime, followed by boosting with a seasonal vaccine, elicits broadly cross-reactive neutralizing antibody responses in mice, ferrets, and nonhuman primates, which were protective in mice and ferrets against heterologous influenza challenge. The neutralizing antibodies were directed against the conserved HA stem region, which indicates the possibility that a more broadly protective vaccine against influenza could be developed.

The pandemic H1N1 virus of 2009 (2009 H1N1) continues to cause illness worldwide, primarily in younger age groups. To better understand the pathogenesis of these viruses in mammals, we used a mouse model to evaluate the relative virulence of selected 2009 H1N1 viruses and compared them to a representative human triple-reassortant swine influenza virus that has circulated in pigs in the United States for over a decade preceding the current pandemic. Additional comparisons were made with the reconstructed 1918 virus, a 1976 H1N1 swine influenza virus, and a highly pathogenic H5N1 virus. Mice were inoculated intranasally with each virus and monitored for morbidity, mortality, viral replication, hemostatic parameters, cytokine production, and lung histology. All 2009 H1N1 viruses replicated efficiently in the lungs of mice and possessed a high degree of infectivity but did not cause lethal disease or exhibit extrapulmonary virus spread. Transient weight loss, lymphopenia, and proinflammatory cytokine and chemokine production were present following 2009 H1N1 virus infection, but these levels were generally muted compared with a triple-reassortant swine virus and the 1918 virus. 2009 H1N1 viruses isolated from fatal cases did not demonstrate enhanced virulence in this model compared with isolates from mild human cases. Histologically, infection with the 2009 viruses resulted in lesions in the lung varying from mild to moderate bronchiolitis with occasional necrosis of bronchiolar epithelium and mild to moderate peribronchiolar alveolitis. Taken together, these studies demonstrate that the 2009 H1N1 viruses exhibited mild to moderate virulence in mice compared with highly pathogenic viruses.

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virusin vitro, and one exerted a marked therapeutic effectin vivo.

The 1918 H1N1 pandemic led to as many as 50 million deaths worldwide, making it the deadliest human influenza virus in recorded history. Poor control measures and the erroneous belief that the culprit was a bacterium called Pfeiffer's bacillus, a gram-negative bacterium now recognized as Haemophilus influenzae, contributed to that dreadful outcome. Subsequent influenza pandemics in 1957 with an H2N2 strain and in 1968 with an H3N2 strain were less severe, offering little to explain the extraordinary virulence of the 1918 strain. As the influenza virus was not isolated in a laboratory until 1930, experts long thought that the 1918 pandemic virus was lost to history.

Purpose: New antiviral agents were prepared by attaching derivatives of sialic acid (1) and of the drug zanamivir (2) to poly(isobutylene-alt-maleic anhydride) (poly-(1 + 2)) or by mixing poly-1 and poly-2, followed by assaying them against wild-type and drug-resistant influenza A Wuhan viruses. Methods: Individually or together, 1 and 2 were covalently bonded to the polymer. The antiviral potencies of the resultant poly-1, poly-2, poly-(1 + 2), and poly-1 + poly-2, as well as 1 and 2, were assessed using plaque reduction assay. Results: Attaching 1 to the polymer improved at best millimolar IC50 values over three orders of magnitude. While 2 exhibited micromolar IC50 values, poly-2 was >100-fold even more potent. The IC50 of poly-(1 + 2) against the wild-type strain was >300-fold and ∼17-fold better than of poly-1 and poly-2, respectively. In contrast, the potency of poly-(1 + 2) vs. poly-2 against the mutant strain merely doubled. The mixture of poly-1 + poly-2 inhibited both viral strains similarly to poly-2. Conclusions: The bifunctional poly-(1 + 2) acts synergistically against the wild-type influenza virus, but not against its drug-resistant mutant, as compared to a physical mixture of the monofunctional poly-1 and poly-2., National Institutes of Health (U.S.) (NIH grant U01-AI074443), Fundación Ramón Areces (postdoctoral fellowship)

The “Spanish influenza” of 1918 claimed an unprecedented number of lives, yet the determinants of virulence for this virus are still not fully understood. Here, we used functional genomics and an in vitro human lung epithelial cell infection model to define the global host transcriptional response to the eight-gene 1918 virus. To better understand the role of the 1918 virus NS1 gene, we also evaluated the host response to a reassortant 1918 virus containing the NS1 gene from A/Texas/36/91 (a seasonal isolate of human influenza virus), as well as the host response to a reassortant of A/Texas/36/91 containing the 1918 NS1 gene. Genomic analyses revealed that the 1918 virus blocked the transcription of multiple interferon-stimulated genes and also downregulated a network of genes associated with lipid metabolism. In contrast, the expression of genes encoding chemokines and cytokines, which serve to attract infiltrating immune cells, was upregulated. Viruses containing the NS1 gene from A/Texas/36/91 induced a significant increase in type I interferon signaling but did not repress lipid metabolism. The 1918 NS1 gene may therefore have contributed to the virulence of the 1918 pandemic virus by disrupting the innate immune response, inducing hypercytokinemia, and by blocking the transcription of certain lipid-based proinflammatory mediators that function as part of the host antiviral response.

The 1918 pandemic influenza virus has demonstrated significant pathogenicity in animal models and is the progenitor of ‘classical’ swine and modern seasonal human H1N1 lineages. Here we characterize the pathogenicity of an early ‘classical’ swine H1N1 influenza A virus isolated in 1931 compared to the pathogenicity of the 1918 pandemic virus and a seasonal H1N1 virus in mice and ferrets. A/Swine/Iowa/31 (Sw31) and the 1918 influenza viruses were uniformly lethal in mice at low doses and produced severe lung pathology. In ferrets, Sw31 and 1918 influenza viruses caused severe clinical disease and lung pathology with necrotizing bronchiolitis and alveolitis. The modern H1N1 virus caused little disease in either animal model. These findings revealed that in these models the virulence factors of the 1918 influenza virus are likely preserved in the Sw31 virus and suggest that early swine viruses may be a good surrogate model to study 1918 virulence and pathogenesis.

Swine influenza was first recognized as a disease entity during the 1918 “Spanish flu” pandemic. The aim of this work was to determine the virulence of a plasmid-derived human 1918 pandemic H1N1 influenza virus (reconstructed 1918, or 1918/rec, virus) in swine using a plasmid-derived A/swine/Iowa/15/1930 H1N1 virus (1930/rec virus), representing the first isolated influenza virus, as a reference. Four-week-old piglets were inoculated intratracheally with either the 1930/rec or the 1918/rec virus or intranasally with the 1918/rec virus. A transient increase in temperature and mild respiratory signs developed postinoculation in all virus-inoculated groups. In contrast to other mammalian hosts (mice, ferrets, and macaques) where infection with the 1918/rec virus was lethal, the pigs did not develop severe respiratory distress or become moribund. Virus titers in the lower respiratory tract as well as macro- and microscopic lesions at 3 and 5 days postinfection (dpi) were comparable between the 1930/rec and 1918/rec virus-inoculated animals. In contrast to the 1930/rec virus-infected animals, at 7 dpi prominent lung lesions were present in only the 1918/rec virus-infected animals, and all the piglets developed antibodies at 7 dpi. Presented data support the hypothesis that the 1918 pandemic influenza virus was able to infect and replicate in swine, causing a respiratory disease, and that the virus was likely introduced into the pig population during the 1918 pandemic, resulting in the current lineage of the classical H1N1 swine influenza viruses.

Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the increasing genetic diversity among these viruses and continued outbreaks in avian species underscore the need for more effective measures for the control and prevention of human H5N1 virus infection. Additional small animal models with which therapeutic approaches against virulent influenza viruses can be evaluated are needed. In this study, we used the guinea pig model to evaluate the relative virulence of selected avian and human influenza A viruses. We demonstrate that guinea pigs can be infected with avian and human influenza viruses, resulting in high titers of virus shedding in nasal washes for up to 5 days postinoculation (p.i.) and in lung tissue of inoculated animals. However, other physiologic indicators typically associated with virulent influenza virus strains were absent in this species. We evaluated the ability of intranasal treatment with human alpha interferon (-IFN) to reduce lung and nasal wash titers in guinea pigs challenged with the reconstructed 1918 pandemic H1N1 virus or a contemporary H5N1 virus. IFN treatment initiated 1 day prior to challenge significantly reduced or prevented infection of guinea pigs by both viruses, as measured by virus titer determination and seroconversion. The expression of the antiviral Mx protein in lung tissue correlated with the reduction of virus titers. We propose that the guinea pig may serve as a useful small animal model for testing the efficacy of antiviral compounds and that -IFN treatment may be a useful antiviral strategy against highly virulent strains with pandemic potential. Since 2003, influenza A viruses of the H5N1 subtype have caused devastating outbreaks in poultry in Asia, Africa, and Europe, resulting in over 400 human infections, with an overall case fatality rate of 60% (1). The increasing persistence and genetic diversity of H5N1 viruses in poultry with concomitant human infection indicate that H5N1 viruses remain a pandemic threat (3). Despite evidence for limited human-to-human transmission, these viruses have yet to exhibit sustained transmission among humans (30, 50, 77). If H5N1 viruses were to acquire this ability, the resulting pandemic could be unusually severe, requiring multiple control measures to limit the morbidity and mortality associated with a pandemic virus. Vaccination remains the primary method of reducing the morbidity associated with seasonal influenza virus infection. Due to the diversity in circulating H5N1 viruses and the overall timeline for manufacturing, antigenically well-matched vaccines may not be available in the initial stages of an H5N1 pandemic (67, 69).

The relationship between immunosenescence and the host response to virus infection is poorly understood at the molecular level. Two different patterns of pulmonary host responses to virus were observed when gene expression profiles from severe acute respiratory syndrome coronavirus (SARS-CoV)-infected young mice that show minimal disease were compared to those from SARS-CoV-infected aged mice that develop pneumonitis. In young mice, genes related to cellular development, cell growth, and cell cycle were downregulated during peak viral replication, and these transcripts returned to basal levels as virus was cleared. In contrast, aged mice had a greater number of upregulated immune response and cell-to-cell signaling genes, and the expression of many genes was sustained even after viral clearance, suggesting an exacerbated host response to virus. Interestingly, in SARS-CoV-infected aged mice, a subset of genes, including Tnfa, Il6, Ccl2, Ccl3, Cxcl10, and Ifng, was induced in a biphasic pattern that correlated with peak viral replication and a subsequent influx of lymphocytes and severe histopathologic changes in the lungs. We provide insight into gene expression profiles and molecular signatures underlying immunosenescence in the context of the host response to viral infection.