The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouseadapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to ␣2,3 together with decreasing binding to ␣2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.

H9 influenza viruses have become endemic in land-based domestic poultry in Asia and have sporadically crossed to pigs and humans. To understand the molecular determinants of their adaptation to land-based birds, we tested the replication and transmission of several 1970s duck H9 viruses in chickens and quail. Quail were more susceptible than chickens to these viruses, and generation of recombinant H9 viruses by reverse genetics showed that changes in the HA gene are sufficient to initiate efficient replication and transmission in quail. Seven amino acid positions on the HA molecule corresponded to adaptation to land-based birds. In quail H9 viruses, the pattern of amino acids at these seven positions is intermediate between those of duck and chicken viruses; this fact may explain the susceptibility of quail to duck H9 viruses. Our findings suggest that quail provide an environment in which the adaptation of influenza viruses from ducks generates novel variants that can cross the species barrier.

BackgroundChickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains.ResultsTranscriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection.ConclusionsTaken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1778-8) contains supplementary material, which is available to authorized users.

H5N1 vaccines may stimulate an immune response that is more cross-protective than what might be predicted by in vitro assays and, thus, hold potential for being stockpiled as "initial" pandemic vaccines.

Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens (natural hosts) is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness.

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.

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC50s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (Vmax, Km and Ki) of the avian-like N1 NA glycoproteins were highly consistent with their IC50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 106 EID50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.

H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Reemergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data show that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cage mates. Markers of mammalian adaptation in hemagglutinin (HA) and PB2 proteins were absent from all isolates, and they retained a preference for avian-like ␣2,3-linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of prepandemic planning.