RESUMO
The sporadic occurrence of human infections with swine-origin influenza A(H3N2) viruses and the continual emergence of novel A(H3N2) viruses in swine herds underscore the necessity for ongoing assessment of the pandemic risk posed by these viruses. Here, we selected 3 recent novel swine-origin A(H3N2) viruses isolated between 2017 to 2020, bearing hemagglutinins from the 1990.1, 2010.1, or 2010.2 clades, and evaluated their ability to cause disease and transmit in a ferret model. We conclude that despite considerable genetic variances, all 3 contemporary swine-origin A(H3N2) viruses displayed a capacity for robust replication in the ferret respiratory tract and were also capable of limited airborne transmission. These findings highlight the continued public health risk of swine-origin A(H3N2) strains, especially in human populations with low cross-reactive immunity.
Assuntos
Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Doenças dos Suínos , Humanos , Animais , Estados Unidos/epidemiologia , Suínos , Vírus da Influenza A Subtipo H3N2/genética , FurõesRESUMO
Despite reports of confirmed human infection following ocular exposure with both influenza A virus (IAV) and SARS-CoV-2, the dynamics of virus spread throughout oculonasal tissues and the relative capacity of virus transmission following ocular inoculation remain poorly understood. Furthermore, the impact of exposure route on subsequent release of airborne viral particles into the air has not been examined previously. To assess this, ferrets were inoculated by the ocular route with A(H1N1)pdm09 and A(H7N9) IAVs and two SARS-CoV-2 (early pandemic Washington/1 and Delta variant) viruses. Virus replication was assessed in both respiratory and ocular specimens, and transmission was evaluated in direct contact or respiratory droplet settings. Viral RNA in aerosols shed by inoculated ferrets was quantified with a two-stage cyclone aerosol sampler (National Institute for Occupational Safety and Health [NIOSH]). All IAV and SARS-CoV-2 viruses mounted a productive and transmissible infection in ferrets following ocular inoculation, with peak viral titers and release of virus-laden aerosols from ferrets indistinguishable from those from ferrets inoculated by previously characterized intranasal inoculation methods. Viral RNA was detected in ferret conjunctival washes from all viruses examined, though infectious virus in this specimen was recovered only following IAV inoculation. Low-dose ocular-only aerosol exposure or inhalation aerosol exposure of ferrets to IAV similarly led to productive infection of ferrets and shedding of aerosolized virus. Viral evolution during infection was comparable between all inoculation routes examined. These data support that both IAV and SARS-CoV-2 can establish a high-titer mammalian infection following ocular exposure that is associated with rapid detection of virus-laden aerosols shed by inoculated animals. IMPORTANCE Documented human infection with influenza viruses and SARS-CoV-2 has been reported among individuals wearing respiratory protection in the absence of eye protection, highlighting the capacity of these respiratory tract-tropic viruses to exploit nonrespiratory routes of exposure to initiate productive infection. However, comprehensive evaluations of how ocular exposure may modulate virus pathogenicity and transmissibility in mammals relative to respiratory exposure are limited and have not investigated multiple virus families side by side. Using the ferret model, we show that ocular exposure with multiple strains of either coronaviruses or influenza A viruses leads to an infection that results in shedding of detectable aerosolized virus from inoculated animals, contributing toward onward transmission of both viruses to susceptible contacts. Collectively, these studies support that the ocular surface represents a susceptible mucosal surface that, if exposed to a sufficient quantity of either virus, permits establishment of an infection which is similarly transmissible as that following respiratory exposure.
Assuntos
COVID-19 , Infecções por Orthomyxoviridae , Animais , Humanos , COVID-19/transmissão , COVID-19/virologia , Modelos Animais de Doenças , Furões , Vírus da Influenza A Subtipo H1N1 , Subtipo H7N9 do Vírus da Influenza A , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Aerossóis e Gotículas Respiratórios , RNA Viral/isolamento & purificação , SARS-CoV-2 , Eliminação de Partículas ViraisRESUMO
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.
Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/virologia , Influenza Humana/virologia , Fenótipo , Replicação Viral/genética , Animais , Ásia , China , Modelos Animais de Doenças , Feminino , Variação Genética , Humanos , Influenza Aviária/imunologia , Influenza Aviária/transmissão , Influenza Humana/imunologia , Influenza Humana/transmissão , Masculino , Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , VietnãRESUMO
Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Tropismo Viral/genética , Animais , Brônquios/virologia , Técnicas de Cultura de Células/métodos , Cílios/virologia , Modelos Animais de Doenças , Células Epiteliais/virologia , Furões/virologia , Células Caliciformes/metabolismo , Células Caliciformes/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Cultura Primária de Células , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Traqueia/virologia , Viroses/genéticaRESUMO
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.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , RNA Viral/genética , Análise de Sequência de RNA/métodos , Animais , Linhagem Celular , China/epidemiologia , Chlorocebus aethiops , Epidemias , Evolução Molecular , Furões , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/epidemiologia , Influenza Humana/transmissão , Camundongos , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Medição de Risco , Células Vero , Tropismo Viral , VirulênciaRESUMO
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.
Assuntos
Hemaglutinação por Vírus/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N2/genética , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/transmissão , Replicação Viral/genética , Animais , Chlorocebus aethiops , Feminino , Furões , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Especificidade da Espécie , Suínos , Células VeroRESUMO
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.
Assuntos
Transmissão de Doença Infecciosa , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Doenças dos Suínos/virologia , Replicação Viral , Animais , Linhagem Celular , Modelos Animais de Doenças , Furões , Humanos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/patologia , Sistema Respiratório/virologia , Suínos , Estados Unidos , Carga ViralRESUMO
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) virus. 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). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. 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.
Assuntos
Furões/virologia , Vírus da Influenza A/patogenicidade , Camundongos/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Animais , Linhagem Celular , Polaridade Celular , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Humanos , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H9N2/patogenicidade , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos BALB C , Polissacarídeos/química , Polissacarídeos/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Sistema Respiratório/citologia , Especificidade por Substrato , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
Vírus da Influenza A Subtipo H7N2/isolamento & purificação , Influenza Humana/virologia , Doenças Profissionais/virologia , Médicos Veterinários , Animais , Gatos , Linhagem Celular , Modelos Animais de Doenças , Transmissão de Doença Infecciosa , Furões , Humanos , Vírus da Influenza A Subtipo H7N2/patogenicidade , Vírus da Influenza A Subtipo H7N2/fisiologia , Camundongos , Cidade de Nova Iorque , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Virulência , Replicação ViralRESUMO
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.
Assuntos
Vírus da Influenza A Subtipo H3N2/metabolismo , Influenza Humana/virologia , Animais , Brônquios/citologia , Criança , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Furões , Humanos , Vírus da Influenza A/metabolismo , Influenza Humana/transmissão , Cinética , Masculino , Polissacarídeos/química , Suínos/virologiaRESUMO
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.
Assuntos
Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Reações Cruzadas , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Testes de Inibição da Hemaglutinação , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Infecções por OrthomyxoviridaeRESUMO
Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.
Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Linfócitos B/imunologia , Surtos de Doenças , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Sobrevida , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/genética , Linhagem Celular , Reações Cruzadas/imunologia , Surtos de Doenças/história , Cães , Feminino , História do Século XX , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
IMPORTANCE: Despite the accumulation of evidence showing that airborne transmissible influenza A virus (IAV) typically has a lower pH threshold for hemagglutinin (HA) fusion activation, the underlying mechanism for such a link remains unclear. In our study, by using a pair of isogenic recombinant A(H9N2) viruses with a phenotypical difference in virus airborne transmission in a ferret model due to an acid-destabilizing mutation (HA1-Y17H) in the HA, we demonstrate that an acid-stable A(H9N2) virus possesses a multitude of advantages over its less stable counterpart, including better fitness in the ferret respiratory tract, more effective aerosol emission from infected animals, and improved host susceptibility. Our study provides supporting evidence for the requirement of acid stability in efficient airborne transmission of IAV and sheds light on fundamental mechanisms for virus airborne transmission.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H9N2 , Influenza Humana , Animais , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/metabolismo , Aerossóis e Gotículas Respiratórios/virologia , Influenza Humana/transmissão , Humanos , Modelos Animais de Doenças , Substituição de AminoácidosRESUMO
Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses have caused large outbreaks within avian populations on five continents, with concurrent spillover into a variety of mammalian species. Mutations associated with mammalian adaptation have been sporadically identified in avian isolates, and more frequently among mammalian isolates following infection. Reports of human infection with A(H5N1) viruses following contact with infected wildlife have been reported on multiple continents, highlighting the need for pandemic risk assessment of these viruses. In this study, the pathogenicity and transmissibility of A/Chile/25945/2023 HPAI A(H5N1) virus, a novel reassortant with four gene segments (PB1, PB2, NP, MP) from North American lineage, isolated from a severe human case in Chile, was evaluated in vitro and using the ferret model. This virus possessed a high capacity to cause fatal disease, characterized by high morbidity and extrapulmonary spread in virus-inoculated ferrets. The virus was capable of transmission to naïve contacts in a direct contact setting, with contact animals similarly exhibiting severe disease, but did not exhibit productive transmission in respiratory droplet or fomite transmission models. Our results indicate that the virus would need to acquire an airborne transmissible phenotype in mammals to potentially cause a pandemic. Nonetheless, this work warrants continuous monitoring of mammalian adaptations in avian viruses, especially in strains isolated from humans, to aid pandemic preparedness efforts.
Assuntos
Furões , Virus da Influenza A Subtipo H5N1 , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Furões/virologia , Humanos , Chile , Influenza Humana/virologia , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/patogenicidade , Vírus Reordenados/classificação , Filogenia , Influenza Aviária/virologia , Influenza Aviária/transmissãoRESUMO
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.
Assuntos
Células Endoteliais/virologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Pulmão/citologia , Síndrome do Desconforto Respiratório/virologia , Replicação Viral , Animais , Linhagem Celular , Embrião de Galinha , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1 , Influenza Humana/genética , Influenza Humana/imunologia , Pulmão/imunologia , Pulmão/virologia , Ratos , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/imunologia , VirulênciaRESUMO
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.
Assuntos
Proteção Cruzada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Reações Cruzadas , Modelos Animais de Doenças , Furões , Febre/prevenção & controle , Humanos , Vacinas contra Influenza/administração & dosagem , Pulmão/patologia , Masculino , Infecções por Orthomyxoviridae/imunologia , Pneumonia Viral/prevenção & controle , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Eliminação de Partículas Virais , Redução de PesoRESUMO
The ferret transmission model is routinely used to evaluate the pandemic potential of newly emerging influenza A viruses. However, concurrent measurement of viral load in the air is typically not a component of such studies. To address this knowledge gap, we measured the levels of virus in ferret nasal washes as well as viral RNA emitted into the air for 14 diverse influenza viruses, encompassing human-, swine-, and avian-origin strains. Here we show that transmissible viruses display robust replication and fast release into the air. In contrast, poorly- and non-transmissible viruses show significantly reduced or delayed replication along with lower detection of airborne viral RNA at early time points post inoculation. These findings indicate that efficient ferret-to-ferret transmission via the air is directly associated with fast emission of virus-laden particles; as such, quantification of viral RNA in the air represents a useful addition to established assessments of new influenza virus strains.
Assuntos
Vírus da Influenza A , Infecções por Orthomyxoviridae , Humanos , Animais , Suínos , Furões , Aves , RNA ViralRESUMO
While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.
Assuntos
Conjuntivite , Vírus da Influenza A , Influenza Humana , Animais , Humanos , Cães , Vírus da Influenza A/genética , Sistema Respiratório , Células Madin Darby de Rim CaninoRESUMO
Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.2) variant displaced the prototype Washington/1/2020 (WA/1) strain. While WA/1 virus exhibited a moderately increased proportion compared to that in the inoculum following co-infection in human respiratory cells, Delta variant possessed a substantial in vivo fitness advantage as this virus becoming predominant in both inoculated and contact animals. This work identifies critical traits of the Delta variant that likely played a role in it becoming a dominant variant and highlights the necessities of employing multiple model systems to assess the fitness of newly emerged SARS-CoV-2 variants.