Transient RNA structures underlie highly pathogenic avian influenza virus genesis.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease and high fatality in poultry1. They emerge exclusively from H5 and H7 low pathogenic avian influenza viruses (LPAIVs)2. Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for LPAIV-to-HPAIV transition3,4, the exact mechanisms underlying said insertion have remained unknown. Here we used an innovative combination of bioinformatic models to predict RNA structures forming around the influenza virus RNA polymerase during replication, and circular sequencing5 to reliably detect nucleotide insertions. We show that transient H5 hemagglutinin RNA structures predicted to trap the polymerase on purine-rich sequences drive nucleotide insertions characteristic of MBCSs, providing the first strong empirical evidence of RNA structure involvement in MBCS acquisition. Insertion frequencies at the H5 cleavage site were strongly affected by substitutions in flanking genomic regions altering predicted transient RNA structures. Introduction of H5-like cleavage site sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions never observed before in H6 viruses. Our results demonstrate that nucleotide insertions that underlie H5 HPAIV emergence result from a previously unknown RNA-structure-driven diversity-generating mechanism, which could be shared with other RNA viruses.

Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe hemorrhagic disease in terrestrial poultry and are a threat to the poultry industry, wild life, and human health. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds. HPAIV emergence is thought to occur in poultry and not wild aquatic birds, but the reason for this species-restriction is not known. We hypothesized that, due to species-specific tropism and replication, intrahost HPAIV selection is favored in poultry and disfavored in wild aquatic birds. We tested this hypothesis by co-inoculating chickens, representative of poultry, and ducks, representative of wild aquatic birds, with a mixture of H7N7 HPAIV and LPAIV, mimicking HPAIV emergence in an experimental setting. Virus selection was monitored in swabs and tissues by RT-qPCR and immunostaining of differential N-terminal epitope tags that were added to the hemagglutinin protein. HPAIV was selected in four of six co-inoculated chickens, whereas LPAIV remained the major population in co-inoculated ducks on the long-term, despite detection of infectious HPAIV in tissues at early time points. Collectively, our data support the hypothesis that HPAIVs are more likely to be selected at the intrahost level in poultry than in wild aquatic birds and point towards species-specific differences in HPAIV and LPAIV tropism and replication levels as possible explanations.

Characterization of A/H7 influenza virus global antigenic diversity and key determinants in the hemagglutinin globular head mediating A/H7N9 antigenic evolution.

IMPORTANCE: A/H7 avian influenza viruses cause outbreaks in poultry globally, resulting in outbreaks with significant socio-economical impact and zoonotic risks. Occasionally, poultry vaccination programs have been implemented to reduce the burden of these viruses, which might result in an increased immune pressure accelerating antigenic evolution. In fact, evidence for antigenic diversification of A/H7 influenza viruses exists, posing challenges to pandemic preparedness and the design of vaccination strategies efficacious against drifted variants. Here, we performed a comprehensive analysis of the global antigenic diversity of A/H7 influenza viruses and identified the main substitutions in the hemagglutinin responsible for antigenic evolution in A/H7N9 viruses isolated between 2013 and 2019. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution add value to A/H7 influenza virus surveillance programs, the design of vaccines and vaccination strategies, and pandemic preparedness.

A Dutch highly pathogenic H5N6 avian influenza virus showed remarkable tropism for extra-respiratory organs and caused severe disease but was not transmissible via air in the ferret model.

Continued circulation of A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage in poultry has resulted in the diversification in multiple genetic and antigenic clades. Since 2009, clade 2.3.4.4 hemagglutinin (HA) containing viruses harboring the internal and neuraminidase (NA) genes of other avian influenza A viruses have been detected. As a result, various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8 have been identified. As of January 2023, 83 humans have been infected with A/H5N6 viruses, thereby posing an apparent risk for public health. Here, as part of a risk assessment, the in vitro and in vivo characterization of A/H5N6 A/black-headed gull/Netherlands/29/2017 is described. This A/H5N6 virus was not transmitted between ferrets via the air but was of unexpectedly high pathogenicity compared to other described A/H5N6 viruses. The virus replicated and caused severe lesions not only in respiratory tissues but also in multiple extra-respiratory tissues, including brain, liver, pancreas, spleen, lymph nodes, and adrenal gland. Sequence analyses demonstrated that the well-known mammalian adaptation substitution D701N was positively selected in almost all ferrets. In the in vitro experiments, no other known viral phenotypic properties associated with mammalian adaptation or increased pathogenicity were identified. The lack of transmission via the air and the absence of mammalian adaptation markers suggest that the public health risk of this virus is low. The high pathogenicity of this virus in ferrets could not be explained by the known mammalian pathogenicity factors and should be further studied. IMPORTANCE Avian influenza A/H5 viruses can cross the species barrier and infect humans. These infections can have a fatal outcome, but fortunately these influenza A/H5 viruses do not spread between humans. However, the extensive circulation and reassortment of A/H5N6 viruses in poultry and wild birds warrant risk assessments of circulating strains. Here an in-depth characterization of the properties of an avian A/H5N6 influenza virus isolated from a black-headed gull in the Netherlands was performed in vitro and in vivo, in ferrets. The virus was not transmissible via the air but caused severe disease and spread to extra-respiratory organs. Apart from the detection in ferrets of a mutation that increased virus replication, no other mammalian adaptation phenotypes were identified. Our results suggest that the risk of this avian A/H5N6 virus for public health is low. The underlying reasons for the high pathogenicity of this virus are unexplained and should be further studied.

Conserved Expression and Functionality of Furin between Chickens and Ducks as an Activating Protease of Highly Pathogenic Avian Influenza Virus Hemagglutinins.

Highly pathogenic avian influenza viruses (HPAIVs) typically emerge from low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes upon spillover from wild aquatic birds into poultry. The conversion from LPAIV to HPAIV is characterized by the acquisition of a multibasic cleavage site (MBCS) at the proteolytic cleavage site in the viral binding and fusion protein, hemagglutinin (HA), resulting in cleavage and activation of HA by ubiquitously expressed furin-like proteases. The ensuing HPAIVs disseminate systemically in gallinaceous poultry, are endotheliotropic, and cause hemorrhagic disease with high mortality. HPAIV infections in wild aquatic birds are generally milder, often asymptomatic, and generally not associated with systemic dissemination nor endotheliotropic. As MBCS cleavage by host proteases is the main virulence determinant of HPAIVs in poultry, we set out to determine whether cleavage of HPAIV HA by host proteases might influence the observed species-specific pathogenesis and tropism. Here, we sequenced, cloned, and characterized the expression and functionality of duck furin. The furin sequence was strongly conserved between chickens and ducks, and duck furin cleaved HPAIV and tetrabasic HA in an overexpression system, confirming its functionality. Furin was expressed ubiquitously and to similar extents in duck and chicken tissues, including in primary duck endothelial cells, which sustained multicycle replication of H5N1 HPAIV but not LPAIVs. In conclusion, differences in furin-like protease biology between wild aquatic birds and gallinaceous poultry are unlikely to largely determine the stark differences observed in species-specific pathogenesis of HPAIVs. IMPORTANCE HPAIV outbreaks are a global concern due to the health risks for poultry, wildlife, and humans and their major economic impact. The number of LPAIV-to-HPAIV conversions, which is associated with spillover from wild birds to poultry, has been increasing over recent decades. Furthermore, H5 HPAIVs from the A/goose/Guangdong/1/96 lineage have been circulating in migratory birds, causing increasingly frequent epizootics in poultry and wild birds. Milder symptoms in migratory birds allow for dispersion of HPAIVs over long distances, justifying the importance of understanding the pathogenesis of HPAIVs in wild birds. Here, we examined whether host proteases are a likely candidate to explain some differences in the degree of HPAIV systemic dissemination between avian species. This is the first report to show that furin function and expression is comparable between chickens and ducks, which renders the hypothesis unlikely that furin-like protease differences influence the HPAIV species-specific pathogenesis and tropism.

Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis.

Highly Pathogenic Avian Influenza Viruses (HPAIVs) arise from low pathogenic precursors following spillover from wild waterfowl into poultry populations. The main virulence determinant of HPAIVs is the presence of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein. The MBCS allows for HA cleavage and, consequently, activation by ubiquitous proteases, which results in systemic dissemination in terrestrial poultry. Since 1959, 51 independent MBCS acquisition events have been documented, virtually all in HA from the H5 and H7 subtypes. In the present article, data from natural LPAIV to HPAIV conversions and experimental in vitro and in vivo studies were reviewed in order to compile recent advances in understanding HA cleavage efficiency, protease usage, and MBCS acquisition mechanisms. Finally, recent hypotheses that might explain the unique predisposition of the H5 and H7 HA sequences to obtain an MBCS in nature are discussed.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis.

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.

Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses.

Highly pathogenic avian influenza viruses (HPAIVs) cause fatal systemic infections in chickens, which are associated with endotheliotropism. HPAIV infections in wild birds are generally milder and not endotheliotropic. Here, we aimed to elucidate the species-specific endotheliotropism of HPAIVs using primary chicken and duck aortic endothelial cells (chAEC and dAEC respectively). Viral replication kinetics and host responses were assessed in chAEC and dAEC upon inoculation with HPAIV H5N1 and compared to embryonic fibroblasts. Although dAEC were susceptible to HPAIV upon inoculation at high multiplicity of infection, HPAIV replicated to lower levels in dAEC than chAEC during multi-cycle replication. The susceptibility of duck embryonic endothelial cells to HPAIV was confirmed in embryos. Innate immune responses upon HPAIV inoculation differed between chAEC, dAEC, and embryonic fibroblasts. Expression of the pro-inflammatory cytokine IL8 increased in chicken cells but decreased in dAEC. Contrastingly, the induction of antiviral responses was stronger in dAEC than in chAEC, and chicken and duck fibroblasts. Taken together, these data demonstrate that although duck endothelial cells are permissive to HPAIV infection, they display markedly different innate immune responses than chAEC and embryonic fibroblasts. These differences may contribute to the species-dependent differences in endotheliotropism and consequently HPAIV pathogenesis.

A paucigranulocytic asthma host environment promotes the emergence of virulent influenza viral variants.

Influenza virus has a high mutation rate, such that within one host different viral variants can emerge. Evidence suggests that influenza virus variants are more prevalent in pregnant and/or obese individuals due to their impaired interferon response. We have recently shown that the non-allergic, paucigranulocytic subtype of asthma is associated with impaired type I interferon production. Here, we seek to address if this is associated with an increased emergence of influenza virus variants. Compared to controls, mice with paucigranulocytic asthma had increased disease severity and an increased emergence of influenza virus variants. Specifically, PB1 mutations exclusively detected in asthmatic mice were associated with increased polymerase activity. Furthermore, asthmatic host-derived virus led to increased disease severity in wild-type mice. Taken together, these data suggest that at least a subset of patients with asthma may be more susceptible to severe influenza and may be a possible source of new influenza virus variants.

Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites.

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Influenza Virus between Mammals.

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.

Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection.

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans.IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

Conserved structural RNA domains in regions coding for cleavage site motifs in hemagglutinin genes of influenza viruses.

The acquisition of a multibasic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein is the main determinant of the conversion of low pathogenic avian influenza viruses into highly pathogenic strains, facilitating HA cleavage and virus replication in a broader range of host cells. In nature, substitutions or insertions in HA RNA genomic segments that code for multiple basic amino acids have been observed only in the HA genes of two out of sixteen subtypes circulating in birds, H5 and H7. Given the compatibility of MBCS motifs with HA proteins of numerous subtypes, this selectivity was hypothesized to be determined by the existence of specific motifs in HA RNA, in particular structured domains. In H5 and H7 HA RNAs, predictions of such domains have yielded alternative conserved stem-loop structures with the cleavage site codons in the hairpin loops. Here, potential RNA secondary structures were analyzed in the cleavage site regions of HA segments of influenza viruses of different types and subtypes. H5- and H7-like stem-loop structures were found in all known influenza A virus subtypes and in influenza B and C viruses with homology modeling. Nucleotide covariations supported this conservation to be determined by RNA structural constraints that are stronger in the domain-closing bottom stems as compared to apical parts. The structured character of this region in (sub-)types other than H5 and H7 indicates its functional importance beyond the ability to evolve toward an MBCS responsible for a highly pathogenic phenotype.

Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System.

Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.IMPORTANCE Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.

H1N1pdm09 Influenza Virus and Its Descendants Lack Extra-epitopic Amino Acid Residues Associated With Reduced Recognition by M158-66-Specific CD8+ T Cells.

Extra-epitopic amino acid residues affect recognition of human influenza A viruses (IAVs) by CD8+ T-lymphocytes (CTLs) specific for the highly conserved HLA-A*0201 restricted M158-66 epitope located in the matrix 1 (M1) protein. These residues are absent in the M1 protein of the 2009-pandemic IAV (H1N1pdm09). Consequently, stimulation with M1 protein of H1N1pdm09 IAV resulted in stronger activation and lytic activity of M158-66-specific CTLs than stimulation with seasonal H3N2 IAVs. During >6 years of circulation in the human population, descendants of the H1N1pdm09 virus had accumulated 4 other amino acid substitutions. However, these did not affect M158-66-specific CTL activation.

Transmission routes of respiratory viruses among humans.

Respiratory tract infections can be caused by a wide variety of viruses. Airborne transmission via droplets and aerosols enables some of these viruses to spread efficiently among humans, causing outbreaks that are difficult to control. Many outbreaks have been investigated retrospectively to study the possible routes of inter-human virus transmission. The results of these studies are often inconclusive and at the same time data from controlled experiments is sparse. Therefore, fundamental knowledge on transmission routes that could be used to improve intervention strategies is still missing. We here present an overview of the available data from experimental and observational studies on the transmission routes of respiratory viruses between humans, identify knowledge gaps, and discuss how the available knowledge is currently implemented in isolation guidelines in health care settings.

Human Clade 2.3.4.4 A/H5N6 Influenza Virus Lacks Mammalian Adaptation Markers and Does Not Transmit via the Airborne Route between Ferrets.

Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low. IMPORTANCE Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus in vitro and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses.

Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes.

The influenza A virus genome consists of eight RNA segments. RNA structures within these segments and complementary (cRNA) and protein-coding mRNAs may play a role in virus replication. Here, conserved putative secondary structures that impose significant evolutionary constraints on the gene segment encoding the surface glycoprotein hemagglutinin (HA) were investigated using available sequence data on tens of thousands of virus strains. Structural constraints were identified by analysis of covariations of nucleotides suggested to be paired by structure prediction algorithms. The significance of covariations was estimated by mutual information calculations and tracing multiple covariation events during virus evolution. Covariation patterns demonstrated that structured domains in HA RNAs were mostly subtype-specific, whereas some structures were conserved in several subtypes. The influence of RNA folding on virus replication was studied by plaque assays of mutant viruses with disrupted structures. The results suggest that over the whole length of the HA segment there are local structured domains which contribute to the virus fitness but individually are not essential for the virus. Existence of subtype-specific structured regions in the segments of the influenza A virus genome is apparently an important factor in virus evolution and reassortment of its genes.

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe.

UNLABELLED: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host. IMPORTANCE: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.