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.

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.

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.

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.

Limited airborne transmission of H7N9 influenza A virus between ferrets.

Wild waterfowl form the main reservoir of influenza A viruses, from which transmission occurs directly or indirectly to various secondary hosts, including humans. Direct avian-to-human transmission has been observed for viruses of subtypes A(H5N1), A(H7N2), A(H7N3), A(H7N7), A(H9N2) and A(H10N7) upon human exposure to poultry, but a lack of sustained human-to-human transmission has prevented these viruses from causing new pandemics. Recently, avian A(H7N9) viruses were transmitted to humans, causing severe respiratory disease and deaths in China. Because transmission via respiratory droplets and aerosols (hereafter referred to as airborne transmission) is the main route for efficient transmission between humans, it is important to gain an insight into airborne transmission of the A(H7N9) virus. Here we show that although the A/Anhui/1/2013 A(H7N9) virus harbours determinants associated with human adaptation and transmissibility between mammals, its airborne transmissibility in ferrets is limited, and it is intermediate between that of typical human and avian influenza viruses. Multiple A(H7N9) virus genetic variants were transmitted. Upon ferret passage, variants with higher avian receptor binding, higher pH of fusion, and lower thermostability were selected, potentially resulting in reduced transmissibility. This A(H7N9) virus outbreak highlights the need for increased understanding of the determinants of efficient airborne transmission of avian influenza viruses between mammals.

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.

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.

Differential Recognition of Influenza A Viruses by M158-66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues.

UNLABELLED: Natural influenza A virus infections elicit both virus-specific antibody and CD4(+) and CD8(+) T cell responses. Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158-66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8(+) T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE: Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8(+) cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

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.

Novel avian-origin influenza A (H7N9) virus attachment to the respiratory tract of five animal models.

We determined the pattern of attachment of the avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs, and guinea pigs and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This information contributes to our knowledge of the different animal models for influenza.

Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.

Genomewide analysis of reassortment and evolution of human influenza A(H3N2) viruses circulating between 1968 and 2011.

UNLABELLED: Influenza A(H3N2) viruses became widespread in humans during the 1968 H3N2 virus pandemic and have been a major cause of influenza epidemics ever since. These viruses evolve continuously by reassortment and genomic evolution. Antigenic drift is the cause for the need to update influenza vaccines frequently. Using two data sets that span the entire period of circulation of human influenza A(H3N2) viruses, it was shown that influenza A(H3N2) virus evolution can be mapped to 13 antigenic clusters. Here we analyzed the full genomes of 286 influenza A(H3N2) viruses from these two data sets to investigate the genomic evolution and reassortment patterns. Numerous reassortment events were found, scattered over the entire period of virus circulation, but most prominently in viruses circulating between 1991 and 1998. Some of these reassortment events persisted over time, and one of these coincided with an antigenic cluster transition. Furthermore, selection pressures and nucleotide and amino acid substitution rates of all proteins were studied, including those of the recently discovered PB1-N40, PA-X, PA-N155, and PA-N182 proteins. Rates of nucleotide and amino acid substitutions were most pronounced for the hemagglutinin, neuraminidase, and PB1-F2 proteins. Selection pressures were highest in hemagglutinin, neuraminidase, matrix 1, and nonstructural protein 1. This study of genotype in relation to antigenic phenotype throughout the period of circulation of human influenza A(H3N2) viruses leads to a better understanding of the evolution of these viruses. IMPORTANCE: Each winter, influenza virus infects approximately 5 to 15% of the world's population, resulting in significant morbidity and mortality. Influenza A(H3N2) viruses evolve continuously by reassortment and genomic evolution. This leads to changes in antigenic recognition (antigenic drift) which make it necessary to update vaccines against influenza A(H3N2) viruses frequently. In this study, the relationship of genetic evolution to antigenic change spanning the entire period of A(H3N2) virus circulation was studied for the first time. The results presented in this study contribute to a better understanding of genetic evolution in correlation with antigenic evolution of influenza A(H3N2) viruses.

Novel avian-origin influenza A (H7N9) virus attaches to epithelium in both upper and lower respiratory tract of humans.

Influenza A viruses from animal reservoirs have the capacity to adapt to humans and cause influenza pandemics. The occurrence of an influenza pandemic requires efficient virus transmission among humans, which is associated with virus attachment to the upper respiratory tract. Pandemic severity depends on virus ability to cause pneumonia, which is associated with virus attachment to the lower respiratory tract. Recently, a novel avian-origin H7N9 influenza A virus with unknown pandemic potential emerged in humans. We determined the pattern of attachment of two genetically engineered viruses containing the hemagglutinin of either influenza virus A/Shanghai/1/13 or A/Anhui/1/13 to formalin-fixed human respiratory tract tissues using histochemical analysis. Our results show that the emerging H7N9 virus attached moderately or abundantly to both upper and lower respiratory tract, a pattern not seen before for avian influenza A viruses. With the caveat that virus attachment is only the first step in the virus replication cycle, these results suggest that the emerging H7N9 virus has the potential both to transmit efficiently among humans and to cause severe pneumonia.

RNA structural constraints in the evolution of the influenza A virus genome NP segment.

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

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.

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.

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.

Genetic evolution of the neuraminidase of influenza A (H3N2) viruses from 1968 to 2009 and its correspondence to haemagglutinin evolution.

Each year, influenza viruses cause epidemics by evading pre-existing humoral immunity through mutations in the major glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). In 2004, the antigenic evolution of HA of human influenza A (H3N2) viruses was mapped (Smith et al., Science 305, 371-376, 2004) from its introduction in humans in 1968 until 2003. The current study focused on the genetic evolution of NA and compared it with HA using the dataset of Smith and colleagues, updated to the epidemic of the 2009/2010 season. Phylogenetic trees and genetic maps were constructed to visualize the genetic evolution of NA and HA. The results revealed multiple reassortment events over the years. Overall rates of evolutionary change were lower for NA than for HA1 at the nucleotide level. Selection pressures were estimated, revealing an abundance of negatively selected sites and sparse positively selected sites. The differences found between the evolution of NA and HA1 warrant further analysis of the evolution of NA at the phenotypic level, as has been done previously for HA.

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.