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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.

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.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

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.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States’ jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies., Hurricane Maria hit Puerto Rico in 2017 and resulted in a complete loss of activity of the Public Health Laboratories. Here, the authors discuss the approach taken and tools developed to re-establish activity in these laboratories using a quality management system and the lessons learned in this process.

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 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.

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.

After their disappearance from the human population in 1968, influenza H2 viruses have continued to circulate in the natural avian reservoir. The isolation of this virus subtype from multiple bird species as well as swine highlights the need to better understand the potential of these viruses to spread and cause disease in humans. Here we analyzed the virulence, transmissibility and receptor-binding preference of two avian influenza H2 viruses (H2N2 and H2N3) and compared them to a swine H2N3 (A/swine/Missouri/2124514/2006 [swMO]), and a human H2N2 (A/England/10/1967 [Eng/67]) virus using the ferret model as a mammalian host. Both avian H2 viruses possessed the capacity to spread efficiently between cohoused ferrets, and the swine (swMO) and human (Eng/67) viruses transmitted to naïve ferrets by respiratory droplets. Further characterization of the swMO hemagglutinin (HA) by x-ray crystallography and glycan microarray array identified receptor-specific adaptive mutations. As influenza virus quasispecies dynamics during transmission have not been well characterized, we sequenced nasal washes collected during transmission studies to better understand experimental adaptation of H2 HA. The avian H2 viruses isolated from ferret nasal washes contained mutations in the HA1, including a Gln226Leu substitution, which is a mutation associated with α2,6 sialic acid (human-like) binding preference. These results suggest that the molecular structure of HA in viruses of the H2 subtype continue to have the potential to adapt to a mammalian host and become transmissible, after acquiring additional genetic markers.

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.

The new H7N9 influenza virus, recently emerged in China, can replicate in human airway cells and in the respiratory tract of ferrets to a higher level than can seasonal H3N2 virus and shows higher lethality in mice than genetically related H7N9 and H9N2 viruses, but shows limited transmission in ferrets by respiratory droplets. Supplementary information The online version of this article (doi:10.1038/nature12391) contains supplementary material, which is available to authorized users., Transmission of emerging H7N9 virus By 20 July 2013, there had been 134 laboratory-confirmed human cases of infection with avian influenza A H7N9 virus infection, including 43 deaths. Yoshihiro Kawaoka and colleagues characterize the biology of two recent isolates of the virus. They provide a wealth of data from infections in mice, pigs, macaques and ferrets. H7N9 virus is shown to be less sensitive to neuraminidase inhibitors than pandemic H1N1 virus, but equally susceptible to an experimental polymerase inhibitor. Terrence Tumpey and colleagues determine the capacity of two clinical H7N9 isolates to cause disease and transmit between mammals. They show that the virus can replicate in human airway cells and in the respiratory tract of ferrets to a higher level than can seasonal H3N2 virus, and show higher lethality in mice than genetically related H7N9 and H9N2 viruses. In transmission studies, the H7N9 virus showed limited transmission in ferrets by respiratory droplets. Ron Fouchier and colleagues investigate the transmissibility of H7N9 virus between ferrets. They show that airborne transmission can occur, but inefficiently. They also show that on passage in ferrets, virus variants that have higher avian receptor binding, higher pH of fusion and lower thermostability are selected, and they suggest that these characteristics may result in reduced transmissibility. Supplementary information The online version of this article (doi:10.1038/nature12391) contains supplementary material, which is available to authorized users., On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus1. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS)2. This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease3. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus. Supplementary information The online version of this article (doi:10.1038/nature12391) contains supplementary material, which is available to authorized users.

Sporadic avian to human transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses necessitates the analysis of currently circulating and evolving clades to assess their potential risk. Following the spread and sustained circulation of clade 2 viruses across multiple continents, numerous subclades and genotypes have been described. To better understand the pathogenesis associated with the continued diversification of clade 2 A(H5N1) influenza viruses, we investigated the relative virulence of eleven human and poultry isolates collected from 2006 to 2013 by determining their ability to cause disease in the ferret model. Numerous clade 2 viruses, including a clade 2.2 avian isolate, a 2.2.2.1 human isolate, and two 2.2.1 human isolates, were found to be of low virulence in the ferret model, though lethality was detected following infection with one 2.2.1 human isolate. In contrast, three of six clade 2.3.2.1 avian isolates tested led to severe disease and death among infected ferrets. Clade 2.3.2.1b and 2.3.2.1c isolates, but not 2.3.2.1a isolates, were associated with ferret lethality. All A(H5N1) viruses replicated efficiently in the respiratory tract of ferrets regardless of their virulence and lethality. However, lethal isolates were characterized by systemic viral dissemination, including detection in the brain and enhanced histopathology in lung tissues. The finding of disparate virulence phenotypes between clade 2 A(H5N1) viruses, notably differences between subclades of 2.3.2.1 viruses, suggests there are distinct molecular determinants present within the established subclades, the identification of which will assist in molecular-based surveillance and public health efforts against A(H5N1) viruses.

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6–sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.

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.

Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced receptor binding domain mutations within the hemagglutinin (HA) gene of two H5N1 viruses and evaluated changes in receptor binding specificity by glycan microarray analysis. The impact of these mutations on replication efficiency was assessed in vitro and in vivo. Although certain mutations switched the receptor binding preference of the H5 HA, the rescued mutant viruses displayed reduced replication in vitro and delayed peak virus shedding in ferrets. An improvement in transmission efficiency was not observed with any of the mutants compared to the parental viruses, indicating that alternative molecular changes are required for H5N1 viruses to fully adapt to humans and to acquire pandemic capability.

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 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.

Immunization against conserved virus components induces broad, heterosubtypic protection against diverse influenza A viruses, providing a strategy for controlling unexpected outbreaks or pandemics until strain-matched vaccines become available. This study characterized immunization to nucleoprotein (NP) and matrix 2 (M2) by DNA priming followed by parenteral or mucosal boosting in mice and ferrets. DNA vaccination followed by boosting with antigen-matched recombinant adenovirus (rAd) or cold-adapted (ca) influenza virus provided robust protection against virulent H1N1 and H5N1 challenges. Compared to other boosts, mucosal rAd induced stronger IgA responses, more virus-specific activated T-cells in the lung, and better protection against morbidity following challenge even eight months post-boost. In ferrets, both mucosal and parenteral rAd boosting protected from lethal H5N1 challenge. These findings demonstrate potent protection by vaccination highly focused on conserved antigens and identify immune response measures in mice that differed among vaccinations and correlated with outcome.

available in PMC 2010 October 12, Recent reports of mild to severe influenza-like illness in humans caused by a novel swine-origin 2009 A(H1N1) influenza virus underscore the need to better understand the pathogenesis and transmission of these viruses in mammals. In this study, selected 2009 A(H1N1) influenza isolates were assessed for their ability to cause disease in mice and ferrets and compared with a contemporary seasonal H1N1 virus for their ability to transmit to naïve ferrets through respiratory droplets. In contrast to seasonal influenza H1N1 virus, 2009 A(H1N1) influenza viruses caused increased morbidity, replicated to higher titers in lung tissue, and were recovered from the intestinal tract of intranasally inoculated ferrets. The 2009 A(H1N1) influenza viruses exhibited less efficient respiratory droplet transmission in ferrets in comparison with the highly transmissible phenotype of a seasonal H1N1 virus. Transmission of the 2009 A(H1N1) influenza viruses was further corroborated by characterizing the binding specificity of the viral hemagglutinin to the sialylated glycan receptors (in the human host) by use of dose-dependent direct receptor-binding and human lung tissue–binding assays.

Generation of Swine Flu As the newly emerged influenza virus starts its journey to infect the world's human population, the genetic secrets of the 2009 outbreak of swine influenza A(H1N1) are being revealed. In extensive phylogenetic analyses, Garten et al. (p. 197 , published online 22 May) confirm that of the eight elements of the virus, the basic components encoded by the hemagglutinin, nucleoprotein, and nonstructural genes originated in birds and transferred to pigs in 1918. Subsequently, these formed a triple reassortant with the RNA polymerase PB1 that transferred from birds in 1968 to humans and then to pigs in 1998, coupled with RNA polymerases PA and PB2 that transferred from birds to pigs in 1998. The neuraminidase and matrix protein genes that complete the virus came from birds and entered pigs in 1979. The analysis offers insights into drug susceptibility and virulence, as well as raising the possibility of hitherto unknown factors determining host specificity. A significant question is, what is the potential for the H1 component of the current seasonal flu vaccine to act as a booster? Apart from the need for ongoing sequencing to monitor for the emergence of new reassortants, future pig populations need to be closely monitored for emerging influenza viruses.

Avian H7 influenza viruses from both the Eurasian and North American lineage have caused outbreaks in poultry since 2002, with confirmed human infection occurring during outbreaks in The Netherlands, British Columbia, and the United Kingdom. The majority of H7 infections have resulted in self-limiting conjunctivitis, whereas probable human-to-human transmission has been rare. Here, we used glycan microarray technology to determine the receptor-binding preference of Eurasian and North American lineage H7 influenza viruses and their transmissibility in the ferret model. We found that highly pathogenic H7N7 viruses from The Netherlands in 2003 maintained the classic avian-binding preference for α2–3-linked sialic acids (SA) and are not readily transmissible in ferrets, as observed previously for highly pathogenic H5N1 viruses. However, H7N3 viruses isolated from Canada in 2004 and H7N2 viruses from the northeastern United States isolated in 2002–2003 possessed an HA with increased affinity toward α2–6-linked SA, the linkage type found prominently on human tracheal epithelial cells. We identified a low pathogenic H7N2 virus isolated from a man in New York in 2003, A/NY/107/03, which replicated efficiently in the upper respiratory tract of ferrets and was capable of transmission in this species by direct contact. These results indicate that H7 influenza viruses from the North American lineage have acquired sialic acid-binding properties that more closely resemble those of human influenza viruses and have the potential to spread to naïve animals.

Influenza epidemics or pandemics can arise for which strain- or subtype-matched vaccines are unavailable. Heterosubtypic immunity (Het-I) targeting conserved influenza A antigens could reduce morbidity and mortality during preparation of matched vaccines. Various vaccines inducing Het-I in animals have been studied separately using different viruses and conditions, but effectiveness for inducing Het-I has not been directly compared. The present studies compared immunization with cold-adapted (ca) viruses to DNA prime-recombinant adenovirus (rAd) boost vaccination to conserved antigens nucleoprotein (NP), matrix-2 (M2), or A/NP+M2. Both ca and DNA-rAd vaccinations induced antibody and T cell responses, and protected against lethal H1N1 challenge. Only A/NP+M2 DNA-rAd protected against challenge with highly pathogenic A/Vietnam/1203/2004 (H5N1); ca vaccine did not. Existing ca vaccines may provide some Het-I, but experimental vaccination focusing on conserved antigens was more effective in this model for protection against a divergent, highly pathogenic virus.

Influenza virus of the H7N2 subtype has been introduced into noncommercial poultry in the United States, and this probably resulted in incidents of transmission of H7N2 virus to humans, documented in 2002 and 2003. This virus could be considered a potential threat to public health if it acquired person-to-person transmissibility. A favored approach for global pandemic preparedness includes development of prepandemic vaccines for any potential pandemic virus. To this end, we created a high-growth reassortant virus (H7N2-PR8) containing the genes for the hemagglutinin and the neuraminidase from a low-pathogenicity (H7N2) virus strain and the remaining six genes from a human vaccine strain (H1N1). The reassortant strain was evaluated to assess its antigenicity, safety, and protective efficacy using a mouse model. Antigenicity studies using ferret antibodies raised against H7N2-PR8 indicated that this virus confers broad cross-reactivity with divergent H7 viruses of different years and lineages. Mice and chickens inoculated with high doses of H7N2-PR8 supported virus replication but survived, indicating that this virus is comparable to other avian viruses of low pathogenicity. To assess the protective efficacy of H7N2-PR8, mice were immunized with two doses of formalin-inactivated H7N2-PR8, alone or with alum. Vaccinated mice subsequently challenged with highly pathogenic viruses from homologous and heterologous lineages A/Canada/444/04 (H7N3) and A/Netherlands/219/03 (H7N7) showed pronounced reduction of wild-type virus replication. These studies indicate that H7N2-PR8 is immunogenic, safe, and protective in animal models; these are the essential attributes to qualify for phase I human clinical trials as a prepandemic vaccine.

The views expressed in this Perspective do not necessarily reflect the views of the journal or of ASM. Zoonotic influenza viruses circulating in poultry and swine pose an ever present threat to human health. In particular, the rapid geographical expansion of highly pathogenic avian influenza (HPAI) A(H5N1) throughout Asia and then into Europe, the Middle East, and Africa during the 2000s galvanized the global community in an attempt to control this rapidly growing threat. Despite successful control efforts in some countries, the virus remains endemic in poultry in at least six countries and continues to cause human illness and deaths as well as countless outbreaks in birds. During the past decade, 668 cases and 393 deaths were detected and reported to the World Health Organization (WHO) (1). During the 17 years since human infections with HPAI A(H5N1) were first identified in Hong Kong, Special Administrative Region, People’s Republic of China, in 1997, these viruses have evolved substantially through mutation and reassortment, resulting in multiple divergent genotypes and clades (2). Ongoing H5N1 circulation has appropriately resulted in a focus on sequencing viral genomes to understand the evolution of these viruses and the significance of observed genetic changes. Expanded laboratory capacity for high-throughput Sanger sequencing and recent technological advances, such as next-generation sequencing and parallel computing, have revolutionized the quantity, quality, and availability of gene sequences and our ability to quickly and accurately analyze these data (3). Consequently, the number of animal and human influenza virus sequences available in publically accessible databases has dramatically increased over the years, as have the bioinformatics …

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.

The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA) to α2→6 sialylated glycan receptors (human receptors). Here we report that a single point mutation (Ile219→Lys; a base pair change) in the glycan receptor-binding site (RBS) of a representative 2009 H1N1 influenza A virus, A/California/04/09 or CA04/09, quantitatively increases its human receptor-binding affinity. The increased human receptor-affinity is in the same range as that of the HA from highly transmissible seasonal and 1918 pandemic H1N1 viruses. Moreover, a 2009 H1N1 virus carrying this mutation in the RBS (generated using reverse genetics) transmits efficiently in ferrets by respiratory droplets thereby reestablishing our previously observed correlation between human receptor-binding affinity and transmission efficiency. These findings are significant in the context of monitoring the evolution of the currently circulating 2009 H1N1 viruses.

Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naïve ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses., Centers for Disease Control and Prevention (U.S.), National Institutes of Health (U.S.) (grant U54 GM62116), Singapore-MIT Alliance for Research and Technology

Influenza viruses with novel hemagglutinin and 1 or more accompanying genes derived from avian influenza viruses sporadically emerge in humans and have the potential to result in a pandemic if the virus causes disease and spreads efficiently in a population that lacks immunity to the novel hemagglutinin. Since 1997, multiple avian influenza virus subtypes have been transmitted directly from domestic poultry to humans and have caused a spectrum of human disease, from asymptomatic to severe and fatal. To assess the pandemic risk that avian influenza viruses pose, we have used multiple strategies to better understand the capacity of avian viruses to infect, cause disease, and transmit among mammals, including humans. Seroepidemiologic studies that evaluate the frequency and risk of human infection with avian influenza viruses in populations with exposure to domestic or wild birds can provide a better understanding of the pandemic potential of avian influenza subtypes. Investigations conducted in Hong Kong following the first H5N1 outbreak in humans in 1997 determined that exposure to poultry in live bird markets was a key risk factor for human disease. Among poultry workers, butchering and exposure to sick poultry were risk factors for antibody to H5 virus, which provided evidence for infection. A second risk assessment tool, the ferret, can be used to evaluate the level of virulence and potential for host-to-host transmission of avian influenza viruses in this naturally susceptible host. Avian viruses isolated from humans exhibit a level of virulence and transmissibility in ferrets that generally reflects that seen in humans. The ferret model thus provides a means to monitor emerging avian influenza viruses for pandemic risk, as well as to evaluate laboratory-generated reassortants and mutants to better understand the molecular basis of influenza virus transmissibility. Taken together, such studies provide valuable information with which we can assess the public health risk of avian influenza viruses.

The influenza virus genes that confer efficient transmission of epidemic and pandemic strains in humans have not been identified. The rapid spread and severe disease caused by the 1918 influenza pandemic virus makes it an ideal virus to study the transmissibility of potentially pandemic influenza strains. Here, we used a series of human 1918-avian H1N1 influenza reassortant viruses to identify the genetic determinants that govern airborne transmission of avian influenza viruses. We have demonstrated that the 1918 HA gene was necessary for efficient direct contact transmission, but did not allow respiratory droplet transmission between ferrets of an avian influenza virus possessing an avian polymerase subunit PB2. The 1918 PB2 protein was found to be both necessary and sufficient for airborne transmission of a virus expressing the 1918 HA and neuraminidase. Also, it was found that influenza viruses that were able to transmit efficiently in ferrets were able to replicate efficiently at the lower temperature (33 °C) found in the environment of mammalian airway. These findings demonstrate that the adaptation of the HA and PB2 proteins are critical for the development of pandemic influenza strains from avian influenza viruses.

The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the 1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain.

The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human alpha-2,6 to the avian alpha-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual alpha-2,6 and alpha-2,3 specificity suggests that a predominant human alpha-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals.

Corrigendum: Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like (α2-6 sialylated glycan) receptors and an increase in the binding to avian-like (α2-3 sialylated glycan) receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types., Centers for Disease Control and Prevention (U.S.), United States. National Institutes of Health (merit award R37 GM057073-13), Singapore-MIT Alliance for Research and Technology