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.

Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission.

Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC50. EC50s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA-BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.

Pleiotropic Effects of Influenza H1, H3, and B Baloxavir-Resistant Substitutions on Replication, Sensitivity to Baloxavir, and Interferon Expression.

Baloxavir is an anti-influenza endonuclease inhibitor that targets the polymerase acidic (PA) protein of influenza A and B viruses. Our knowledge regarding the pleiotropic effects of baloxavir resistance-associated substitutions is limited. We generated recombinant A/California/04/09 (H1N1)-, A/Hong Kong/218849/2006 (H3N2)-, and B/Victoria/504/2000-like viruses that contained PA substitutions identified in baloxavir clinical trials and surveillance that could potentially be associated with baloxavir resistance. We characterized their susceptibility to baloxavir, impact on polymerase activity, viral growth, and ability to induce interferon (IFN) and IFN-stimulated genes expression in vitro. Four PA substitutions, H1N1 I38L/T, E199D, and B G199R, significantly reduced the sensitivity of the recombinant viruses to baloxavir (14.1-fold). We confirmed our findings by using the luciferase-based ribonucleoprotein minigenome assay and by using virus yield reduction assay in Calu-3 and normal human bronchial epithelial (NHBE) cells. We observed that I38L and E199D resulted in decreased viral replication of the H1N1 wild-type virus (1.4-fold) but the H1N1 I38T and B G199R substitutions did not significantly alter replication capacity in Calu-3 cells. In addition, H1N1 variants with PA I38L/T and E199D induced significantly higher levels of IFNB1 gene expression compared to the wild-type virus (4.2-fold). In contrast, the B variant, G199R, triggered the lowest levels of IFN genes in Calu-3 cells (1.6-fold). Because baloxavir is a novel anti-influenza therapeutic agent, identifying and characterizing substitutions associated with reduced sensitivity to baloxavir, as well as the impact of these substitutions on viral fitness, is paramount to the strategic implementation of this novel countermeasure.

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.

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.

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.

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.

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.

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.

Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors.

The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC50]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC50). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PAN target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs.IMPORTANCE The effectiveness of antiviral drugs can be severely compromised by the emergence of resistant viruses. Therefore, determination of the mechanisms by which viruses become resistant is critical for drug development and clinical use. RO-7 is a compound that potently inhibits influenza virus replication and belongs to a new class of drugs in late-stage clinical trials for treatment of influenza virus infection. Here we demonstrate that a single amino acid change acquired under prolonged virus exposure to RO-7 renders influenza viruses significantly less susceptible to its inhibitory effects. We have discovered how the mutation can simultaneously interfere with drug activity and still maintain efficient virus replication. These findings have important implications for the development of more effective derivatives of RO-7-like drugs and provide guidance for how to monitor the emergence of resistance.

Molecular basis of mammalian transmissibility of avian H1N1 influenza viruses and their pandemic potential.

North American wild birds are an important reservoir of influenza A viruses, yet the potential of viruses in this reservoir to transmit and cause disease in mammals is not well understood. Our surveillance of avian influenza viruses (AIVs) at Delaware Bay, USA, revealed a group of similar H1N1 AIVs isolated in 2009, some of which were airborne-transmissible in the ferret model without prior adaptation. Comparison of the genomes of these viruses revealed genetic markers of airborne transmissibility in the Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and Nuclear Export Protein (NEP) genes. We studied the role of NS1 in airborne transmission and found that NS1 mutants that were not airborne-transmissible caused limited tissue pathology in the upper respiratory tract (URT). Viral maturation was also delayed, evident as strong intranuclear staining and little virus at the mucosa. Our study of this naturally occurring constellation of genetic markers has provided insights into the poorly understood phenomenon of AIV airborne transmissibility by revealing a role for NS1 and characteristics of viral replication in the URT that were associated with airborne transmission. The transmissibility of these viruses further highlights the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.

Role of domestic ducks in the emergence of a new genotype of highly pathogenic H5N1 avian influenza A viruses in Bangladesh.

Highly pathogenic avian influenza H5N1 viruses were first isolated in Bangladesh in February 2007. Subsequently, clades 2.2.2, 2.3.4.2 and 2.3.2.1a were identified in Bangladesh, and our previous surveillance data revealed that by the end of 2014, the circulating viruses exclusively comprised clade 2.3.2.1a. We recently determined the status of circulating avian influenza viruses in Bangladesh by conducting surveillance of live poultry markets and waterfowl in wetland areas from February 2015 through February 2016. Until April 2015, clade 2.3.2.1a persisted without any change in genotype. However, in June 2015, we identified a new genotype of H5N1 viruses, clade 2.3.2.1a, which quickly became predominant. These newly emerged H5N1 viruses contained the hemagglutinin, neuraminidase and matrix genes of circulating 2.3.2.1a Bangladeshi H5N1 viruses and five other genes of low pathogenic Eurasian-lineage avian influenza A viruses. Some of these internal genes were closely related to those of low pathogenic viruses isolated from ducks in free-range farms and wild birds in a wetland region of northeastern Bangladesh, where commercially raised domestic ducks have frequent contact with migratory birds. These findings indicate that migratory birds of the Central Asian flyway and domestic ducks in the free-range farms in Tanguar haor-like wetlands played an important role in the emergence of this novel genotype of highly pathogenic H5N1 viruses.

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.

Manipulation of neuraminidase packaging signals and hemagglutinin residues improves the growth of A/Anhui/1/2013 (H7N9) influenza vaccine virus yield in eggs.

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8's hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5' and 3' packaging signals of PR8's HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity. IMPORTANCE: Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

Highly Pathogenic Reassortant Avian Influenza A(H5N1) Virus Clade 2.3.2.1a in Poultry, Bhutan.

Highly pathogenic avian influenza A(H5N1), clade 2.3.2.1a, with an H9-like polymerase basic protein 1 gene, isolated in Bhutan in 2012, replicated faster in vitro than its H5N1 parental genotype and was transmitted more efficiently in a chicken model. These properties likely help limit/eradicate outbreaks, combined with strict control measures.

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein.

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5-6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2-5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009-2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.