Dissecting the role of the HA1-226 leucine residue in the fitness and airborne transmission of an A(H9N2) avian influenza virus.

Unlabelled: A better understanding of viral factors that contribute to influenza A virus (IAV) airborne transmission is crucial for pandemic preparedness. A limited capacity for airborne transmission was recently observed in a human A(H9N2) virus isolate (A/Anhui-Lujiang/39/2018, AL/39) that possesses a leucine (L) residue at position HA1-226 (H3 numbering), indicative of human-like receptor binding potential. To evaluate the roles of the residue at this position in virus fitness and airborne transmission, a wild-type AL/39 (AL/39-wt) and a mutant virus (AL/39-HA1-L226Q) with a single substitution at position HA1-226 from leucine to glutamine (Q), a consensus residue in avian influenza viruses, were rescued and assessed in the ferret model.

Transmission of a human isolate of clade 2.3.4.4b A(H5N1) virus in ferrets.

Since 2020, there has been unprecedented global spread of highly pathogenic avian influenza A(H5N1) in wild bird populations with spillover into a variety of mammalian species and sporadically humans. In March 2024, clade 2.3.

An aggregated dataset of serial morbidity and titer measurements from influenza A virus-infected ferrets.

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible.

Key considerations to improve the normalization, interpretation and reproducibility of morbidity data in mammalian models of viral disease.

Viral pathogenesis and therapeutic screening studies that utilize small mammalian models rely on the accurate quantification and interpretation of morbidity measurements, such as weight and body temperature, which can vary depending on the model, agent and/or experimental design used. As a result, morbidity-related data are frequently normalized within and across screening studies to aid with their interpretation. However, such data normalization can be performed in a variety of ways, leading to differences in conclusions drawn and making comparisons between studies challenging.

Redirecting antibody responses from egg-adapted epitopes following repeat vaccination with recombinant or cell culture-based versus egg-based influenza vaccines.

Repeat vaccination with egg-based influenza vaccines could preferentially boost antibodies targeting the egg-adapted epitopes and reduce immunogenicity to circulating viruses. In this randomized trial (Clinicaltrials.gov: NCT03722589), sera pre- and post-vaccination with quadrivalent inactivated egg-based (IIV4), cell culture-based (ccIIV4), and recombinant (RIV4) influenza vaccines were collected from healthcare personnel (18-64 years) in 2018-19 (N = 723) and 2019-20 (N = 684) influenza seasons.

A naturally occurring HA-stabilizing amino acid (HA1-Y17) in an A(H9N2) low-pathogenic influenza virus contributes to airborne transmission.

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.

Antibody-Mediated Suppression Regulates the Humoral Immune Response to Influenza Vaccination in Humans.

Background: Preexisting immunity, including memory B cells and preexisting antibodies, can modulate antibody responses to influenza in vivo to antigenically related antigens. We investigated whether preexisting hemagglutination inhibition (HAI) antibodies targeting the K163 epitope on the hemagglutinin (K163 antibodies) could affect antibody responses following vaccination with A/California/07/2009-like A(H1N1)pdm09 influenza viruses in humans.

Methods: Pre- and postvaccination sera collected from 300 adults (birth years, 1961-1998) in 6 seasons (2010-2016) were analyzed by HAI assays with 2 reverse genetics viruses and A(H1N1) viruses circulated from 1977 to 2018.

Pathogenesis and Transmission Assessment of 3 Swine-Origin Influenza A(H3N2) Viruses With Zoonotic Risk to Humans Isolated in the United States, 2017-2020.

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.

Enhanced fitness of SARS-CoV-2 B.1.617.2 Delta variant in ferrets.

Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.

Kinetics and magnitude of viral RNA shedding as indicators for Influenza A virus transmissibility in ferrets.

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.

Utility of Human Data in Risk Assessments of Influenza A Virus Using the Ferret Model.

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. pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of infections are predictive or correlates of nasal wash and tissue measurements for IAV infections .

Detection of Airborne Influenza A and SARS-CoV-2 Virus Shedding following Ocular Inoculation of Ferrets.

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.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1.

Comparative Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Variants in the Ferret Model.

The continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans necessitates evaluation of variants for enhanced virulence and transmission. We used the ferret model to perform a comparative analysis of four SARS-CoV-2 strains, including an early pandemic isolate from the United States (WA1), and representatives of the Alpha, Beta, and Delta lineages. While Beta virus was not capable of pronounced replication in ferrets, WA1, Alpha, and Delta viruses productively replicated in the ferret upper respiratory tract, despite causing only mild disease with no overt histopathological changes.

Low quality antibody responses in critically ill patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection.

Although some adults infected with influenza 2009 A(H1N1)pdm09 viruses mounted high hemagglutination inhibition (HAI) antibody response, they still suffered from severe disease, or even death. Here, we analyzed antibody profiles in patients (n = 31, 17-65 years) admitted to intensive care units (ICUs) with lung failure and invasive mechanical ventilation use due to infection with A(H1N1)pdm09 viruses during 2009-2011. We performed a comprehensive analysis of the quality and quantity of antibody responses using HAI, virus neutralization, biolayer interferometry, enzyme-linked-lectin and enzyme-linked immunosorbent assays.

Pathogenesis and Transmissibility of North American Highly Pathogenic Avian Influenza A(H5N1) Virus in Ferrets.

Highly pathogenic avian influenza A(H5N1) viruses have spread rapidly throughout North American flyways in recent months, affecting wild birds in over 40 states. We evaluated the pathogenicity and transmissibility of a representative virus using a ferret model and examined replication kinetics of this virus in human respiratory tract cells.

Multiplex Detection of Antibody Landscapes to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/Influenza/Common Human Coronaviruses Following Vaccination or Infection With SARS-CoV-2 and Influenza.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to co-circulate, representing 2 major public health threats from respiratory infections with similar clinical presentations. SARS-CoV-2 and influenza vaccines can also now be co-administered. However, data on antibody responses to SARS-CoV-2 and influenza coinfection and vaccine co-administration remain limited.

Pathogenesis and transmission of human seasonal and swine-origin A(H1) influenza viruses in the ferret model.

Influenza A viruses (IAVs) in the swine reservoir constantly evolve, resulting in expanding genetic and antigenic diversity of strains that occasionally cause infections in humans and pose a threat of emerging as a strain capable of human-to-human transmission. For these reasons, there is an ongoing need for surveillance and characterization of newly emerging strains to aid pandemic preparedness efforts, particularly for the selection of candidate vaccine viruses and conducting risk assessments. Here, we performed a parallel comparison of the pathogenesis and transmission of genetically and antigenically diverse swine-origin A(H1N1) variant (v) and A(H1N2)v, and human seasonal A(H1N1)pdm09 IAVs using the ferret model.

Inherent Heterogeneity of Influenza A Virus Stability following Aerosolization.

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.

Antibody Landscape Analysis following Influenza Vaccination and Natural Infection in Humans with a High-Throughput Multiplex Influenza Antibody Detection Assay.

To better understand the antibody landscape changes following influenza virus natural infection and vaccination, we developed a high-throughput multiplex influenza antibody detection assay (MIADA) containing 42 recombinant hemagglutinins (rHAs) (ectodomain and/or globular head domain) from pre-2009 A(H1N1), A(H1N1)pdm09, A(H2N2), A(H3N2), A(H5N1), A(H7N7), A(H7N9), A(H7N2), A(H9N2), A(H13N9), and influenza B viruses. Panels of ferret antisera, 227 paired human sera from vaccinees (children and adults) in 5 influenza seasons (2010 to 2018), and 17 paired human sera collected from real-time reverse transcription-PCR (rRT-PCR)-confirmed influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B virus-infected adults were analyzed by the MIADA. Ferret antisera demonstrated clear strain-specific antibody responses to exposed subtype HA.

Characterization of highly pathogenic avian influenza H5Nx viruses in the ferret model.

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.

Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

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.

Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018.

Low pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes.

Identification of novel influenza A virus exposures by an improved high-throughput multiplex MAGPIX platform and serum adsorption.

Background: The development of serologic assays that can rapidly assess human exposure to novel influenza viruses remains a public health need. Previously, we developed an 11-plex magnetic fluorescence microsphere immunoassay (MAGPIX) by using globular head domain recombinant hemagglutinins (rHAs) with serum adsorption using two ectodomain rHAs.

Methods: We compared sera collected from two cohorts with novel influenza exposures: animal shelter staff during an A(H7N2) outbreak in New York City in 2016-2017 (n = 119 single sera) and poultry workers from a live bird market in Bangladesh in 2012-2014 (n = 29 pairs).