Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-20 (of 20 Records) |
Query Trace: Creager HM [original query] |
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Utility of human in vitro data in risk assessments of influenza a virus using the ferret model
Creager HM , Kieran TJ , Zeng H , Sun X , Pulit-Penaloza JA , Holmes KE , Johnson AF , Tumpey TM , Maines TR , Beauchemin CAA , Belser JA . J Virol 2023 97 (1) e0153622 As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone. |
Inherent heterogeneity of influenza A virus stability following aerosolization
Belser JA , Pulit-Penaloza JA , Brock N , Creager HM , Gustin KM , Tumpey TM , Maines TR . Appl Environ Microbiol 2022 88 (4) aem0227121 Efficient human-to-human transmission represents a necessary adaptation for a zoonotic influenza A virus (IAV) to cause a pandemic. As such, many emerging IAVs are characterized for transmissibility phenotypes in mammalian models, with an emphasis on elucidating viral determinants of transmission and the role host immune responses contribute to mammalian adaptation. Investigations of virus infectivity and stability in aerosols concurrent with transmission assessments have increased in recent years, enhancing our understanding of this dynamic process. Here, we employ a diverse panel of 17 human and zoonotic IAVs, inclusive of seasonally circulating H1N1 and H3N2 viruses, and avian and swine viruses associated with human infection, to evaluate differences in spray factor (a value that assesses efficiency of the aerosolization process), stability, and infectivity following aerosolization. While most seasonal influenza viruses did not exhibit substantial variability within these parameters, there was more heterogeneity among zoonotic influenza viruses, which possess a diverse range of transmission phenotypes. Aging of aerosols at different relative humidities identified strain-specific levels of stability with different profiles identified between zoonotic H3, H5, and H7 subtype viruses associated with human infection. As studies continue to elucidate the complex components governing virus transmissibility, notably aerosol matrices and environmental parameters, considering the relative role of subtype- and strain-specific factors to modulate these parameters will improve our understanding of the pandemic potential of zoonotic influenza A viruses. Importance Transmission of respiratory pathogens through the air can facilitate the rapid and expansive spread of infection and disease through a susceptible population. While seasonal influenza viruses are quite capable of airborne spread, there is a lack of knowledge regarding how well influenza viruses remain viable after aerosolization, and if influenza viruses capable of jumping species barriers to cause human infection differ in this property from seasonal strains. We evaluated a diverse panel of influenza viruses associated with human infection (originating from human, avian, and swine reservoirs) for their ability to remain viable after aerosolization in the laboratory under a range of conditions. We found greater diversity among avian and swine-origin viruses compared with seasonal influenza viruses; strain-specific stability was also noted. Although influenza virus stability in aerosols is an underreported property, if molecular markers associated with enhanced stability are identified, we will be able to quickly recognize emerging strains of influenza that present the greatest pandemic threat. |
Genetically and antigenically divergent influenza A(H9N2) viruses exhibit differential replication and transmission phenotypes in mammalian models.
Belser JA , Sun X , Brock N , Pappas C , Pulit-Penaloza JA , Zeng H , Jang Y , Jones J , Carney PJ , Chang J , Van Long N , Diep NT , Thor S , Di H , Yang G , Cook PW , Creager HM , Wang D , McFarland J , Van Dong P , Wentworth DE , Tumpey TM , Barnes JR , Stevens J , Davis CT , Maines TR . J Virol 2020 94 (17) Low pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Co-circulation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world, and for over twenty years, have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported and was found to exhibit properties of adaptation to humans and, importantly, show similarities to strains isolated from the live bird markets of Vietnam. |
Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses.
Sun X , Belser JA , Yang H , Pulit-Penaloza JA , Pappas C , Brock N , Zeng H , Creager HM , Stevens J , Maines TR . Virology 2019 535 232-240 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. |
Risk assessment of fifth-wave H7N9 influenza A viruses in mammalian models
Sun X , Belser JA , Pappas C , Pulit-Penaloza JA , Brock N , Zeng H , Creager HM , Le S , Wilson M , Lewis A , Stark TJ , Shieh WJ , Barnes J , Tumpey TM , Maines TR . J Virol 2018 93 (1) The fifth-wave of the H7N9 influenza epidemic in China was distinguished by a sudden increase in human infections, an extended geographic distribution, and the emergence of highly pathogenic avian influenza (HPAI) viruses. Genetically, some H7N9 viruses from the fifth-wave have acquired novel amino acid changes at positions involved in mammalian adaptation, antigenicity, and HA cleavability. Here, several low pathogenic avian influenza (LPAI) and HPAI H7N9 human isolates from the fifth epidemic wave were assessed for their pathogenicity and transmissibility in mammalian models, as well as their ability to replicate in human airway epithelial cells. We found that a LPAI virus exhibited a similar capacity to replicate and cause disease in two animal species as viruses from previous waves. In contrast, HPAI H7N9 viruses possessed enhanced virulence, causing greater lethargy and mortality, with an extended tropism for brain tissues in both ferret and mouse models. These HPAI viruses also showed signs of adaptation to mammalian hosts by acquiring the ability to fuse at a lower pH threshold compared with other H7N9 viruses. All of the fifth-wave H7N9 viruses were able to transmit among cohoused ferrets, but exhibited a limited capacity to transmit by respiratory droplets and deep sequencing analysis revealed that the H7N9 viruses sampled after transmission showed a reduced amount of minor variants. Taken together, we conclude that the fifth-wave HPAI H7N9 viruses have gained the ability to cause enhanced disease in mammalian models, and with further adaptation may acquire the ability to cause an H7N9 pandemic.ImportanceThe potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increased number of human infections and the emergence of antigenically distinct LPAI and HPAI H7N9 viruses. In this study, a group of fifth-wave HPAI and LPAI viruses were evaluated for their ability to infect, cause disease, and transmit in small animal models. The ability of HPAI H7N9 viruses to cause more severe disease and to replicate in brain tissues in animal models as well as their ability to fuse at a lower pH threshold compared to LPAI H7N9 viruses suggest that the fifth-wave H7N9 viruses have evolved to acquire novel traits with the potential to pose a higher risk to humans. Although the fifth-wave H7N9 viruses have not yet gained the ability to transmit efficiently by air, continuous surveillance and risk assessment remain essential parts of our pandemic preparedness efforts. |
Infection of cultured mammalian cells with aerosolized influenza virus
Creager HM , Tumpey TM , Maines TR , Belser JA . Methods Mol Biol 2018 1836 387-399 Inoculation of animals via inhaled aerosols has long been used to study the infectivity and pathogenesis of both influenza virus and other respiratory pathogens in a context that mimics natural infection. In contrast, traditional in vitro studies of cellular tropism have been limited to the use of liquid inocula. We have recently shown that cultured cells can become successfully infected after exposure to aerosolized influenza virus. In this chapter, we describe the methodology employed, including the operation of aerosolization instrumentation and calculation of infectious dose, both in experimental planning and after exposure occurs. |
Pathogenesis and transmission of genetically diverse swine-origin H3N2v influenza A viruses from multiple lineages isolated in the United States, 2011-2016.
Sun X , Pulit-Penaloza JA , Belser JA , Pappas C , Pearce MB , Brock N , Zeng H , Creager HM , Zanders N , Jang Y , Tumpey TM , Davis T , Maines TR . J Virol 2018 92 (16) While several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011-2016 were evaluated for their replicative ability in both in vitro and in vivo mammalian models, as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacity in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.Importance: Swine-origin influenza viruses of the H3N2 subtype, with the HA and NA derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the U.S between 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the ability of these viruses to replicate, caused disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlights their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy. |
Antigenically diverse swine-origin H1N1 variant influenza viruses exhibit differential ferret pathogenesis and transmission phenotypes.
Pulit-Penaloza JA , Jones J , Sun X , Jang Y , Thor S , Belser JA , Zanders N , Creager HM , Ridenour C , Wang L , Stark TJ , Garten R , Chen LM , Barnes J , Tumpey TM , Wentworth DE , Maines TR , Davis CT . J Virol 2018 92 (11) Influenza A(H1) viruses circulating in swine represent an emerging virus threat as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus (A/Iowa/39/2015), as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Pre-existing immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a non-fatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with pre-existing immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015 demonstrating decreased protection. This data illustrates the potential for currently circulating swine influenza viruses to infect and cause illness in humans with pre-existing immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness. |
Infection and replication of influenza virus at the ocular surface
Creager HM , Kumar A , Zeng H , Maines TR , Tumpey TM , Belser JA . J Virol 2018 92 (7) Though influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subject to aerosol inoculation. The temperature at which cells were cultured post-inoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33 degrees C relative to 37 degrees C and this effect was slightly greater for the conjunctivitis isolates than the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multi-layer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited hemagglutination of both ocular and non-ocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of non-ocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells, but that this is more likely for ocular tropic viruses as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza viral tropism have yet to be fully elucidated. Here, we sought to identify factors which limit the ability of most influenza viruses to cause ocular infection. Though ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors which influence infection and replication in this non-canonical site may point toward novel determinants of tropism in the respiratory tract. |
Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.
Pulit-Penaloza JA , Simpson N , Yang H , Creager HM , Jones J , Carney P , Belser JA , Yang G , Chang J , Zeng H , Thor S , Jang Y , Killian ML , Jenkins-Moore M , Janas-Martindale A , Dubovi E , Wentworth DE , Stevens J , Tumpey TM , Davis CT , Maines TR . J Infect Dis 2017 216 S499-s507 Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health. |
Pathogenesis, transmissibility, and tropism of a highly pathogenic avian influenza A(H7N7) virus associated with human conjunctivitis in Italy, 2013
Belser JA , Creager HM , Zeng H , Maines TR , Tumpey TM . J Infect Dis 2017 216 S508-s511 H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses. |
Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets.
Smith GE , Sun X , Bai Y , Liu YV , Massare MJ , Pearce MB , Belser JA , Maines TR , Creager HM , Glenn GM , Flyer D , Pushko P , Levine MZ , Tumpey TM . Virology 2017 509 90-97 Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses. |
Stockpiled pre-pandemic H5N1 influenza virus vaccines with AS03 adjuvant provide cross-protection from H5N2 clade 2.3.4.4 virus challenge in ferrets
Sun X , Belser JA , Pulit-Penaloza JA , Creager HM , Guo Z , Jefferson SN , Liu F , York IA , Stevens J , Maines TR , Jernigan DB , Katz JM , Levine MZ , Tumpey TM . Virology 2017 508 164-169 Avian influenza viruses, notably H5 subtype viruses, pose a continuous threat to public health due to their pandemic potential. In recent years, influenza virus H5 subtype split vaccines with novel oil-in-water emulsion based adjuvants (e.g. AS03, MF59) have been shown to be safe, immunogenic, and able to induce broad immune responses in clinical trials, providing strong scientific support for vaccine stockpiling. However, whether such vaccines can provide protection from infection with emerging, antigenically distinct clades of H5 viruses has not been adequately addressed. Here, we selected two AS03-adjuvanted H5N1 vaccines from the US national pre-pandemic influenza vaccine stockpile and assessed whether the 2004-05 vaccines could provide protection against a 2014 highly pathogenic avian influenza (HPAI) H5N2 virus (A/northern pintail/Washington/40964/2014), a clade 2.3.4.4 virus responsible for mass culling of poultry in North America. Ferrets received two doses of adjuvanted vaccine containing 7.5microg of hemagglutinin (HA) from A/Vietnam/1203/2004 (clade 1) or A/Anhui/1/2005 (clade 2.3.4) virus either in a homologous or heterologous prime-boost vaccination regime. We found that both vaccination regimens elicited robust antibody responses against the 2004-05 vaccine viruses and could reduce virus-induced morbidity and viral replication in the lower respiratory tract upon heterologous challenge despite the low level of cross-reactive antibody titers to the challenge H5N2 virus. This study supports the value of existing stockpiled 2004-05 influenza H5N1 vaccines, combined with AS03-adjuvant for early use in the event of an emerging pandemic with H5N2-like clade 2.3.4.4 viruses. |
A novel A(H7N2) influenza virus isolated from a veterinarian caring for cats in a New York City animal shelter causes mild disease and transmits poorly in the ferret model
Belser JA , Pulit-Penaloza JA , Sun X , Brock N , Pappas C , Creager HM , Zeng H , Tumpey TM , Maines TR . J Virol 2017 91 (15) In December 2016, a low pathogenic avian influenza (LPAI) A(H7N2) virus was identified as the causative source of an outbreak in a cat shelter in New York City, which subsequently spread to multiple shelters in New York and Pennsylvania. One person with occupational exposure to infected cats became infected with the virus, representing the first LPAI H7N2 human infection 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. 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 co-housed ferrets following traditional or aerosol-based inoculation methods. Environmental persistence of NY/108 virus was generally comparable to other LPAI H7N2 viruses. However, NY/108 virus replicated with increased efficiency in human bronchial epithelial cells compared with previously isolated H7N2 viruses. Furthermore, the novel H7N2 virus was found to utilize a relatively lower pH for HA 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 a 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 New York animal shelters, which was the first detection of this virus in the Northeastern U.S. in over a decade and the first documented infection of H7N2 virus in a felid. A veterinarian became infected following occupational exposure to H7N2 virus-infected cats, necessitating the evaluation of this virus for its capacity to cause mammalian disease. 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 mammalian virulence. 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. |
Role of H7 hemagglutinin in murine infectivity of influenza viruses following ocular inoculation
Belser JA , Sun X , Creager HM , Johnson A , Ridenour C , Chen LM , Tumpey TM , Maines TR . Virology 2016 502 13-19 H7 subtype influenza viruses have demonstrated an ocular tropism in humans, causing conjunctivitis and not respiratory symptoms in many infected individuals. However, the molecular determinants which confer ocular tropism are still poorly understood. Here, we used a murine model of ocular inoculation to demonstrate that H7 influenza viruses are more likely to cause infection following ocular exposure than are non-H7 subtype viruses. We included investigation regarding the potential role of several properties of influenza viruses with murine infectivity following ocular inoculation, including virus lineage, pathogenicity, and HA cleavage site composition. Furthermore, we examined the potential contribution of internal proteins to murine ocular infectivity. These studies establish a link between H7 subtype viruses and the risk of heightened infectivity in a mammalian species following ocular exposure, and support the development of non-traditional inoculation methods and models to best understand the human risk posed by influenza viruses of all subtypes. |
In vitro exposure system for study of aerosolized influenza virus
Creager HM , Zeng H , Pulit-Penaloza JA , Maines TR , Tumpey TM , Belser JA . Virology 2016 500 62-70 Infection of adherent cell monolayers using a liquid inoculum represents an established method to reliably and quantitatively study virus infection, but poorly recapitulates the exposure and infection of cells in the respiratory tract that occurs during infection with aerosolized pathogens. To better simulate natural infection in vitro, we adapted a system that generates viral aerosols similar to those exhaled by infected humans to the inoculation of epithelial cell monolayers. Procedures for cellular infection and calculation of exposure dose were developed and tested using viruses characterized by distinct transmission and pathogenicity phenotypes: an HPAI H5N1, an LPAI H7N9, and a seasonal H3N2 virus. While all three aerosolized viruses were highly infectious in a human bronchial epithelial cell line (Calu-3) cultured submerged in media, differences between the viruses were observed in primary human alveolar epithelial cells and in Calu-3 cells cultured at air-liquid interface. This system provides a novel enhancement to traditional in vitro experiments, particularly those focused on the early stages of infection. |
Mammalian pathogenesis and transmission of H7N9 influenza viruses from three waves, 2013-2015.
Belser JA , Creager HM , Sun X , Gustin KM , Jones T , Shieh WJ , Maines TR , Tumpey TM . J Virol 2016 90 (9) 4647-4657 Three waves of human infection with H7N9 influenza viruses have concluded to date, but only viruses within the first wave (isolated between March-September 2013) have been extensively studied in mammalian models. While second- and third-wave viruses remain closely linked phylogenetically and antigenically, even subtle molecular changes can impart critical shifts in mammalian virulence. To determine if H7N9 viruses isolated from humans during 2013-15 have maintained the phenotype first identified among 2013 isolates, we assessed the ability of first-, second-, and third-wave H7N9 viruses isolated from humans to cause disease in mice and ferrets and to transmit among ferrets. Similar to first-wave viruses, H7N9 viruses from 2013-15 were highly infectious in mice, with comparable lethality to the well-studied A/Anhui/1/2013 virus. Second- and third-wave viruses caused moderate disease in ferrets, transmitted efficiently to cohoused, naive contact animals, and demonstrated limited transmissibility by respiratory droplets. All H7N9 viruses replicated efficiently in human bronchial epithelial cells, with subtle changes in pH fusion threshold identified between H7N9 viruses examined. Our results indicate that despite increased genetic diversity and geographical distribution since their initial detection in 2013, H7N9 viruses have maintained a pathogenic phenotype in mammals and continue to represent an immediate threat to public health. IMPORTANCE: H7N9 influenza viruses, first isolated in 2013, continue to cause human infection and represent an ongoing public health threat. Now entering the fourth wave of human infection, H7N9 viruses continue to exhibit genetic diversity in avian hosts, necessitating continuous efforts to monitor their pandemic potential. However, viruses isolated post-2013 have not been extensively studied, limiting our understanding of potential changes in virus-host adaptation. In order to ensure that current research with first-wave H7N9 viruses still pertains to more recently isolated strains, we compared the relative virulence and transmissibility of H7N9 viruses isolated during the second and third waves, through 2015, in the mouse and ferret models. Our finding that second and third wave viruses generally exhibit comparable disease in mammals as first-wave viruses strengthens our ability to extrapolate research from the 2013 viruses to current public health efforts. These data further contribute to our understanding of molecular determinants of pathogenicity, transmissibility, and tropism. |
Pathogenesis and Transmission of Novel Highly Pathogenic Avian Influenza H5N2 and H5N8 Viruses in Ferrets and Mice
Pulit-Penaloza JA , Sun X , Creager HM , Zeng H , Belser JA , Maines TR , Tumpey TM . J Virol 2015 A novel highly pathogenic avian influenza (HPAI) H5N8 virus, first detected in January 2014 in poultry and wild birds in South Korea, has spread throughout Asia and Europe, and caused outbreaks in Canada and the United States by the end of the year. The spread of H5N8 and the novel reassortant viruses, H5N2 and H5N1 (H5Nx), in domestic poultry across multiple states in the U.S. pose a potential public health risk. To evaluate the potential of cross-species infection, we determined the pathogenesis and transmissibility of two Asian-origin H5Nx viruses in mammalian animal models. The newly isolated H5N2 and H5N8 viruses were able to cause severe disease in mice only at high doses. Both viruses replicated efficiently in the upper and lower respiratory tracts of ferrets; however clinical symptoms were generally mild and there was no evidence of systemic dissemination of virus to multiple organs. Moreover, these influenza H5Nx viruses lacked the ability to transmit between ferrets in a direct contact setting. We further assessed viral replication kinetics of the novel H5Nx viruses in a human bronchial epithelium cell line, Calu-3. Both H5Nx viruses replicated to a level comparable to a human seasonal H1N1 virus, but significantly lower than a virulent Asian-lineage H5N1 HPAI virus. Although the recently isolated H5N2 and H5N8 viruses displayed moderate pathogenicity in mammalian models, their ability to rapidly spread among avian species, reassort, and generate novel strains underscores the need for continued risk assessment in mammals. IMPORTANCE: In 2015, highly pathogenic avian influenza (HPAI) H5 viruses have caused outbreaks in domestic poultry in multiple U.S. states. The economic losses incurred with H5N8 and H5N2 subtype virus infection have raised serious concerns for the poultry industry and the general public due to the potential risk of human infection. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals, which is an essential component of pandemic risk assessment. This study demonstrates that the newly isolated H5N2 and H5N8 viruses lacked the ability to transmit between ferrets and exhibited low to moderate virulence in mammals. In human bronchial epithelial (Calu-3) cells, both H5N8 and H5N2 viruses replicated to a level comparable to a human seasonal virus, but significantly lower than a virulent Asian-lineage H5N1 (A/Thailand/16/2004) virus. The results of this study are important for the evaluation of public health risk. |
Oseltamivir inhibits influenza virus replication and transmission following ocular-only aerosol inoculation of ferrets
Belser JA , Maines TR , Creager HM , Katz JM , Tumpey TM . Virology 2015 484 305-312 Ocular exposure to influenza virus represents an alternate route of virus entry capable of establishing a respiratory infection in mammals, but the effectiveness of currently available antiviral treatments to limit virus replication within ocular tissue or inhibit virus spread from ocular sites to the respiratory tract is poorly understood. Using an inoculation method that delivers an aerosol inoculum exclusively to the ocular surface, we demonstrate that oral oseltamivir administration following ocular-only aerosol inoculation with multiple avian and human influenza viruses protected ferrets from a fatal and systemic infection, reduced clinical signs and symptoms of illness, and decreased virus transmissibility to susceptible contacts when a respiratory infection was initiated. The presence of oseltamivir further inhibited influenza virus replication in primary human corneal epithelial cells. These findings provide critical experimental evidence supporting the use of neuraminidase inhibitors during outbreaks of influenza virus resulting in ocular disease or following ocular exposure. |
Recombinant virus-like particles elicit protective immunity against avian influenza A(H7N9) virus infection in ferrets
Liu YV , Massare MJ , Pearce MB , Sun X , Belser JA , Maines TR , Creager HM , Glenn GM , Pushko P , Smith GE , Tumpey TM . Vaccine 2015 33 (18) 2152-8 In March 2013, diagnosis of the first reported case of human infection with a novel avian-origin influenza A(H7N9) virus occurred in eastern China. Most human cases have resulted in severe respiratory illness and, in some instances, death. Currently there are no licensed vaccines against H7N9 virus, which continues to cause sporadic human infections. Recombinant virus-like particles (VLPs) have been previously shown to be safe and effective vaccines for influenza. In this study, we evaluated the immunogenicity and protective efficacy of a H7N9 VLP vaccine in the ferret challenge model. Purified recombinant H7N9 VLPs morphologically resembled influenza virions and elicited high-titer serum hemagglutination inhibition (HI) and neutralizing antibodies specific for A/Anhui/1/2013 (H7N9) virus. H7N9 VLP-immunized ferrets subsequently challenged with homologous virus displayed reductions in fever, weight loss, and virus shedding compared to these parameters in unimmunized control ferrets. H7N9 VLP was also effective in protecting against lung and tracheal infection. The addition of either ISCOMATRIX or Matrix-M1 adjuvant improved immunogenicity and protection of the VLP vaccine against H7N9 virus. These results provide support for the development of a safe and effective human VLP vaccine with potent adjuvants against avian influenza H7N9 virus with pandemic potential. |
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