N-Glycolylneuraminic Acid Binding of Avian and Equine H7 Influenza A Viruses.

Influenza A viruses (IAV) initiate infection by binding to glycans with terminal sialic acids on the cell surface. Hosts of IAV variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in most mammals, including horses and pigs, but is absent in humans, ferrets, and birds. The only known naturally occurring IAV that exclusively bind NeuGc are extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with NeuGc and observed high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs and an H15 HA. We found that the A135E mutation is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. The additional mutations S128T, I130V, T189A, and K193R converted the specificity from NeuAc to NeuGc. We investigated the residues at positions 128, 130, 135, 189, and 193 in a phylogenetic analysis of avian and equine H7 HAs. This analysis revealed a clear distinction between equine and avian residues. The highest variability was observed at key position 135, of which only the equine glutamic acid led to NeuGc binding. These results demonstrate that genetically distinct H7 and H15 HAs can be switched from NeuAc to NeuGc binding and vice versa after the introduction of several mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. IMPORTANCE Influenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A viruses, these "keys" (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority bind NeuGc. NeuGc is present in species like horses, pigs, and mice but not in humans, ferrets, and birds. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

The Effector Domain of the Influenza A Virus Nonstructural Protein NS1 Triggers Host Shutoff by Mediating Inhibition and Global Deregulation of Host Transcription When Associated with Specific Structures in the Nucleus.

Host shutoff in influenza A virus (IAV) infection is a key process contributing to viral takeover of the cellular machinery and resulting in the downregulation of host gene expression. Analysis of nascently transcribed RNA in a cellular model that allows the functional induction of NS1 demonstrates that NS1 suppresses host transcription. NS1 inhibits the expression of genes driven by RNA polymerase II as well as RNA polymerase I-driven promoters, but not by the noneukaryotic T7 polymerase. Additionally, transcriptional termination is deregulated in cells infected with wild-type IAV. The NS1 effector domain alone is able to mediate both effects, whereas NS1 mutant GLEWN184-188RFKRY (184-188) is not. Overexpression of CPSF30 counteracts NS1-mediated inhibition of RNA polymerase II-driven reporter gene expression, but knockdown of CPSF30 expression does not attenuate gene expression. Although NS1 is associated with nuclear chromatin, superresolution microscopy demonstrates that NS1 does not colocalize with genomic DNA. Moreover, NS1 mutants and NS1 fusion proteins, unable to associate with nuclear chromatin and displaying an altered subcellular distribution are still able to attenuate reporter gene expression. However, tethering NS1 artificially to the cytoskeleton results in the loss of reporter gene inhibition. A NS1 deficient in both native nuclear localization signals (NLS) is able to inhibit gene expression as effective as wild-type NS1 when a synthetic NLS relocates it to specific structures of the nucleus. Colocalization experiments and reporter gene cotransfection experiments with a NS1 fusion guiding it to nuclear speckles suggest that the presence of NS1 in nuclear speckles seems to be essential for host shutoff. IMPORTANCE We investigated the role of IAV nonstructural protein 1 NS1 in host gene shutoff-a central feature of IAV replication. We demonstrate that the effector domain of NS1 alone mediates host gene shutoff by inhibition of host transcription and by deregulation of the polyadenylation (polyA) signal-mediated 3' termination of host transcription. NS1 mutated in amino acids 184 to 188 fails to shut off host gene expression. Knockdown of CPSF30 does not result in transcriptional attenuation. By analyzing the subcellular localization of modified NS1 proteins and relating these data to their ability to inhibit reporter gene expression, we show for the first time that the presence of NS1 in granular structures of the nucleus-representing most likely nuclear speckles-seems to be essential to mediate host gene shutoff. Thus, our data present so far unknown insights into the molecular and spatial requirements needed for IAV-NS1-mediated host shutoff.

Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain.

Highly pathogenic avian influenza viruses (HPAIV) differ from all other strains by a polybasic cleavage site in their hemagglutinin. All these HPAIV share the H5 or H7 subtype. In order to investigate whether the acquisition of a polybasic cleavage site by an avirulent avian influenza virus strain with a hemagglutinin other than H5 or H7 is sufficient for immediate transformation into an HPAIV, we adapted the hemagglutinin cleavage site of A/Duck/Ukraine/1/1963 (H3N8) to that of the HPAIV A/Chicken/Italy/8/98 (H5N2), A/Chicken/HongKong/220/97 (H5N1), or A/Chicken/Germany/R28/03 (H7N7) and generated the recombinant wild-type and cleavage site mutants. In contrast to the wild type, multicycle replication of these mutants in tissue culture was demonstrated by positive plaque assays and viral multiplication in the absence of exogenous trypsin. Therefore, in vitro all cleavage site mutants resemble an HPAIV. However, in chicken they did not exhibit high pathogenicity, although they could be reisolated from cloacal swabs to some extent, indicating enhanced replication in vivo. These results demonstrate that beyond the polybasic hemagglutinin cleavage site, the virulence of HPAIV in chicken is based on additional pathogenicity determinants within the hemagglutinin itself or in the other viral proteins. Taken together, these observations support the notion that acquisition of a polybasic hemagglutinin cleavage site by an avirulent strain with a non-H5/H7 subtype is only one among several alterations necessary for evolution into an HPAIV.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model.

Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses.

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts.

An aptamer that binds efficiently to the hemagglutinins of highly pathogenic avian influenza viruses (H5N1 and H7N7) and inhibits hemagglutinin-glycan interactions.

Highly pathogenic avian influenza (HPAI) H5 and H7 viruses have ravaged the poultry industry in numerous countries in Asia, Europe, Africa and the Middle East, and have resulted in the deaths of millions of birds. Although HPAI H5N1 viruses currently remain avian viruses, they are continuously evolving and have the potential to become pandemic-type viruses capable of human-human transmission. To develop specific reagents to allow better preparedness against this threat, we selected an aptamer (8-3) from a completely random RNA pool that binds with high affinity (∼ KD 170pM) to the hemagglutinins (HAs) derived from HPAI H5N1 (A/H5N1/Vietnam/1194/2004 and A/H5N1/Indonesia/05/2005) and H7N7 (A/H7N7/Netherlands/219/2003) influenza A viruses. Aptamer 8-3 was able to efficiently distinguish HAs derived from subtypes of influenza A virus other than H5 and H7. Aptamer 8-3 was analyzed further to assess its ability to interfere with HA-glycan interactions using our previously established SPR-based competitive assay, and we found that aptamer 8-3 efficiently interferes with HA-glycan binding (EC50 ∼ 25 nM). To derive shorter variants for other applications, aptamer 8-3 was shortened to a 44-mer by deletion analyses. The shortened aptamer, 8-3S, retains the full-length aptamer's affinity and specificity for its cognate Has, and also interferes with HA-glycan interactions. These studies suggest that aptamer 8-3S should be studied further to explore its potential applications not only in surveillance and diagnosis, but also in the development of H5N1- and H7N7-specific virucidal products that interfere with virus-host interactions to contain future H5N1 and H7N7 pandemics.

Evaluation of live attenuated H7N3 and H7N7 vaccine viruses for their receptor binding preferences, immunogenicity in ferrets and cross reactivity to the novel H7N9 virus.

Live attenuated influenza vaccine (LAIV) candidates of the H7 subtype, A/Netherlands/219/03 (H7N7, NL03 ca) and A/chicken/British Columbia/CN-6/2004 (H7N3, BC04 ca), were evaluated for their receptor binding specificity and immunogenicity in ferrets. The BC04 ca virus exhibited α2,3-SA and α2,6-SA dual receptor binding preference while the NL03 ca virus preferentially bound to α2,3-SA. Substitution of the Q226 and G228 (Q-G) by the L226 and S228 (L-S) residues in the HA improved binding to α2,6-SA for NL03 ca. The vaccine viruses with L-S retained the attenuation phenotype. NL03 L-S ca replicated more efficiently than the original NL03 ca virus in the upper respiratory tract of ferrets, and induced higher levels of humoral and cellular immune responses. Prior vaccination with seasonal LAIV reduced H7-specific antibody responses, but did not reduce the H7N7 vaccine mediated protection against a heterologous H7N3 BC04 wt virus infection in ferrets. In addition, the H7N3 and H7N7 vaccine immunized ferret sera cross reacted with the newly emerged H7N9 virus. These data, in combination with the safety data from previously conducted Phase 1 studies, suggest that these vaccines may have a role in responding to the threat posed by the H7N9 virus.

Structural differences between the avian and human H7N9 hemagglutinin proteins are attributable to modifications in salt bridge formation: a computational study with implications in viral evolution.

Influenza A hemagglutinin (HA) is a homotrimeric glycoprotein composed of a fibrous globular stem supporting a globular head containing three sialic acid binding sites responsible for infection. The H7N9 strain has consistently infected an avian host, however, the novel 2013 strain is now capable of infecting a human host which would imply that the HA in both strains structurally differ. A better understanding of the structural differences between the avian and human H7N9 strains may shed light into viral evolution and transmissibility. In this study, we elucidated the structural differences between the avian and human H7N9 strains. Throughout the study, we generated HA homology models, verified the quality of each model, superimposed HA homology models to determine structural differences, and, likewise, elucidated the probable cause for these structural differences. We detected two different types of structural differences between the novel H7N9 human and representative avian strains, wherein, one type (Pattern-1) showed three non-overlapping regions while the other type (Pattern-2) showed only one non-overlapping region. In addition, we found that superimposed HA homology models exhibiting Pattern-1 contain three non-overlapping regions designated as: Region-1 (S1571-A1601); Region-3 (R2621-S2651); and Region-4 (S2701-D2811), whereas, superimposed HA homology models showing Pattern-2 only contain one non-overlapping region designated as Region-2 (S1371-S1451). We attributed the two patterns we observed to either the presence of salt bridges involving the E1141 residue or absence of the R1411:D771 salt bridge. Interestingly, comparison between the human H7N7 and H7N9 HA homology models showed high structural similarity. We propose that the putative absence of the R1411:D771 salt bridge coupled with the putative presence of the E1141:R2621 and E1141:K2641 salt bridges found in the 2013 H7N9 HA homology model is associated to human-type receptor binding. This highlights the possible significance of HA salt bridge formation modifications in viral infectivity, immune escape, transmissibility and evolution.

Quantitative description of glycan-receptor binding of influenza A virus H7 hemagglutinin.

In the context of recently emerged novel influenza strains through reassortment, avian influenza subtypes such as H5N1, H7N7, H7N2, H7N3 and H9N2 pose a constant threat in terms of their adaptation to the human host. Among these subtypes, it was recently demonstrated that mutations in H5 and H9 hemagglutinin (HA) in the context of lab-generated reassorted viruses conferred aerosol transmissibility in ferrets (a property shared by human adapted viruses). We previously demonstrated that the quantitative binding affinity of HA to α2→6 sialylated glycans (human receptors) is one of the important factors governing human adaptation of HA. Although the H7 subtype has infected humans causing varied clinical outcomes from mild conjunctivitis to severe respiratory illnesses, it is not clear where the HA of these subtypes stand in regard to human adaptation since its binding affinity to glycan receptors has not yet been quantified. In this study, we have quantitatively characterized the glycan receptor-binding specificity of HAs from representative strains of Eurasian (H7N7) and North American (H7N2) lineages that have caused human infection. Furthermore, we have demonstrated for the first time that two specific mutations; Gln226→Leu and Gly228→Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptor. Our findings contribute to a framework for monitoring the evolution of H7 HA to be able to adapt to human host.