Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Circulating Globally in 2022-2023.

The antiviral susceptibility of currently circulating (2022-2023) highly pathogenic avian influenza (HPAI) A(H5N1) viruses was assessed by genotypic and phenotypic approaches. The frequency of neuraminidase (NA) and polymerase acidic (PA) substitutions associated with reduced inhibition by NA inhibitors (NAIs) (21/2698, 0.78%) or by the PA inhibitor baloxavir (14/2600, 0.54%) was low. Phenotypic testing of 22 clade 2.3.2.1a and 2.3.4.4b viruses revealed broad susceptibility to NAIs and baloxavir concluding that most contemporary HPAI A(H5N1) viruses retain susceptibility to antiviral drugs. Novel NA-K432E and NA-T438I substitutions (N2 numbering) were identified at elevated frequencies (104/2698, 3.85%) and caused reduced zanamivir and peramivir inhibition.

Emergence of a new genotype of clade 2.3.4.4b H5N1 highly pathogenic avian influenza A viruses in Bangladesh.

Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Distinct but connected avian influenza virus activities in wetlands and live poultry markets in Bangladesh, 2018-2019.

From April 2018 to October 2019, we continued active surveillance for influenza viruses in Bangladeshi live poultry markets (LPMs) and in Tanguar Haor, a wetland region of Bangladesh where domestic ducks have frequent contact with migratory birds. The predominant virus subtypes circulating in the LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses of the H5N1-R1 genotype, like those found in previous years. Viruses of the H5N1-R2 genotype, which were previously reported as co-circulating with H5N1-R1 genotype viruses in LPM, were not detected. In addition to H9N2 viruses, which were primarily found in chicken and quail, H2N2, H3N8 and H11N3 LPAI viruses were detected in LPMs, exclusively in ducks. Viruses in domestic ducks and/or wild birds in Tanguar Haor were more diverse, with H1N1, H4N6, H7N1, H7N3, H7N4, H7N6, H8N4, H10N3, H10N4 and H11N3 detected. Phylogenetic analyses of these LPAI viruses suggested that some were new to Bangladesh (H2N2, H7N6, H8N4, H10N3 and H10N4), likely introduced by migratory birds of the Central Asian flyway. Our results show a complex dynamic of viral evolution and diversity in Bangladesh based on factors such as host populations and geography. The LPM environment was characterised by maintenance of viruses with demonstrated zoonotic potential and H5N1 genotype turnover. The wetland environment was characterised by greater viral gene pool diversity but a lower overall influenza virus detection rate. The genetic similarity of H11N3 viruses in both environments demonstrates that LPM and wetlands are connected despite their having distinct influenza ecologies.

It's the Wrong Virus: Rapid Adaptation of Operations Inside a Highly Pathogenic Avian Influenza Select Agent Laboratory in Response to the 2019 SARS-CoV-2 Pandemic.

Background: The Animal Biosafety Level 3 Enhanced (ABSL-3+) laboratory at St. Jude Children's Research Hospital has a long history of influenza pandemic preparedness. The emergence of SARS-CoV-2 and subsequent expansion into a pandemic has put new and unanticipated demands on laboratory operations since April 2020. Administrative changes, investigative methods requiring increased demand for inactivation and validation of sample removal, and the adoption of a new animal model into the space required all arms of our Biorisk Management System (BMS) to respond with speed and innovation. Results: In this report, we describe the outcomes of three major operational changes that were implemented to adapt the ABSL-3+ select agent space into a multipathogen laboratory. First were administrative controls that were revised and developed with new Institutional Biosafety Committee protocols, laboratory space segregation, training of staff, and occupational health changes for potential exposure to SARS-CoV-2 inside the laboratory. Second were extensive inactivation and validation experiments performed for both highly pathogenic avian influenza and SARS-CoV-2 to meet the demands for sample removal to a lower biosafety level. Third was the establishment of a new caging system to house Syrian Golden hamsters for SARS-CoV-2 risk assessment modeling. Summary: The demands placed on biocontainment laboratories for response to SARS-CoV-2 has highlighted the importance of a robust BMS. In a relatively short time, the ABSL-3+ was able to adapt from a single select agent space to a multipathogen laboratory and expand our pandemic response capacity.

Detection of a Novel Reassortant H9N9 Avian Influenza Virus in Free-Range Ducks in Bangladesh.

Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs). In this study, an A(H9N9) influenza A virus (A/duck/Bangladesh/44493/2020) was identified via routine surveillance in free-range domestic ducks in Bangladesh. Phylogenetic analysis of hemagglutinin showed that the H9N9 virus belonged to the Y439-like lineage. The HA gene had the highest nucleotide identity to A/Bean Goose (Anser fabalis)/South Korea/KNU 2019-16/2019 (H9N2). The other seven gene segments clustered within the Eurasian lineage.

Baloxavir Treatment Delays Influenza B Virus Transmission in Ferrets and Results in Limited Generation of Drug-Resistant Variants.

Clinical efficacy of the influenza antiviral baloxavir marboxil (baloxavir) is compromised by treatment-emergent variants harboring a polymerase acidic protein I38T (isoleucine-38-threonine) substitution. However, the fitness of I38T-containing influenza B viruses (IBVs) remains inadequately defined. After the pharmacokinetics of the compound were confirmed in ferrets, animals were injected subcutaneously with 8 mg/kg of baloxavir acid (BXA) at 24 h postinoculation with recombinant BXA-sensitive (BXA-Sen, I38) or BXA-resistant (BXA-Res, I38T) B/Brisbane/60/2008 (Victoria lineage) virus. BXA treatment of donor ferrets reduced virus replication and delayed transmission of the BXA-Sen but not the BXA-Res IBV. The I38 genotype remained dominant in the BXA-Sen-infected animals, even with BXA treatment. In competitive-mixture experiments, no transmission to aerosol contacts was seen from BXA-treated donors coinfected with the BXA-Sen and BXA-Res B/Brisbane/60/2008 viruses. However, in parallel mixed infections with the B/Phuket/3073/2013 (Yamagata lineage) virus background, BXA treatment failed to block airborne transmission of the BXA-Res virus, and the I38T genotype generally predominated. Therefore, the relative fitness of BXA-Res IBVs is complex and dependent on the virus backbone and within-host virus competition. BXA treatment of single-virus-infected ferrets hampers aerosol transmission of the BXA-Sen virus and does not readily generate BXA-Res variants, whereas mixed infections may result in propagation of BXA-Res IBVs of the Yamagata lineage. Our findings confirm the antiviral potency of baloxavir against IBVs, while supporting optimization of the dosing regimen to maximize clinical benefit.

Highly Pathogenic Avian Influenza A(H5N6) Virus Clade 2.3.4.4h in Wild Birds and Live Poultry Markets, Bangladesh.

Migratory birds play a major role in spreading influenza viruses over long distances. We report highly pathogenic avian influenza A(H5N6) viruses in migratory and resident ducks in Bangladesh. The viruses were genetically similar to viruses detected in wild birds in China and Mongolia, suggesting migration-associated dissemination of these zoonotic pathogens.

Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals.

To optimize the public health response to coronavirus disease 2019 (COVID-19), we must first understand the antibody response to individual proteins on the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the antibody's cross reactivity to other coronaviruses. Using a panel of 37 convalescent COVID-19 human serum samples, we showed that the magnitude and specificity of responses varied across individuals, independent of their reactivity to seasonal human coronaviruses (HCoVs). These data suggest that COVID-19 vaccines will elicit primary humoral immune responses in naïve individuals and variable responses in those previously exposed to SARS-CoV-2. Unlike the limited cross-coronavirus reactivities in humans, serum samples from 96 dogs and 10 cats showed SARS-CoV-2 protein-specific responses focused on non-S1 proteins. The correlation of this response with those to other coronaviruses suggests that the antibodies are cross-reactive and generated to endemic viruses within these hosts, which must be considered in seroepidemiologic studies. We conclude that substantial variation in antibody generation against coronavirus proteins will influence interpretations of serologic data in the clinical and veterinary settings.

Continued Evolution of H5Nx Avian Influenza Viruses in Bangladeshi Live Poultry Markets: Pathogenic Potential in Poultry and Mammalian Models.

The genesis of novel influenza viruses through reassortment poses a continuing risk to public health. This is of particular concern in Bangladesh, where highly pathogenic avian influenza viruses of the A(H5N1) subtype are endemic and cocirculate with other influenza viruses. Active surveillance of avian influenza viruses in Bangladeshi live poultry markets detected three A(H5) genotypes, designated H5N1-R1, H5N1-R2, and H5N2-R3, that arose from reassortment of A(H5N1) clade 2.3.2.1a viruses. The H5N1-R1 and H5N1-R2 viruses contained HA, NA, and M genes from the A(H5N1) clade 2.3.2.1a viruses and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. H5N2-R3 viruses contained the HA gene from circulating A(H5N1) clade 2.3.2.1a viruses, NA and M genes from concurrently circulating A(H9N2) influenza viruses, and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. Representative viruses of all three genotypes and a parental clade 2.3.2.1a strain (H5N1-R0) infected and replicated in mice without prior adaptation; the H5N2-R3 virus replicated to the highest titers in the lung. All viruses efficiently infected and killed chickens. All viruses replicated in inoculated ferrets, but no airborne transmission was detected, and only H5N2-R3 showed limited direct-contact transmission. Our findings demonstrate that although the A(H5N1) viruses circulating in Bangladesh have the capacity to infect and replicate in mammals, they show very limited capacity for transmission. However, reassortment does generate viruses of distinct phenotypes.IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have circulated continuously in Bangladesh since 2007, and active surveillance has detected viral evolution driven by mutation and reassortment. Recently, three genetically distinct A(H5N1) reassortant viruses were detected in live poultry markets in Bangladesh. Currently, we cannot assign pandemic risk by only sequencing viruses; it must be conducted empirically. We found that the H5Nx highly pathogenic avian influenza viruses exhibited high virulence in mice and chickens, and one virus had limited capacity to transmit between ferrets, a property considered consistent with a higher zoonotic risk.

Transmission experiments support clade-level differences in the transmission and pathogenicity of Cambodian influenza A/H5N1 viruses.

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more "avian-like," with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings.

Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility.

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

A(H9N2) influenza viruses associated with chicken mortality in outbreaks in Algeria 2017.

In late 2017, increased mortality was detected in chicken farms in Algeria undergoing A(H9N2) influenza outbreaks. Analysis of viruses isolated from affected farms showed that they were monophyletic, were of the G1 hemagglutinin (HA) lineage, and were antigenically and genetically similar to viruses detected contemporaneously in other countries in Northern Africa and the Middle East. The virus was able to spread via contact transmission between ferrets but did not cause disease in intravenously inoculated chickens.

Continuing evolution of highly pathogenic H5N1 viruses in Bangladeshi live poultry markets.

Since November 2008, we have conducted active avian influenza surveillance in Bangladesh. Clades 2.2.2, 2.3.4.2, and 2.3.2.1a of highly pathogenic avian influenza H5N1 viruses have all been identified in Bangladeshi live poultry markets (LPMs), although, since the end of 2014, H5N1 viruses have been exclusively from clade 2.3.2.1a. In June 2015, a new reassortant H5N1 virus (H5N1-R1) from clade 2.3.2.1a was identified, containing haemagglutinin, neuraminidase, and matrix genes of H5N1 viruses circulating in Bangladesh since 2011, plus five other genes of Eurasian-lineage low pathogenic avian influenza A (LPAI) viruses. Here we report the status of circulating avian influenza A viruses in Bangladeshi LPMs from March 2016 to January 2018. Until April 2017, H5N1 viruses exclusively belonged to H5N1-R1 clade 2.3.2.1a. However, in May 2017, we identified another reassortant H5N1 (H5N1-R2), also of clade 2.3.2.1a, wherein the PA gene segment of H5N1-R1 was replaced by that of another Eurasian-lineage LPAI virus related to A/duck/Bangladesh/30828/2016 (H3N8), detected in Bangladeshi LPM in September 2016. Currently, both reassortant H5N1-R1 and H5N1-R2 co-circulate in Bangladeshi LPMs. Furthermore, some LPAI viruses isolated from LPMs during 2016-2017 were closely related to those from ducks in free-range farms and wild birds in Tanguar haor, a wetland region of Bangladesh where ducks have frequent contact with migratory birds. These data support a hypothesis where Tanguar haor-like ecosystems provide a mechanism for movement of LPAI viruses to LPMs where reassortment with poultry viruses occurs adding to the diversity of viruses at this human-animal interface.

H9N2 influenza viruses from Bangladesh: Transmission in chicken and New World quail.

The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10 days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets.

Genesis of Influenza A(H5N8) Viruses.

Highly pathogenic avian influenza A(H5N8) clade 2.3.4.4 virus emerged in 2016 and spread to Russia, Europe, and Africa. Our analysis of viruses from domestic ducks at Tanguar haor, Bangladesh, showed genetic similarities with other viruses from wild birds in central Asia, suggesting their potential role in the genesis of A(H5N8).

Insight into live bird markets of Bangladesh: an overview of the dynamics of transmission of H5N1 and H9N2 avian influenza viruses.

Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been recognized as threats to public health in Bangladesh since 2007. Although live bird markets (LBMs) have been implicated in the transmission, dissemination, and circulation of these viruses, an in-depth analysis of the dynamics of avian transmission of H5N1 and H9N2 viruses at the human-animal interface has been lacking. Here we present and evaluate epidemiological findings from active surveillance conducted among poultry in various production sectors in Bangladesh from 2008 to 2016. Overall, the prevalence of avian influenza viruses (AIVs) in collected samples was 24%. Our data show that AIVs are more prevalent in domestic birds within LBMs (30.4%) than in farms (9.6%). Quail, chickens and ducks showed a high prevalence of AIVs (>20%). The vast majority of AIVs detected (99.7%) have come from apparently healthy birds and poultry drinking water served as a reservoir of AIVs with a prevalence of 32.5% in collected samples. HPAI H5N1 was more frequently detected in ducks while H9N2 was more common in chickens and quail. LBMs, particularly wholesale markets, have become a potential reservoir for various types of AIVs, including HPAI H5N1 and LPAI H9N2. The persistence of AIVs in LBMs is of great concern to public health, and this study highlights the importance of regularly reviewing and implementing infection control procedures as a means of reducing the exposure of the general public to AIVs.Emerging Microbes & Infections (2017) 6, e12; doi:10.1038/emi.2016.142; published online 8 March 2017.

The Continuing Evolution of H5N1 and H9N2 Influenza Viruses in Bangladesh Between 2013 and 2014.

In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra-and interclade reassortant; its HA, polymerase basic 1 (PB1), polymerase (PA), and nonstructural (NS) genes come from subclade 2.3.2.1a; the polymerase basic 2 (PB2) comes from subclade 2.3.2.1c; and the NA, nucleocapsid protein (NP), and matrix (M) gene from clade 2.3.4.2. The H9N2 influenza viruses cocirculating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from an HPAI H7N3 virus previously isolated in Pakistan. Despite frequent coinfection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried seven genes from the local HPAI H5N1 lineage and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although the live birds we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans.

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.