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Enabling the 'host jump': Structural determinants of receptor-binding specificity in influenza A viruses
Abstract
The recent emergence of the H7N9 avian influenza A virus and its ability to infect humans emphasize the epidemic and pandemic potential of these viruses. Interspecies transmission is the result of many factors, which ultimately lead to a change in the host tropism of the virus. One of the key factors involved is a shift in the receptor-binding specificity of the virus, which is mostly determined by mutations in the viral haemagglutinin (HA). In this Review, we discuss recent crystallographic studies that provide molecular insights into HA-host receptor interactions that have enabled several influenza A virus subtypes to 'jump' from avian to human hosts.
... Concerning the viral proteins on the infectious capacity of viruses, it is necessary to consider the surface glycoproteins of AIV at the beginning of this description, HA and NA, referred to as HxNy [47,48]. The HxNy profile determines the virus subtype according to the type of HA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and NA (1-11) (Fig. 1) [9,42,[49][50][51][52][53], along with the regulation of the range of interaction with the host, affecting the virus transmissibility to new hosts and binding and releasing specific receptors [26,42,54,55]. The role of the HA protein is to specifically bind to sialic acid (SA) in cellular receptors [25,50,51,56], specifically 5-N-acetylneuraminic acid (Neu5Ac) and recently demonstrated binding to 5-N-glycolylneuraminic acid (Neu5Gc) [51,[57][58][59][60]. ...
... The HxNy profile determines the virus subtype according to the type of HA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and NA (1-11) (Fig. 1) [9,42,[49][50][51][52][53], along with the regulation of the range of interaction with the host, affecting the virus transmissibility to new hosts and binding and releasing specific receptors [26,42,54,55]. The role of the HA protein is to specifically bind to sialic acid (SA) in cellular receptors [25,50,51,56], specifically 5-N-acetylneuraminic acid (Neu5Ac) and recently demonstrated binding to 5-N-glycolylneuraminic acid (Neu5Gc) [51,[57][58][59][60]. For Neu5Ac receptors, it has been named galactose-linked SA receptors (SA α−2,6 Gal) as mammalian receptors and SA α−2,3 Gal as avian receptors [61]. ...
... The HxNy profile determines the virus subtype according to the type of HA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and NA (1-11) (Fig. 1) [9,42,[49][50][51][52][53], along with the regulation of the range of interaction with the host, affecting the virus transmissibility to new hosts and binding and releasing specific receptors [26,42,54,55]. The role of the HA protein is to specifically bind to sialic acid (SA) in cellular receptors [25,50,51,56], specifically 5-N-acetylneuraminic acid (Neu5Ac) and recently demonstrated binding to 5-N-glycolylneuraminic acid (Neu5Gc) [51,[57][58][59][60]. For Neu5Ac receptors, it has been named galactose-linked SA receptors (SA α−2,6 Gal) as mammalian receptors and SA α−2,3 Gal as avian receptors [61]. ...
The high pathogenicity avian influenza virus H5N1, which first emerged in the winter of 2021, has resulted in multiple outbreaks across the American continent through the summer of 2023 and they continue based on early 2025 records, presenting significant challenges for global health and food security. The viruses causing the outbreaks belong to clade 2.3.4.4b, which are descendants of the lineage A/Goose/Guangdong/1/1996 (Gs/Gd) through genetic reassortments with several low pathogenicity avian influenza viruses present in populations of Anseriformes and Charadriiformes orders. This review addresses these issues by thoroughly analysing available epidemiological databases and specialized literature reviews. This project explores the mechanisms behind the resurgence of the H5N1 virus. It provides a comprehensive overview of the origin, timeline and factors contributing to its prevalence among wild bird populations on the American continent.
... Surprisingly, the AA 137 residue is mutated into Ser in HN/4-10 H3N8 strain and the strains nearby, unlikely H3N8 of other species, but the same to A/Finland/486/2004 (2004 H3N2), A/Victoria/361/2011 (2011 H3N2) and A/Hunan-Yuhua/11022/2022 (2022 H3N2). Previous research has shown that AA substitutions at sites 186, 190, 225, 226, and 228 (H3 numbering) are significant for receptor-binding adaptations in a variety of HA subtypes, including H1, H2, H3, H4, H5, H6, and H7 (9). AA 186 and AA 190 sites are conserved in all H3N8 and 1968 H3N2. ...
... As for the 222 and 225 sites, which may affect the sialic acid-binding affinity in 2004 H3N2 (19), HN/4-10 H3N8 is partial conserved as Trp and Gly as other H3N8. L226 and S228 are often considered signatures for H2 and H3 AIV that change receptor-binding preference from avian receptor to human receptor (9). The presence of Q226 in HN/4-10 H3N8 indicates that the virus may be still a typical AIV strain. ...
... Although the HN/4-10 H3N8 strain already possesses the adaptive mutation G228S, it retains the Q226 residue. For IAVs such as H3N2 and H4N6, L226 has been identified as favorable for binding to α2-6 linked sialic acid (SA), and the Q226L mutation is commonly observed in the HA of influenza viruses adapted for human infection (9,38). To investi gate whether the Q226L mutation alters the receptor-binding properties of H3N8 HA, we introduced this mutation into both the S228 and G228 HA. ...
Recent avian-origin H3N8 influenza A virus (IAV) that have infected humans pose a potential public health concern. Alterations in the viral surface glycoprotein, hemagglutinin (HA), are typically required for IAVs to cross the species barrier for adaptation to a new host, but whether H3N8 has adapted to infect humans remains elusive. The observation of a degenerative codon in position 228 of HA in human H3N8 A/Henan/4-10/2022 protein sequence, which could be residue G or S, suggests a dynamic viral adaptation for human infection. Previously, we found this human-isolated virus has shown the ability to transmit between ferrets via respiratory droplets, with the HA-G228S substitution mutation emerging as a critical determinant for the airborne transmission of the virus in ferrets. Here, we investigated the receptor-binding properties of these two H3N8 HAs. Our results showed H3N8 HAs have dual receptor-binding properties with a preference for avian receptor binding, and G228S slightly increased binding to human receptors. Cryo-electron microscopy structures of the two H3N8 HAs with avian and human receptor analogs revealed the basis for dual receptor binding. Mutagenesis studies reveal that the Q226L mutation shifts H3N8 HA’s receptor preference from avian to human, while the G228S substitution enhances binding to both receptor types. H3N8 exhibits distinct antigenic sites compared to H3N2, prompting concerns regarding vaccine efficacy. These findings suggest that the current H3N8 human isolates are yet to adapt for efficient human-to-human transmission and further continuous surveillance should be implemented.
IMPORTANCE
Influenza virus transmission remains a public health concern currently. H3N8 subtype influenza A viruses infect humans and their HAs acquire the ability to bind to both human and avian receptors, posing a threat to human health. We have solved and analyzed the structural basis of dual receptor binding of recently human-infecting H3N8 HA, and we demonstrate that the G228S enhances human receptor binding and adaptation. We also found that HN/4-10 H3N8 HA has distinct antigenic sites, which challenges vaccine efficacy. Taken together, our work is critical to the prevention and control of human H3 influenza virus infection.
... The viral surface glycoprotein hemagglutinin (HA), a trimeric class I transmembrane protein that binds sialic acid (SA) receptors to initiate virus infection, is a major determinant of host tropism, pathogenicity, and transmission 19 . Generally, the HA of AIVs preferentially binds avian-type α2,3-linked sialic acid (α2-3-SA) receptor, while humanadapted viruses preferentially bind to the human-type α2,6-linked sialic acid (α2-6-SA) receptor. ...
... It is well known that the Q226L and G228S substitutions on the HA protein are critical for binding human-type receptor in H2, H3, H4, H5, and H9 subtype viruses 19,26 . To study the synergistic effects of the N144S-Q226L-G228S substitution, we further introduced substitution combinations L226/G228 and L226/S228 into the Dk/ FJ/2021 HA protein (S144-Q226-G228). ...
... To overcome host barriers, influenza A viruses should be able to bind receptors on the surface of new host cells 19 . In the present study, several H2Ny LPAIVs were identified, where one WT H2N2 AIV (Dk/FJ/ 2021) possessed the ability to bind both avian-and human-type receptors but preferred to avian-type receptor. ...
The 1957 H2N2 influenza pandemic virus [A(H2N2)pdm1957] has disappeared from humans since 1968, while H2N2 avian influenza viruses (AIVs) are still circulating in birds. It is necessary to reveal the recurrence risk and potential cross-species infection of these AIVs from avian to mammals. We find that H2 AIVs circulating in domestic poultry in China have genetic and antigenic differences compared to the A(H2N2)pdm1957. One H2N2 AIV has a dual receptor-binding property similar to that of the A(H2N2)pdm1957. Molecular and structural studies reveal that the N144S, and N144E or R137M substitutions in hemagglutinin (HA) enable H2N2 avian or human viruses to bind or preferentially bind human-type receptor. The H2N2 AIV rapidly adapts to mice (female) and acquires mammalian-adapted mutations that facilitated transmission in guinea pigs and ferrets (female). These findings on the receptor-binding, infectivity, transmission, and mammalian-adaptation characteristics of H2N2 AIVs provide a reference for early-warning and prevention for this subtype.
... Influenza viruses have two major surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA). HA mediates the binding of viruses to the cellular sialic acid receptors and fusion of the virion membrane with the intracellular endosome membranes 5 . NA removes decoy receptors from mucins and cleaves off the sialic acid, allowing the release of progeny viruses from the infected cells 6,7 . ...
... In addition to seasonal H1N1, H3N2 influenza A virus and influenza B virus, the recent rise in the frequency of avian influenza viruses (AIVs) infection has also raised serious public health concerns 4,5,29 . Among them, H5N6 AIV has obtained global attention. ...
The influenza neuraminidase (NA) is a potential target for the development of a next-generation influenza vaccine, but its antigenicity is not well understood. Here, we isolate an anti-N6 human monoclonal antibody, named 18_14D, from an H5N6 avian influenza virus (AIV) infected patient. The antibody weakly inhibits enzymatic activity but confers protection in female mice, mainly via ADCC function. The cryo-EM structure shows that 18_14D binds to a unique epitope on the lateral surface of the N6 tetramer, preventing the formation of tightly closed NA tetramers. These findings contribute to the molecular understanding of protective immune responses to NA of AIVs in humans and open an avenue for the rational design of NA-based vaccines.
... Influenza A virus (IAV) is a zoonotic pathogen that remains an imminent global threat to public health and, even more so, to livestock welfare worldwide [1,2]. Due to the segmented nature of their RNA genome and their error-prone RNA replication machinery, IAVs are highly genetically flexible, resulting in antigenically drifted viruses with a rapid evolution in surface proteins (antigenic drift) and rapid gene reassortment (antigenic shift), which occurs when two or more IAVs infect a cell or host and viral gene segments are reshuffled [1,2,3]. Surveillance of IAVs within the swine population is important because pigs are susceptible to avian, swine, and human IAVs and are considered "mixing vessels" in the generation of influenza viruses with pandemic potential [3,4]. ...
... Due to the segmented nature of their RNA genome and their error-prone RNA replication machinery, IAVs are highly genetically flexible, resulting in antigenically drifted viruses with a rapid evolution in surface proteins (antigenic drift) and rapid gene reassortment (antigenic shift), which occurs when two or more IAVs infect a cell or host and viral gene segments are reshuffled [1,2,3]. Surveillance of IAVs within the swine population is important because pigs are susceptible to avian, swine, and human IAVs and are considered "mixing vessels" in the generation of influenza viruses with pandemic potential [3,4]. The first IAV pandemic of the twenty-first century, originating in swine in Mexico during March-May 2009 and killing approximately 575,000 people worldwide, was caused by a reassortant H1N1 virus termed "A(H1N1)pdm09" (pdm09) [5], which contained segments from avian, human, and swine viruses that had evolved in swine populations for over 90 years before virus emergence in humans [6,7]. ...
The 2009 influenza A H1N1 pandemic (pdm09) originated from the influenza A virus of swine (IAV-S) through multiple reassortment events with avian and human IAVs. The pdm09 reportedly reintroduced the virus to pigs, contributing to the evolution and diversity of IAV-S through frequent reassortment and drifts. Surveillance and whole-genome sequencing of IAV-S from conventional pig farms in Korea during 2021–2022 revealed that the genetic diversity of H1 and H3 IAV-S was continuously enriched after human-to-swine spillover of pdm09 viruses with long-term maintenance, persistence, and reassortment of virus lineages. Evidence of additional human-to-swine spillover of viruses that are different from the 2009 virus but close to that of the recent H1N1pdm09 human vaccine was identified in this study. The identification of swine-adapted pdm09 viruses, which have accumulated amino acid mutations with potentially altered antigenicity and a unique potential N-glycosylation site within the haemagglutinin (HA) gene, suggests the distinctive evolution of spillover pdm09 viruses in swine. The genetic constellation of the recently emerging Eurasian avian-like swine lineage and the preexisting classical swine lineage H1 viruses in Korea has been expanded through reassortment with cocirculating pdm09 viruses and/or H3N2 IAV-S harboring the pdm09 M gene (H3N2pM). Collectively, after the major shift of Korean IAV-S from the classical swine lineage to the pdm09 lineage in 2009, the frequent spillover of pdm09 viruses and the circulation of IAV-S harboring pdm09 gene segments led to the continuous diversification of IAV-S through antigenic drift and shift, raising concerns about the potential reintroduction of these viruses to humans.
... Mutational Effects. As indicated in Shi et al. (2014), there are various mutations in the HA1 receptor binding domain that enable 'host jumping'. Our results show several statistically significant differences in binding affinity of antibodies given the presence of these mutations. ...
... The distribution of van der Waals energies and HADDOCK score docking metrics broken out by antigen mutations. The first amino acid shown on the left of each plot in gold represents the reference residue at that position as described inShi et al. (2014). Statistical comparisons shown are significant Wilcoxon Rank Sum test p-values at the α<0.05 level.4 ...
The United States Department of Agriculture has recently released reports that show samples from 2022-2024 of highly pathogenic avian influenza (H5N1) have been detected in mammals and birds (1). To date, the United States Centers for Disease Control reports that there have been 27 humans infected with H5N1 in 2024 (2). The broader potential impact on human health remains unclear. In this study, we computationally model 1,804 protein complexes consisting of various H5 isolates from 1959 to 2024 against 11 hemagglutinin domain 1 (HA1)-neutralizing antibodies. This study shows a trend of weakening binding affinity of existing antibodies against H5 isolates over time, indicating that the H5N1 virus is evolving immune escape of our medical defenses. We also found that based on the wide variety of host species and geographic locations in which H5N1 was observed to have been transmitted from birds to mammals, there is not a single central reservoir host species or location associated with H5N1’s spread. These results indicate that the virus has potential to move from epidemic to pandemic status in the near future. This study illustrates the value of high-performance computing to rapidly model protein-protein interactions and viral genomic sequence data at-scale for functional insights into medical preparedness.
Research in Context
Evidence before this study
Previous studies have shown cases of avian influenza transmissions to mammals that are increasing in frequency, which is of concern to human health. Since 1997, nearly a thousand H5N1 cases have been reported in humans with a 52% fatality rate. Previous analyses have indicated specific mutations on the hemagglutinin protein that allow for this “host jumping” between birds and mammals (3). There are also existing evidence of recent viral strains with reduced neutralization to sera (4).
Added value of this study
This study provides a comprehensive look at the mutational space of hemagglutinin of H5N1 influenza and presents computational predictions of the binding between various HA1-neutralizing antibodies derived from infected vaccinated patients and humanized mice and 1,804 representative H5 HA1 proteins. These analyses show a weakening trend of existing antibodies. We also confirm that the mutations found in other studies that enable zoonosis also affect binding affinities of the antibodies tested. Furthermore, through phylogenetic analyses, we quantify the avian-to-mammalian transmissions from 1959 to 2024 and show a persistent circulation of isolates between North America and Europe.
Taken together, the continuous transmission of H5N1 from birds to mammals and the increase in immuno-evasive HA strains in mammals sampled over time suggest that antigenic drift is a source of spillover risk.
Implications of all the available evidence
Our findings indicate that the worsening in antibody binding, along with the increase in of avian-to-mammalian H5N1 influenza transmissions are risks to public health.
Through the findings of previous studies along with the predictions reported in this study, we can now monitor specific mutations of interest, quantified by their potential impact on antibody evasion, and inform public health monitoring of circulating isolates in 2024 and beyond.
In addition, these findings may help to guide future vaccine and therapeutic development in the fight against H5N1 influenza infections in humans.
... Notably, the R467Q mutation in the HA protein enhances the virus's ability to bind to human receptors. Although it is generally believed that alterations in the virus's receptor-binding ability are primarily caused by variations near the HA pocket structure (the 130-loop, 190-helix, and 220-loop) [32], both our hemagglutination assay and solid-phase binding assay confirmed that the R467Q mutation enhances the binding of H4N1 AIV to human receptors. This finding indicates that changes distant from the pocket structure can also impact the virus's receptor-binding ability. ...
Background
Avian influenza virus (AIV), a zoonotic pathogen found worldwide, includes multiple subtypes, one of which is the H4 subtype frequently detected in wild birds and poultry. Despite its prevalence, research on H4 subtype AIV has been scarce, with a focus predominantly on the H4N2 and H4N6 subtypes. The zoonotic potential of H4N1 has not been investigated to date.
Methods
In this study, we used gene sequencing in conjunction with bioinformatics methodologies to analyze wild-type H4N1 AIV strain and mutant strains emerging from serial passaging in cell culture. Furthermore, we assessed the zoonotic potential of H4N1 and the alterations caused by mutations via a series of phenotype assays, including evaluation of receptor binding affinity, immunofluorescence assays, analyses of growth kinetics across different animal cell cultures, and in vivo pathogenicity studies.
Results
Our research reveals that H4N1 AIV can bind to human receptors and exhibits an affinity for human lung and tracheal tissues. In vitro experiments demonstrate that H4N1 replicates efficiently in human cell lines. Furthermore, animal studies demonstrate that H4N1 can induce pneumonia in mice without the need for prior adaptation to the host. Notably, during passage in cell culture, H4N1 rapidly acquired two previously unreported mutations. These mutations significantly enhanced the virus’s ability to attach to human receptors and its capacity for replication.
Conclusions
In summary, our study provides preliminary experimental evidence for the emerging zoonotic potential of H4N1 AIV. These findings expand our knowledge of the H4 subtype AIV and reinforce the critical need for continued surveillance of AIV to prevent and prepare for potential outbreaks affecting human health.
... Previous analyses have indicated specific mutations in the haemagglutinin protein that allow for this "host jumping" between birds and mammals. 3 There is also existing evidence of recent viral strains with reduced neutralisation by sera. 4 Added value of this study This study provides a comprehensive look at the mutational space of haemagglutinin of H5N1 influenza and presents computational predictions of the binding between various HA1-neutralising antibodies derived from infected vaccinated patients and humanised mice, and 1804 representative H5 HA1 proteins. These analyses show a weakening trend of existing antibodies. ...
Background: The United States Department of Agriculture has recently released reports that show samples collected from 2022 to 2025 of highly pathogenic avian influenza (H5N1) have been detected in mammals and birds. Up to February 2025, the United States Centres for Disease Control and Prevention reports that there have been 67 humans infected with H5N1 since 2024 with 1 death. The broader potential impact on human health remains unclear.
Methods: In this study, we computationally model 1804 protein complexes consisting of various H5 isolates from 1959 to 2024 against 11 haemagglutinin domain 1 (HA1)-neutralising antibodies. This was performed using AI-based protein folding and physics-based simulations of the antibody-antigen interactions. We analysed binding affinity changes over time and across various antibodies using multiple biochemical and biophysical binding metrics.
Findings: This study shows a trend of weakening binding affinity of existing antibodies against H5 isolates over time, indicating that the H5N1 virus is evolving immune escape from our therapeutic and immunological defences. We also found that based on the wide variety of host species and geographic locations in which H5N1 was observed to have been transmitted from birds to mammals, there is not a single central reservoir host species or location associated with H5N1's spread.
Interpretation: These results indicate that the virus has potential to move from epidemic to pandemic status. This study illustrates the value of high-performance computing to rapidly model protein–protein interactions and viral genomic sequence data at-scale for functional insights into medical preparedness.
... The changes in the H3N2 CIV have included previously identified mammalian-adaptive sites [23], some experimentally confirmed as having biological effects [47], and the H3N2 CIV appears to have lost the host range for at least some avian hosts [78]. Other concerning adaptive changes that underlie known human adaptation and pandemic risk (including NP changes to evade MxA/BTN3A3 restriction factors, and HA RBS changes to the 226/228 residues) are absent, as the H3N2 CIV remains 'avian-like' [79][80][81]. Changes have occurred in NA domains related to ligand binding, but the constellation of changes does not include any known inhibitor-resistance markers, suggesting antiviral use in zoonotic exposure would be efficacious [82]. ...
The H3N2 canine influenza virus (CIV) emerged from an avian reservoir in Asia to circulate entirely among dogs for the last 20 years. The virus was first seen circulating outside Asian dog populations in 2015, in North America. Utilizing viral genomic data in addition to clinical reports and diagnostic testing data, we provide an updated analysis of the evolution and epidemiology of the virus in its canine host. CIV in dogs in North America is marked by a complex life history – including local outbreaks, regional lineage die-outs, and repeated reintroductions of the virus (with diverse genotypes) from different regions of Asia. Phylogenetic and Bayesian analysis reveal multiple CIV clades, and viruses from China have seeded recent North American outbreaks, with 2 or 3 introductions in the past 3 years. Genomic epidemiology confirms that within North America the virus spreads very rapidly among dogs in kennels and shelters in different regions – but then dies out locally. The overall epidemic therefore requires longer-distance dispersal of virus to maintain outbreaks over the long term. With a constant evolutionary rate over 20 years, CIV still appears best adapted to transmission in dense populations and has not gained properties for prolonged circulation among dogs.
... Based on the antigenic differences of its hemagglutinin (HA) and neuraminidase (NA) molecules, IAV could be classified into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11), most of which spread among wild birds [4]. The presence of both avian α-2,3 and human α-2,6 sialic acid receptors in the pig respiratory tract allows avian influenza and human influenza viruses to infect pigs [5]. This susceptibility enables viruses from different sources to acquire adaptive mutations or undergo genetic reassortment within pigs, thereby generating novel viruses with pandemic potential [6,7]. ...
Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China, 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants, possessing internal genes of identical origins (PB2, PB1, PA, NP, M: pdm09/H1N1 origin, NS: North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mice. Taken together, our study indicates that the H1 subtype SIVs recently circulating in China pose a potential threat to human health and emphasizes the importance of continuing to closely monitor their evolution and spread.
... Due to their vulnerability to avian, swine, and human influenza A viruses (IAVs), pigs are regarded as "mixing vessels" for the creation of influenza viruses with the potential to cause a pandemic (1)(2)(3)(4). Moreover, these reassortants may evolve mutations to adapt to humans, resulting in a human influenza pandemic (5). ...
Introduction
Animal influenza viruses pose a danger to the general public. Eurasian avian-like H1N1 (EA H1N1) viruses have recently infected humans in several different countries and are often found in pigs in China, indicating that they have the potential to cause a pandemic. Therefore, there is an urgent need to develop a potent vaccine against EA H1N1.
Methods
In this study, we report the effective intramuscular (i.m.) and/or intranasal (i.n.) vaccination of mice with a subunit influenza vaccine utilizing safe adjuvant gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis. The hemagglutinin (HA)-protein anchor (PA) subunit vaccine can be simply mixed with GEM particles to produce vaccines.
Results
After two booster injections, the i.m.+i.n. administered GEM subunit vaccine achieved hemagglutination inhibition titers in the serum that were equivalent to those observed using the conventional i.m. method. The mucosal and Th1-biased immune responses generated by the i.m. administered subunit vaccine alone were inferior to those induced by the i.n. and i.m.+i.n. administered subunit vaccines. Vaccinated mice were challenged with live viruses (G4 EA H1N1 and A/PR/8/34) to determine whether the adjuvant combination protected against the virus after vaccination with the influenza subunit vaccine. Compared to mice inoculated with HA alone, mice immunized with i.m.+i.n. or i.n. HA-PA-GEM displayed undetectable viral titers in the lungs, at 5 d after challenge.
Discussion
Overall, this study not only offers other potential platforms for the generation of swine influenza vaccines, but also a theoretical foundation for vaccine vector platforms that can be utilized for future research on other infections.
... вирусы 126 линии B/Yamagata впервые появились на Тайване, тогда как викторианские 127 вирусы преобладали в Китае и циркулировали еще в некоторых регионах мира 128 [144]. Похожая ситуация наблюдалась в том же сезоне в Австралии, где 129 доминировала викторианская линия, и в Новой Зеландии, где в то же время 130 доминировала линия B/Yamagata [138]. 131 Позднее во многих странах продолжала доминировать линия B/Yamagata 132 [39], а в сезон 2017-2018 гг. ...
Although the history of the influenza virus existence goes back thousands of years, the first human influenza A virus was discovered only in 1933, when proper models and substrates for virus isolation became available; then the influenza B virus was isolated and, some later, the influenza C virus. Influenza A viruses evolve most rapidly. Influenza B viruses mutate 2–3 times slower, with influenza C viruses being most conservative. From the moment of isolation until the end of the 1970s, the antigenic evolution of influenza B viruses proceeded smoothly; the isolates were genetically quite homogeneous. In the 1970s–1980s, influenza B viruses diverged into two genetic lineages, “B/Victoria/2/87-like virus lineage” and the “B/Yamagata/16/88-like virus lineage.” For some time, B/Yamagata lineage viruses were widespread throughout the world, while the circulation area of B/Victoria viruses was limited to East Asia. Then the Victorian lineage began its triumphal march across the globe. From this moment on, both lineages of influenza B virus circulated together, with dominance of one or the other lineage in different geographic regions and different epidemiological seasons. Later, the B/Yamagata lineage dominated in many countries, but by the onset of the COVID–19 pandemic, Victorian viruses were already dominant. At the same time, the last representatives of the B/Yamagata lineage were identified. Today, the B/Yamagata lineage has disappeared from circulation and the WHO has concluded that its inclusion in influenza vaccine strains is no longer necessary. Antigenic variability undoubtedly plays a decisive role in the virus evolution. It is accompanied by changes in biological characteristics that, to one degree or another, determine the virus’s ability to self-preserve. No matter how antigenically new a next influenza virus variant is, it will bear a certain set of biological properties, the combination of which will allow the pathogen to best survive in sensitive host. In this review, we have summarized information on the most striking biological properties of influenza B viruses, such as sensitivity to nonspecific blood serum inhibitors, hemagglutinin receptor specificity, its thermostability, sensitivity to low pH values, and temperature sensitivity of reproduction.
... For molecular docking between HA and SA receptors, structural analogs of avian and human receptors were downloaded from PubChem. The molecules treated as HA ligands for the analysis were: Alpha-N-acetylneuraminyl-(2,6)-beta-D-galactosyl-(1,4)-N-acetyl-beta-D-glucosamine (PubChem CID 125128) (human receptor analog) and N-acetylalpha-neuraminyl-(2,3)-beta-D-galactosyl-(1,4)-N-acetyl-beta-D-glucosamine (PubChem CID 44611401) (avian receptor analog), as previously reported [26]. ...
Avian influenza subtype H5N1 has caused outbreaks worldwide since 1996, with the emergence of the Guandong lineage in China. The current clade 2.3.4.4b has evolved from this lineage, with increased virulence and mass mortality events in birds and mammals. The objective of this study was the analysis of 17 viral genomes of H5N1 avian influenza isolated in Venezuela during the 2022–2023 outbreak. The eight viral genomic segments were amplified using universal primers and sequenced via next-generation sequencing. The sequences were analyzed to confirm the H5 hemagglutinin clade, identify possible genetic reassortments, and perform a phylogenetic and docking analysis of the viral isolates. The viruses found in Venezuela belonged, as expected, to clade 2.3.4.4b and formed a monophyletic clade with North American influenza viruses, with no evidence of further reassortment. The introduction of the virus in South America is associated with bird migration through the Atlantic (Venezuela), Atlantic/Mississippi (Choco, Colombia), and Pacific migratory flyways, with the emergence of several viral lineages. Several mutations were found in all segments of the genome, although none of the key mutations was involved in mammalian adaptation. Moreover, in silico structural analysis suggests, as expected, that the viral hemagglutinin maintained a predilection for avian α2,3-linked sialic acid. The unprecedented pathogenic outbreak of avian influenza disease in South America was associated with the circulation of three different lineages, which maintain a lower affinity for the mammalian receptor.
... Analysis of receptor binding specificity markers in HA protein revealed the presence of a QRG amino acid sequence at positions 222-224 (H5 numbering), which is associated with virus HA affinity to avian α-2.3-linked receptors 19,20 . At the same time, there were amino acids markers 123P, 133A, 156A which were previously reported as associated with the increased binding to mammalian α-2.6-linked ...
Currently A(H5Nx) avian influenza viruses are globally widespread and continue to evolve. Since their emergence in 2020 novel highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b reassortant viruses have become predominant in the world and caused multiple infections in mammals. It was shown that some of A(H5N1) viruses mostly isolated from mammals contain an E627K mutation in the PB2 protein which can lead to adaptation of influenza viruses to mammalian cells. In 2023 in Russia we have isolated two highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses from birds one of which contained an E627K mutation in the PB2 protein. This virus had increased virulence in mice. Limited airborne transmission of the virus with the PB2-E627K mutation was observed between ferrets, in which infectious virus was detected in the nasal washings of the three of the twelve recipient ferrets, and clinical symptoms of the disease were observed in one case. Both viruses showed dominant binding to avian-type sialoside receptors, which was most likely the reason for the limited transmissibility. Thus, this study indicates a possible limited increase in the pandemic potential of A(H5N1) 2.3.4.4b viruses and highlights the importance of continuous avian influenza surveillance for pandemic preparedness and response.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-024-78175-y.
... The analysis of the full HA gene segments of the three HPAI H5N8 viruses showed that they all belonged to phylogenetic clade 2.3.4.4b. The three HA segments showed avian-like receptor preferences (α-2,3-sialic acid linkage) in 186E, 216R, 221G, 222Q, and 224G in the hemagglutinin gene using H5 numbering [29,30]. The multi-basic cleavage site of the HA gene was also maintained as PLREKRRKR/GLF. ...
Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021–2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine’s effectiveness, and identify zoonotic potential.
... In general, avian influenza viruses have a higher affinity for α2-3 SAs, whereas influenza viruses of human or mammalian origin have a higher affinity for α2-6 SAs [17,18]. Acquisition of high affinity for α2-6 SAs indicates the potential for avian influenza viruses to cause cross-species transmission and infection in mammals or humans [19]. ...
Newcastle disease (ND) is a major burden for the poultry industry worldwide, especially in developing countries. The virus that causes this disease, Newcastle disease virus (NDV), is also an effective vector for the development of novel human and animal vaccines and a promising oncolytic virus for cancer therapy. The mechanism of entry of NDV into host cells is of particular interest because it has a significant impact on the infectivity, host range, and pathogenicity of the virus. Here, we present an overview of the entry of NDV into cells, focusing on the interplay among viral and host factors involved in this process. In particular, recent research revealing novel features of NDV attachment to cells, the identification of viral and cellular components that regulate binding of the virus to cells, and the emerging role of novel cellular routes of NDV entry are discussed. More importantly, some of the remaining gaps in our understanding of NDV entry and some fundamental questions for research efforts in the future are also highlighted.
... Due to its central role in the infection process, the HA protein is considered the primary driver of adaptation to a new host species. This adaptation often involves the acquisition of mutations in the receptor binding site (RBS) that enhance affinity for host-specific SA conformations [5][6][7] . Previous studies have demonstrated that adaptation of FLUAVs isolated from humans to pigs results in amino acid changes in the HA RBS, increasing affinity for swine airway epithelial cells 8 and improving transmission efficiency compared to the original human virus 9 . ...
Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA’s role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca²⁺) binding in the low-affinity Ca²⁺-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca²⁺-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca²⁺ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca²⁺ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca²⁺ binding in the low-affinity calcium-binding pocket.
... Therefore, The primary goal of this study was to train and validate classifiers for the classification of the most likely host species using the hemagglutinin gene segment of H3 viruses. The HA segment was used because it is part of the viral envelope and a large contributor to a host switch event (Shi et al., 2014). Additionally, it has been noted that HA was highly significant in species differentiation and showed many markers for species specificity (Qiang et al., 2018). ...
... Numerous studies have identified several key determinants of host adaptation, which were responsible for increasing the host range as well as their virulence. For example, the receptor binding specificity of hemagglutinin (HA), the changes of species-specific amino acids of the ribonucleoprotein complex (RNP) in their mammalian hosts and the immune antagonists induced by the non-structural protein 1 (NS1) [8][9][10]. ...
The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-β in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.
... However, the molecular features of these cases are different, and our case has some different mutations. The Q226L mutation makes the virus more adept at binding to human a-2,6-sialic acid receptors, significantly increasing the likelihood of human infection (Shi et al., 2014). The mutation D701N in the PB2 protein has been shown to enhance the replication activity of avian influenza RNA polymerase within the human body. ...
In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People’s Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient’s sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.
... While environmental factors play a considerable role in influencing the seasonality of influenza outbreaks (5), other non-environmental factors can also affect seasonal epidemics (6). Indeed, the influenza A virus has a high mutation rate, which allows it to evolve rapidly and overcome host barriers (7). While all eight segments evolve continuously, the HA and NA glycoprotein segments evolve more rapidly. ...
Outbreaks of influenza A viruses are generally seasonal and cause annual epidemics worldwide. Due to their frequent reassortment and evolution, annual surveillance is of paramount importance to guide vaccine strategies. The aim of this study was to explore the molecular epidemiology of influenza A virus and nasopharyngeal microbiota composition in infected patients in Saudi Arabia. A total of 103 nasopharyngeal samples from 2015 and 12 samples from 2022 were collected from patients positive for influenza A. Sequencing of influenza A as well as metatranscriptomic analysis of the nasopharyngeal microbiota was conducted using Oxford Nanopore sequencing. Phylogenetic analysis of hemagglutinin, neuraminidase segments, and concatenated influenza A genomes was performed using MEGA7. Whole-genome sequencing analysis revealed changing clades of influenza A virus: from 6B.1 in 2015 to 5a.2a in 2022. One sample containing the antiviral resistance-mediating mutation S247N toward oseltamivir and zanamivir was found. Phylogenetic analysis showed the clustering of influenza A strains with the corresponding vaccine strains in each period, thus suggesting vaccine effectiveness. Principal component analysis and alpha diversity revealed the absence of a relationship between hospital admission status, age, or gender of infected patients and the nasopharyngeal microbial composition, except for the infecting clade 5a.2a. The opportunistic pathogens Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were the most common species detected. The molecular epidemiology appears to be changing in Saudi Arabia after the COVID-19 pandemic. Antiviral resistance should be carefully monitored in future studies. In addition, the disease severity of patients as well as the composition of the nasopharyngeal microbiota in patients infected with different clades should also be assessed.
IMPORTANCE
In this work, we have found that the clade of influenza A virus circulating in Riyadh, KSA, has changed over the last few years from 6B.1 to 5a.2a. Influenza strains clustered with the corresponding vaccine strains in our population, thus emphasizing vaccine effectiveness. Metatranscriptomic analysis showed no correlation between the nasopharyngeal microbiome and the clinical and/or demographic characteristics of infected patients. This is except for the 5a.2a strains isolated post-COVID-19 pandemic. The influenza virus is among the continuously evolving viruses that can cause severe respiratory infections. Continuous surveillance of its molecular diversity and the monitoring of anti-viral-resistant strains are thus of vital importance. Furthermore, exploring potential microbial markers and/or dysbiosis of the nasopharyngeal microbiota during infection could assist in the better management of patients in severe cases.
... The only difference between LSTc and 6′SLNLN is the C2 substituent in the fifth glycan moiety, which pointed away from and did not interact with HA. Vic20 HA-bound LSTc exhibited an energetically favorable foldedback conformation 30 , which is typically adopted by HA-bound humantype receptor analogs (Fig. 1a, b) 31 . Nevertheless, the human-type receptor analog in Vic20 was tilted upward starting from GlcNAc-3, as compared to that observed in HK68 and Ecu16 HAs (Fig. 1a). ...
The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.
... RBS has a pocket-like shape with a sialic acid binding site, and amino acid changes in and around it are essential for the specificity of receptor binding and determining the host range of the virus [41]. The molecular mechanisms by which avianspecific HAs gain human receptor-binding avidity vary among different subtypes [42]. E190D and G225D for H1, G226L and G228S for H2 and H3 (HA residues here follow H3 numbering unless otherwise stated) not only confer α-2,6 binding and infection of humans but also successfully spread between human hosts [43][44][45][46]. ...
Influenza A viruses continue to be a serious health risk to people and result in a large-scale socio-economic loss. Avian influenza viruses typically do not replicate efficiently in mammals, but through the accumulation of mutations or genetic reassortment, they can overcome interspecies barriers, adapt to new hosts, and spread among them. Zoonotic influenza A viruses sporadically infect humans and exhibit limited human-to-human transmission. However, further adaptation of these viruses to humans may result in airborne transmissible viruses with pandemic potential. Therefore, we are beginning to understand genetic changes and mechanisms that may influence interspecific adaptation, cross-species transmission, and the pandemic potential of influenza A viruses. We also discuss the genetic and phenotypic traits associated with the airborne transmission of influenza A viruses in order to provide theoretical guidance for the surveillance of new strains with pandemic potential and the prevention of pandemics.
... This indicates that cs2301 has the potential for cross-species transmission. Notably, the natural hosts for H4N1 include poultry and pigs, which serve as conduits for potential human transmission of the virus [27]. Given the frequent and intimate human-poultry interactions within LPMs, cs2301 may cause human disease. ...
Background
Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent need for vigilance.
Methods
During the period from December 2021 to November 2023, we collected AIV-related environmental samples and assessed them using a comprehensive protocol that included nucleic acid testing, gene sequencing, isolation culture, and resequencing.
Results
In this study, a total of 934 environmental samples were assessed, revealing a remarkably high detection rate (43.66%, 289/662) of AIV in the live poultry market. Notably, the H4N1 subtype AIV (cs2301) was isolated from the live poultry market and its complete genome sequence was successfully determined. Subsequent analysis revealed that cs2301, resulting from a reassortment event between wild and domesticated waterfowl, exhibits multiple mutations and demonstrates potential for host transfer.
Conclusions
Our research once again demonstrates the significant role of wild and domesticated waterfowl in the reassortment process of avian influenza virus, enriching the research on the H4 subtype of AIV, and emphasizing the importance of proactive monitoring the environment related to avian influenza virus.
... Speci cally, the H10N3 strain from this patient showed a closer genetic relationship to a chicken (GISAID#EPIISL15737164) from Jiangsu Province. Molecular characterization revealed a mutation at the 226th amino acid residue in the receptor binding site of the HA protein, where the amino acid changed from Q to L. This mutation makes the virus more adept at binding to human α-2,6-sialic acid receptors, signi cantly increasing the likelihood of human infection 10 . The mutation D701N in the PB2 protein has been shown to enhance the replication activity of avian in uenza RNA polymerase within the human body. ...
In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. Despite this, only two cases of human infection with the H10N3 strain have been documented. Here, we present the initial instance of human infection with avian influenza virus H10N3 in Yunnan Province, Southwest China. The patient, a previously healthy 51-year-old male, presented with recurrent fever peaking at 39℃, accompanied by symptoms such as cough, expectoration, chest tightness, and shortness of breath. Diagnosis revealed severe pneumonia, type I respiratory failure, and infection with avian influenza virus H10N3. Additionally, the patient experienced complications from Candida albicans and Staphylococcus epidermidis infections. Following treatment with appropriate antiviral drugs and antibiotics, the patient's condition improved. Molecular analysis of the viral strain identified four mutations potentially hazardous to human health. This underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus.
... Even though the Gs/GD-like viruses (A/Goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses) have been circulating for 28 years, the key genetic changes in the HA gene known to induce a complete switch of the receptor specificity from avian to human receptors have not yet been identified. Several adaptive mutations in the receptor-binding site of avian HAs have been shown to cause a switch of binding specificity, exemplified by E190D/G225D and A138S in H1 HA and Q226L/ G228S in the HAs of H2 and H3 subtypes (Shi et al., 2014). These mutations enhance the virus' ability to bind to the human receptor while diminishing its affinity for the avian receptor. ...
Avian influenza viruses (AIV) remain prevalent among wild bird populations in the European Union and European Economic Area (EU/EEA), leading to significant illness in and death of birds. Transmission between bird and mammal species has been observed, particularly in fur animal farms, where outbreaks have been reported. While transmission from infected birds to humans is rare, there have been instances of exposure to these viruses since 2020 without any symptomatic infections reported in the EU/EEA. However, these viruses continue to evolve globally, and with the migration of wild birds, new strains carrying potential mutations for mammalian adaptation could be selected. If avian A(H5N1) influenza viruses acquire the ability to spread efficiently among humans, large‐scale transmission could occur due to the lack of immune defences against H5 viruses in humans. The emergence of AIV capable of infecting mammals, including humans, can be facilitated by various drivers. Some intrinsic drivers are related to virus characteristics or host susceptibility. Other drivers are extrinsic and may increase exposure of mammals and humans to AIV thereby stimulating mutation and adaptation to mammals. Extrinsic drivers include the ecology of host species, such as including wildlife, human activities like farming practices and the use of natural resources, climatic and environmental factors. One Health measures to mitigate the risk of AIV adapting to mammals and humans focus on limiting exposure and preventing spread. Key options for actions include enhancing surveillance targeting humans and animals, ensuring access to rapid diagnostics, promoting collaboration between animal and human sectors, and implementing preventive measures such as vaccination. Effective communication to different involved target audiences should be emphasised, as well as strengthening veterinary infrastructure, enforcing biosecurity measures at farms, and reducing wildlife contact with domestic animals. Careful planning of poultry and fur animal farming, especially in areas with high waterfowl density, is highlighted for effective risk reduction.
Sporadic human infections of avian influenza virus (AIV) raise significant public health concerns. A critical factor limiting the transmission of AIVs is the shift in receptor-binding preference from Siaα2,3 to Siaα2,6. To reveal the adaptive selection dynamics during the host adaptation process of AIVs, this study generated a viral library with random mutations in the HA gene of the H9N2 strain. Upon passaging the viral library in chickens and mice, the predominantly selected variants exhibited a preference for Siaα2,3 receptors. Notably, the wild-type strain remained dominant in both inoculated and direct-contact chickens, while variants with the ΔL226/R229I substitutions were preferentially selected in mice. Ferrets have a predominance of Siaα2,6 in their respiratory tract. As expected, the variant harboring the N289D mutation, which prefers Siaα2,6 binding, was enriched during in vivo passaging in ferrets. The mice-adapted variant with the ΔL226/R229I mutations causes reduced levels of TNF-α in the early days post-infection in mice, which correlated with an increase in its viral titers. Conversely, elevated levels of IL-6 and IL-1β at five dpi may contribute to the development of the cytokine release syndrome, potentially elucidating the higher fatality rate observed. In conclusion, based on the mutant spectra of the HA gene, this study elucidates the distinct quasispecies dynamics during the adaptation of H9N2 to different hosts, with receptor availability serving as one of the driving factors. Furthermore, a series of critical substitutions that influence the interspecific transmission potential of H9N2 AIVs were identified.
IMPORTANCE
The mutation of viruses creates a quasispecies reservoir. In this study, we aimed to investigate the dynamics of quasispecies during the host adaptation of AIVs. We generated a viral library with random mutations in the HA gene of H9N2 and conducted serial passaging in chickens, mice, and ferrets for five generations, respectively. The wild-type strain was dominant in chickens, while mice selected viruses with the ΔL226/R229I substitutions. Both variants showed a preference for binding to Siaα2,3, which aligned with the abundance of Siaα2,3 found in the respiratory tract epithelial cells of chickens and mice. In ferrets, where Siaα2,6 is more prevalent, the variant with the N289D mutation, which prefers Siaα2,6, was found to be enriched. In summary, this study revealed the adaptive selection of H9N2 quasispecies in various hosts, contributing to our understanding of AIV host adaptation.
In 2018, a single detection of a novel reassortant swine influenza A virus (swIAV) was made in Denmark. The hemagglutinin (HA) of the virus was from the H1N1 pandemic 2009 (H1N1pdm09) lineage and the neuraminidase (NA) from the H1N1 Eurasian avian-like swine lineage (H1N1av). By 2022, the novel reassortant virus (H1pdm09N1av) constituted 27% of swIAVs identified through the Danish passive swIAV surveillance program. Sequencing detected two H1pdm09N1av genotypes; Genotype 1 contained an entire internal gene cassette of H1N1pdm09 origin, Genotype 2 differed by carrying an NS gene segment of H1N1av origin. The internal gene cassette of Genotype 2 became increasingly dominant, not only in the H1pdm09N1av population, but also in other Danish enzootic swIAV subtypes. Phylogenetic analysis of the HA genes from H1pdm09N1av viruses revealed a monophyletic source, a higher substitution rate compared to other H1N1pdm09 viruses and genetic differences with human seasonal and other swine adapted H1N1pdm09 viruses. Correspondingly, H1pdm09N1av viruses were antigenically distinct from human H1N1pdm09 vaccine viruses. Both H1pdm09N1av genotypes transmitted between ferrets by direct contact, but only Genotype 1 was capable of efficient aerosol transmission. The rapid spread of H1pdm09N1av viruses in Danish swine herds is concerning for swine and human health. Their zoonotic threat is highlighted by the limited pre-existing immunity observed in the human population, aerosol transmission in ferrets and the finding that the internal gene cassette of Genotype 2 was present in the first two zoonotic influenza infections ever detected in Denmark.
The ongoing circulation of highly pathogenic avian influenza (HPAI) A(H5N1) viruses, particularly clade 2.3.4.4b strains, poses a significant threat to animal and public health. Recent outbreaks in cattle highlight concerns about cross-species transmission and zoonotic spillover. Here, we found that the hemagglutinin (HA) protein from a cattle-infecting H5N1 virus has acquired slight binding to human-like α2-6-linked receptors, while still exhibiting a strong preference for avian-like α2-3-linked sialic acid receptors. Immunohistochemical staining revealed HA binding to bovine pulmonary and mammary tissues, aligning with clinical observations. HA also binds effectively to human conjunctival, tracheal, and mammary tissues, indicating a risk for human transmission, notably in cases of conjunctivitis. High-resolution cryo-electron microscopy (cryo-EM) structures of this H5 HA in complex with either α2-3 or α2-6 receptors elucidated the molecular mechanisms underlying its receptor binding properties. These findings provide critical insights into the tropism and transmission potential of this emerging pathogen.
The H5N6 avian influenza virus (AIV) is constantly undergoing recombination and evolution with other subtypes of AIV, resulting in various types of recombinant H5N6 viruses. However, the risk to human public health of different recombinant types of H5N6 viruses remains unclear. Recently, two types of different recombinant H5N6 viruses were isolated from chickens. One of the viruses possessed six internal genes originating from H9N2, named A/Chicken/Hubei/112/2020 (H5N6) (abbreviated 112); the other virus possessed PB2, PB1, PA, and NP originating from H5N1, while the M and NS genes were derived from H9N2, named A/Chicken/Hubei/125/2020 (H5N6) (abbreviated 125). Here, we investigated the receptor binding properties, pathogenicity, and transmissibility of the two H5N6 AIVs. The results showed that 112 and 125 could bind α-2,3-linked sialic acid receptor (avian-like receptor) and α-2,6-linked sialic acid receptor (human-like receptor). However, 125 and 112 showed different pathogenicity in mice. Mice infected with 125 lost only a slight body weight and all survived, while mice infected with 112 lost weight rapidly and all died within a week of infection. Furthermore, in the transmission experiment, 125 could only transmit through direct contact, while 112 could transmit not only by direct contact but also by aerosol. The above results indicated that 112 exhibited enhanced pathogenicity and transmissibility compared to 125, suggesting that the H5N6 virus, whose internal genes were derived from H9N2, could pose a greater threat to human health. Therefore, continuous monitoring of different recombinant H5N6 viruses in poultry should be carried out to prevent their transmission to humans.
Respiratory viral infections continue to cause pandemic and epidemic outbreaks in humans and animals. Under steady-state conditions, alveolar macrophages (AlvMϕ) fulfill a multitude of tasks in order to maintain tissue homeostasis. Due to their anatomic localization within the deep lung, AlvMϕ are prone to detect and react to inhaled viruses and thus play a role in the early pathogenesis of several respiratory viral infections. Here, detection of viral pathogens causes diverse antiviral and proinflammatory reactions. This fact not only makes them promising research targets, but also suggests them as potential targets for therapeutic and prophylactic approaches. This review aims to give a comprehensive overview of the current knowledge about the role of AlvMϕ in respiratory viral infections of humans and animals.
Cross-species transmission of animal viruses poses a threat to human health. However, systematic experimental assessments of these risks remain scarce. A critical step in viral infection is cellular internalization mediated by viral receptor-binding proteins (RBPs). Here we constructed viral pseudotypes bearing the RBPs of 102 enveloped RNA viruses and assayed their infectivity across 5,202 RBP–cell combinations. This showed that most of the tested viruses have the potential to enter human cells. Pseudotype infectivity varied widely among the 14 viral families examined and was influenced by RBP characteristics, host of origin and target cell type. Cellular gene expression data revealed that the availability of specific cell-surface receptors is not necessarily the main factor limiting viral entry and that additional host factors must be considered. Altogether, these results suggest weak interspecies barriers in the early stages of infection and advance our understanding of the molecular interactions driving viral zoonosis.
A switch from avian-type α-2,3 to human-type α-2,6 receptors is an essential element for the initiation of a pandemic from an avian influenza virus. Some H9N2 viruses exhibit a preference for binding to human-type α-2,6 receptors. This identifies their potential threat to public health. However, our understanding of the molecular basis for the switch of receptor preference is still limited. In this study, we employed the random forest algorithm to identify the potentially key amino acid sites within hemagglutinin (HA), which are associated with the receptor binding ability of H9N2 avian influenza virus (AIV). Subsequently, these sites were further verified by receptor binding assays. A total of 12 substitutions in the HA protein (N158D, N158S, A160 N, A160D, A160T, T163I, T163V, V190T, V190A, D193 N, D193G, and N231D) were predicted to prefer binding to α-2,6 receptors. Except for the V190T substitution, the other substitutions were demonstrated to display an affinity for preferential binding to α-2,6 receptors by receptor binding assays. Especially, the A160T substitution caused a significant upregulation of immune-response genes and an increased mortality rate in mice. Our findings provide novel insights into understanding the genetic basis of receptor preference of the H9N2 AIV.
Genetic Diversity of Coronaviruses (Volume 1) provides a comprehensive analysis of the genetic mutations and host interactions across three major coronaviruses—SARS-CoV, MERS-CoV, and SARS-CoV-2. This volume explores the evolutionary history, mutations, and emerging variants of these viruses, with a focus on understanding how they adapt to different hosts. The book is organized into three parts: Part I covers SARS-CoV, detailing its genetic mutations, host genetic diversity, and new variants. Part II focuses on MERS-CoV, offering insights into mutations and host adaptations. Part III addresses SARS-CoV-2, discussing its evolving variants and the role of host proteins. The book also discusses the connections between coronaviruses and neurological, epigenetic, and AI-related issues. Key Features: - In-depth analysis of genetic mutations in coronaviruses. - Exploration of host genetic diversity and virus adaptation. - Insight into emerging variants of SARS-CoV, MERS-CoV, and SARS-CoV-2. - Examination of host proteins' role in viral infections. - Discussion on the impact of AI and epigenetics on coronavirus research.
Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods but we uncovered that avian species lack KHNYN. We found that chicken ZAP does not affect IAV (PR8) or CpG enriched IAV. Human ZAP or KHNYN independently restricted CpG enriched IAV by overexpression in chicken cells or knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of direct restriction of multiple diverse viruses. We suggest that mammalian KHNYN may be a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN.
SIGNIFICANCE
Viruses adapt to hosts to replicate successfully. We show that two mammalian proteins, ZAP and KHNYN, restrict CpG-enriched avian viruses. Mammalian KHNYN may be a bona fide restriction factor with cell-autonomous activity. We also identified a platypus KHNYN with potent and broad antiviral activity highlighting a significant need to investigate antiviral mechanisms in novel and understudied species. Ongoing efforts to understand viruses with zoonotic potential will benefit from further identification of species-/class-specific restriction factors and their antiviral preferences. Furthermore, we speculate that evolving viral nucleotide composition indicates zoonotic potential and adaptation to mammals requires dinucleotide and amino acid changes.
HIGHLIGHTS
CpG content is depleted in human and swine IAV relative to avian IAV
Human ZAP-S and KHNYN but not chicken ZAP independently restrict CpG-rich IAV.
Mammalian ZAP-L and KHNYN but not chicken ZAP independently restrict ROSV.
Platypus KHNYN potently restricts retroviruses, including IAV, HIV-1, MLV, and ROSV.
In 2018, a single detection of a novel reassortant swine influenza A virus (swIAV) was made in Denmark. The hemagglutinin (HA) of the virus was from the H1N1 pandemic 2009 (H1N1pdm09) lineage and the neuraminidase (NA) from the H1N1 Eurasian avian-like swine lineage (H1N1av). By 2022, the novel reassortant virus (H1pdm09N1av) constituted 27 % of swIAVs identified through the Danish passive swIAV surveillance program. Sequencing 2 detected two H1pdm09N1av genotypes; Genotype 1 contained an internal gene cassette of H1N1pdm09 origin, Genotype 2 differed by carrying an NS gene segment of H1N1av origin. The internal gene cassette of Genotype 2 became increasingly dominant, not only in the H1pdm09N1av population, but also in other Danish enzootic swIAV subtypes. Phylogenetic analysis of the HA genes from H1pdm09N1av viruses revealed a monophyletic source, a higher substitution rate compared to other H1N1pdm09 viruses and genetic differences with human seasonal and other swine adapted H1N1pdm09 viruses. Correspondingly, H1pdm09N1av viruses were antigenically distinct from human H1N1pdm09 vaccine viruses. Both H1pdm09N1av genotypes transmitted between ferrets by direct contact, but only Genotype 1 was capable of efficient aerosol transmission. The rapid spread of H1pdm09N1av viruses in Danish swine herds is concerning for swine and human health. Their zoonotic threat is highlighted by the limited pre-existing immunity observed in the human population, aerosol transmission in ferrets and the finding that the internal gene cassette of Genotype 2 was present in the first two zoonotic infections ever detected in Denmark.
Influenza is a global epidemic infectious disease that causes a significant number of illnesses and deaths annually. Influenza exhibits high variability and infectivity, constantly jumping from birds to mammals. Genomic mutations of the influenza virus are a central mechanism leading to viral variation and antigenic evolution. These are crucial in facilitating the virus to cross species barriers and cause human infection. This review summarizes the types of genomic mutations in the influenza virus, their roles and mechanisms in crossing species barriers, and analyzes the impact of these mutations on human health.
Current highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses in bovine and other mammals have been posing unprecedented risks to public health. It’s vital and urgent to assess the pandemic potential of the HPAI H5N1 virus, and the risk degree posed by the virus infection or the genome reassortment with human influenza A viruses (IAVs). An attentional deep learning framework here was constructed of H uman A daptive I nfluenza virus R eassortment using A ttentional N etworks based on G enome E mbedding (HAIRANGE), to predict high-risk reassortment between avian and human IAVs. HAIRANGE embedded genomic contextual codons covering both RNA and protein information, biologically interpretable on viral adaptive codon contexts of IAVs, predicted accurately adaptive IAV genes and adaptive reassortment between avian and human IAVs on independent validation data sets of RNA polymerase-related genes. A high adaptive reassortment risk was predicted by HAIRANGE of the current bovine HPAI H5N1 viruses with human H3N2 IAVs, as has been in vitro validated with polymerase reporter assay. In summary, the present study provides an intelligent tool to predict high-risk IAV reassortment based on genome embedding. Current bovine HPAI H5N1 is posing high pandemic potential via possible genomic reassortment with human IAVs.
The H3N2 canine influenza virus (CIV) emerged from an avian reservoir in Asia around 2004. As the virus has now been circulating entirely among dogs for 20 years, we here update our understanding of the evolution of virus in its new host. As a host-switched virus, H3N2 CIV will also reveal any host-adaptive changes arising during thousands of infections within its new host, and our analysis showed that the virus has evolved at a constant rate. CIV was first introduced into North America in 2015 from Korea, and we specifically examined the epidemiology of the virus among dogs in North America since then, including local outbreaks, regional die-outs, and repeated reintroduction from Asia. The H3N2 CIV now appears endemic only in China after dying out in South Korea around 2017. Virus lineages circulating in China appear to have seeded the most recent US outbreaks – with 2 or 3 introductions into North America during the past 3 years. Combining clinical reports, diagnostic testing data, and analysis of viral genomes we show that the virus spreads rapidly among dogs in kennels and shelters in different regions – likely dying out locally after all those animals become infected and immune. The overall epidemic therefore requires longer-distance dispersal of virus to initiate outbreaks in new locations. Patterns of spread in the USA may select viruses most adapted to those dense populations, which may lack the properties required for efficient long-distance transfers to other dog populations that would keep the virus in prolonged circulation.
IMPORTANCE
Viruses occasionally jump into new hosts to cause epidemics and may spread widely due to movement of humans or animals, or their viruses, with profound consequences for global health. The emergence and epidemiology of new epidemic viruses in companion animals provides a model for understanding disease dynamics and evolution. The H3N2 canine influenza virus arose from an avian virus, and infected dogs provide many opportunities for human exposure. H3N2 CIV transmission is dominated by fast-moving outbreaks within dense populations in animal shelters or kennels, while sustaining the epidemic likely requires movement of virus to more distant dog populations. Viral spread within North Americahas only been sustained for a few years at a time after which the virus dies out. The epidemiological and evolutionary dynamics of this virus in this structured host population shows how an acute respiratory pathogen can emerge and spread in a new host and population.
The H10 subtype of avian influenza virus (AIV) poses an ongoing threat to both birds and humans. Notably, fatal human cases of H10N3 and H10N8 infections have drawn public attention. In 2022, we isolated two H10N3 strains (A/chicken/Shandong/0101/2022 and A/chicken/Shandong/0603/2022) from diseased chickens in China. Genome analysis revealed that these strains were genetically associated with human-origin H10N3 virus, with internal genes originating from local H9N2 viruses. Compared to the H10N8 strain (A/chicken/Jiangxi/102/2013), the H10N3 strains exhibited enhanced thermostability, increased viral release from erythrocytes, and accumulation of hemagglutinin (HA) protein. Additionally, we evaluated the pathogenicity of both H10N3 and H10N8 viruses in mice. We found that viral titers could be detected in the lungs and nasal turbinates of mice infected with the two H10N3 viruses, whereas H10N8 virus titers were detectable in the lungs and brains of mice. Notably, the proportion of double HA Q222R and G228S mutations in H10N3 viruses has increased since 2019. However, the functional roles of the Q222R and G228S double mutations in the HA gene of H10N3 viruses remain unknown and warrant further investigation. Our study highlights the potential public health risk posed by the H10N3 virus. A spillover event of AIV to humans could be a foretaste of a looming pandemic. Therefore, it is imperative to continuously monitor the evolution of the H10N3 influenza virus to ensure targeted prevention and control measures against influenza outbreaks.
Influenza A (H7N9) virus has been causing human infections in China since February 2013, raising serious concerns of potential pandemics. Previous studies demonstrate that human infection is directly linked to live animal markets, and that the internal genes of the virus are derived from H9N2 viruses circulating in the Yangtze River Delta area in Eastern China. Here following analysis of 109 viruses, we show a much higher genetic heterogeneity of the H7N9 viruses than previously reported, with a total of 27 newly designated genotypes. Phylogenetic and genealogical inferences reveal that genotypes G0 and G2.6 dominantly co-circulate within poultry, with most human isolates belonging to the genotype G0. G0 viruses are also responsible for the inter- and intra-province transmissions, leading to the genesis of novel genotypes. These observations suggest the province-specific H9N2 virus gene pools increase the genetic diversity of H7N9 via dynamic reassortments and also imply that G0 has not gained overwhelming fitness and the virus continues to undergo reassortment.
Author Summary
Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.
Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet ("airborne transmission") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.
In March 2013, the Chinese Center for Disease Control and Prevention reported human infections with an H7N9 influenza virus,
and by 20 July 2013, the numbers of laboratory-confirmed cases had climbed to 134, including 43 fatalities and 127 hospitalizations.
The newly emerging H7N9 viruses constitute an obvious public health concern because of the apparent severity of this outbreak.
Here we focus on the hemagglutinins (HAs) of these viruses and assess their receptor binding phenotype in relation to previous
HAs studied. Glycan microarray and kinetic analyses of recombinant A(H7N9) HAs were performed to compare the receptor binding
profile of wild-type receptor binding site variants at position 217, a residue analogous to one of two positions known to
switch avian to human receptor preference in H2N2 and H3N2 viruses. Two recombinant A(H7N9) HAs were structurally characterized,
and a mutational study of the receptor binding site was performed to analyze important residues that can affect receptor preference
and affinity. Results highlight a weak human receptor preference of the H7N9 HAs, suggesting that these viruses require further
adaptation in order to adapt fully to humans.
Two Viruses to Bind
Structural studies of two different H7N9 influenza viruses isolated from humans—A/Shanghai/1/2013 and A/Anhui/1/2013—which have different amino acid sequences in the receptor binding site, provide data indicating that the virus is in transition with respect to host adaptation. The Shanghai virus was one of the first isolated in humans that binds avian receptor glycans with high affinity, but binds poorly to human receptors. However, the later Anhui isolates can bind both avian and human receptors at high affinity. Shi et al. (p. 243 , published online 5 September) show that four hydrophobic mutations contribute to acquisition of affinity for the human receptor by the virus hemagglutinin (HA) and confirm this effect in binding studies with virus particles. Further comparison of a mutant H7N9 A/Anhui/1/2013 HA with the bird flu H5N1 virus revealed the significance of some of the naturally occurring changes observed in circulating H7N9 viruses, which helps to explain how these viruses have been able to cause many severe human infections in a short time.
A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.
On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.
Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.
The emergence of the H7N9 influenza virus in humans in Eastern China has raised concerns that a new influenza pandemic could
occur. Here, we used a ferret model to evaluate the infectivity and transmissibility of A/Shanghai/2/2013 (SH2), a human H7N9
virus isolate. This virus replicated in the upper and lower respiratory tracts of the ferrets and was shed at high titers
for 6 to 7 days, with ferrets showing relatively mild clinical signs. SH2 was efficiently transmitted between ferrets via
direct contact, but less efficiently by airborne exposure. Pigs were productively infected by SH2 and shed virus for 6 days
but were unable to transmit the virus to naïve pigs or ferrets. Under appropriate conditions, human-to-human transmission
of the H7N9 virus may be possible.
A novel H7N9 influenza A virus has been discovered as the causative identity of the emerging acute respiratory infection cases in Shanghai, China. This virus has also been identified in cases of infection in the neighboring area Hangzhou City in Zhejiang Province. In this study, epidemiologic, clinical, and virological data from three patients in Hangzhou who were confirmed to be infected by the novel H7N9 influenza A virus were collected and analyzed. Human respiratory specimens and chicken feces from a contacted free market were tested for influenza virus by real-time reverse transcription PCR (RT-PCR) and sequencing. The clinical features of the three cases were similar featured with high fever and severe respiratory symptoms; however, only one of the patients died. A certain degree of diversity was observed among the three Hangzhou viruses sequenced from human samples compared with other reported H7N9 influenza A viruses. The sequences of the novel avian-origin H7N9 influenza viruses from Hangzhou City contained important amino acid substitutions related to human adaptation. One of the Hangzhou viruses had gained a novel amino acid substitution (Q226I) in the receptor binding region of hemagglutinin. More importantly, the virus sequenced from the chicken feces had a 627E substitution in the PB2 protein instead of the mammalian-adapted 627K substitution that was found in the PB2 proteins from the Hangzhou viruses from the three patients. Therefore, the newly-emerging H7N9 virus might be under adaptation pressure that will help it "jump" from avian to human hosts. The significance of these substitutions needs further exploration, with both laboratory experiments and extensive field surveillance.
Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.
Background:
Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus.
Methods:
We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses.
Results:
A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died.
Conclusions:
Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).
Influenza A virus uses sialic acids as cell entry receptors, and there are two main receptor forms, α2,6 linkage or α2,3 linkage
to galactose, that determine virus host ranges (mammalian or avian). The receptor binding hemagglutinins (HAs) of both 1918
and 2009 pandemic H1N1 (18H1 and 09H1, respectively) influenza A viruses preferentially bind to the human α2,6 linkage receptor.
A single D225G mutation in both H1s switches receptor binding specificity from α2,6 linkage binding to dual receptor binding.
However, the molecular basis for this specificity switch is not fully understood. Here, we show via H1-ligand complex structures
that the D225G substitution results in a loss of a salt bridge between amino acids D225 and K222, enabling the key residue
Q226 to interact with the avian receptor, thereby obtaining dual receptor binding. This is further confirmed by a D225E mutant
that retains human receptor binding specificity with the salt bridge intact.
Cyclophilin A (CypA) is the main member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity. CypA participates in protein folding, cell signaling, inflammation and tumorigenesis. Further, CypA plays critical roles in the replication of several viruses. Upon influenza virus infection, CypA inhibits viral replication by interacting with the M1 protein. In addition, CypA is incorporated into the influenza virus virions. Finally, Cyclosporin A (CsA), the main inhibitor of CypA, inhibits influenza virus replication through CypA-dependent and -independent pathways. This review briefly summarizes recent advances in understanding the roles of CypA during influenza virus infection.
The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA-receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.
Many replication events are involved in the influenza A virus life cycle, which are accomplished by different virus proteins with specific functions. However, because the size of influenza virus genome is limited, the virus uses different mechanisms to express multiple viral proteins from a single gene segment. The M2 and NS2 proteins are produced by splicing and several novel influenza A virus proteins, such as PB1-F2, PB1-N40, and PA-X have recently been identified. Here, we identified novel PA-related proteins in influenza A virus-infected cells. These newly identified proteins are translated from the eleventh and thirteenth in-frame AUG codons in the PA mRNA and are, therefore, N-terminally truncated forms of PA, which we named PA-N155 and PA-N182, respectively. The eleventh and thirteenth AUG codons are highly conserved among influenza A viruses, and the PA-N155 and PA-N182 proteins were detected in cells infected with various influenza A viruses isolated from different host species, suggesting the expression of these N-truncated PAs is universal in nature among influenza A viruses. These N-truncated PAs did not show polymerase activity when expressed together with PB1 and PB2; however, mutant viruses lacking the N-truncated PAs replicated more slowly in cell culture and had lower pathogenicity in mice than did wild-type virus. These results suggest that these novel PA-related proteins likely possess important functions in the replication cycle of influenza A virus.
Influenza's Cryptic Constraint
Because of the well-known pandemic potential of influenza viruses, it is important to understand the range of molecular interactions between the virus and its host. Despite years of intensive research on the virus, Jagger et al. (p. 199 , published online 28 June; see the Perspective by Yewdell and Ince ) have found that the influenza A virus has been hiding a gene in its small negative-sense RNA genome. An overlapping open reading frame was found contained in the PA viral RNA polymerase gene, which is accessed by ribosomal frameshifting to produce a fusion protein containing the N-terminal messenger RNA (mRNA) endonuclease domain of PA and an alternative C-terminal X domain. The resulting polypeptide, PA-X, selectively degrades host mRNAs and, in a mouse model of infection, modulated cellular immune responses, thus limiting viral pathogenesis.
Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment,
might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that
two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions
to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution
to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus
has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains,
make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are
needed for assessing, and potentially averting, this threat.
Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.
Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153-18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.
Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.
In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.
Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits
membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza
viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before
widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex
with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However,
favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225,
which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal
sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures
with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human
and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009
pandemic swine flu virus.
Influenza A viruses are zoonotic pathogens that continuously circulate and change in several animal hosts, including birds, pigs, horses and humans. The emergence of novel virus strains that are capable of causing human epidemics or pandemics is a serious possibility. Here, we discuss the value of surveillance and characterization of naturally occurring influenza viruses, and review the impact that new developments in the laboratory have had on our understanding of the host tropism and virulence of viruses. We also revise the lessons that have been learnt from the pandemic viruses of the past 100 years.
Nucleotide sequences of the 3' 20% of the hemagglutinin gene of 32 influenza A virus strains from the 12 known hemagglutinin subtypes have been determined. Although the sequences of hemagglutinin genes and proteins of different subtypes differ greatly, cysteine and some other amino acid residues are totally conserved, presumably reflecting evolution of the 12 different hemagglutinins from a single gene. When viruses of one subtype, isolated over a period of time, are compared, the hemagglutinin gene and protein sequences show a slow accumulation of nucleotide changes and some amino acid changes. Since sequence data from the genes coding for the matrix and nonstructural proteins also show an accumulation of changes with time, it seems that antigenic selection (of the surface antigens) does not contribute significantly to the rate of change on influenza gene sequences. Although the rate of nucleotide change during drift is more than sufficient to account for the amino acid sequence differences observed in the 12 subtypes, there is a clear distinction, by antigenic as well as sequence analyses, between viruses of one subtype (0-9% amino acid variation) and viruses of other subtypes (20-74% amino acid variation). No virus has yet been found that is intermediate between subtypes.
Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.
Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naïve ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.
Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface
glycoproteins of influenza A virus with their common ligand/substrate: carbohydrate chains terminating in sialic acid. The
specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its
receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid
substrate of the virus neuraminidase enzyme but utilize the much tighter binding of the drugs for efficacy. Resistance to
one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence
of structural differences in the enzyme active site between the two phylogenetic groups.
Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.
Since 2003, H5N1 influenza viruses have caused over 400 known cases of human infection with a mortality rate greater than 60%. Most of these cases resulted from direct contact with virus-contaminated poultry or poultry products. Although only limited human-to-human transmission has been reported to date, it is feared that efficient human-to-human transmission of H5N1 viruses has the potential to cause a pandemic of disastrous proportions. The genetic basis for H5N1 viral transmission among humans is largely unknown. In this study, we used guinea pigs as a mammalian model to study the transmission of six different H5N1 avian influenza viruses. We found that two viruses, A/duck/Guangxi/35/2001 (DKGX/35) and A/bar-headed goose/Qinghai/3/2005(BHGQH/05), were transmitted from inoculated animals to naïve contact animals. Our mutagenesis analysis revealed that the amino acid asparagine (Asn) at position 701 in the PB2 protein was a prerequisite for DKGX/35 transmission in guinea pigs. In addition, an amino acid change in the hemagglutinin (HA) protein (Thr160Ala), resulting in the loss of glycosylation at 158-160, was responsible for HA binding to sialylated glycans and was critical for H5N1 virus transmission in guinea pigs. These amino acids changes in PB2 and HA could serve as important molecular markers for assessing the pandemic potential of H5N1 field isolates.
The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the "Asian Influenza" of 1957. We compare it with the 1918 "Spanish Influenza" hemagglutinin, H1, and the 1968 "Hong Kong Influenza" hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human-specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.
In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.
Representatives of three families of enveloped viruses were shown to fuse tissue culture cells together. These were: Semliki Forest virus (SFV, a togavirus), vesicular stomatitis virus (a rhabdovirus), and two myxoviruses, fowl plaque virus and Japan influenza virus (Japan)/A/305/57). Unlike paramyxoviruses, whose fusion activity is known to occur over a broad pH range, fusion by these viruses was restricted to mildly acidic pH. The pH thresholds for the four viruses were 6.0, 6.1, 5.5, and 5.1, respectively. The precursor form of Japan influenza, which is not infectious and which contains the uncleaved hemagglutinin, had no fusion activity. This result suggested a role for the influenza hemagglutinin in the low-pH-dependent membrane fusion activity. Taken together, our results show that low-pH-induced fusion is a widespread property of enveloped animal viruses and that it may play a role in the infective process. The fusion reactions with all four viruses were fast, efficient, and easy to induce. With UV-inactivated SFV, the fusion was shown to be nonlytic and the polykaryons were viable for at least 12 h. 30 ng of SFV/1 x 10(6) BHK-21 cells were required for 50% fusion, and 250 ng sufficed to fuse the entire culture into a single polykaryon.
Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.
The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glycans present on the apical surface of host cells can function as receptors for the influenza virus hemagglutinin (HA) protein. Avian and human influenza viruses typically have a different sialic acid (SA)-binding preference and only few amino acid changes in the HA protein can cause a switch from avian to human receptor specificity. Recent experiments using glycan arrays, virus histochemistry, animal models, and structural analyses of HA have added a wealth of knowledge on receptor binding specificity. Here, we review recent data on the interaction between influenza virus HA and SA receptors of the host, and the impact on virus host range, pathogenesis, and transmission. Remaining challenges and future research priorities are also discussed.
Shorebirds and waterfowls are believed to be the reservoir hosts for influenza viruses, whereas swine putatively act as mixing vessels. The recent identification of two influenza-like virus genomes (designated H17N10 and H18N11) from bats has challenged this notion. A crucial question concerns the role bats might play in influenza virus ecology. Structural and functional studies of the two major surface envelope proteins, hemagglutinin (HA) and neuraminidase (NA), demonstrate that neither has canonical HA or NA functions found in influenza viruses. However, putative functional modules and domains in other encoded proteins are conserved, and the N-terminal domain of the H17N10 polymerase subunit PA has a classical structure and function. Therefore, potential genomic reassortments of such influenza-like viruses with canonical influenza viruses cannot be excluded at this point and should be assessed.
The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the
human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu226, a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the
H7 hemagglutinin reveals negligible binding to humanlike α2-6–linked receptors and strong preference for a subset of avian-like
α2-3–linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan
complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest
that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.
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.
Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.
Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.
To the Editor: A recent outbreak of a previously unrecognized novel reassortant avian influenza A (H7N9) virus in China(1) had resulted in 131 documented cases and 36 deaths as of May 16.(2) Although some patients had a history of contact with live poultry or visiting live-animal markets before the onset of illness,(3) the source of infection remains unclear. A 45-year-old woman was admitted to a hospital on March 23, 2013, with a 5-day history of fever (temperature, 41.0°C), cough, expectoration, malaise, and shortness of breath. Computed tomography of the chest that was performed 2 days before admission showed infection in . . .
In the past, avian influenza viruses have crossed species barriers to trigger human pandemics by reassorting with mammal-infective
viruses in intermediate livestock hosts. H5N1 viruses are able to infect pigs, and some of them have affinity for the mammalian
type α-2,6-linked sialic acid airway receptor. Using reverse genetics, we systematically created 127 reassortant viruses between
a duck isolate of H5N1, specifically retaining its hemagglutinin (HA) gene throughout, and a highly transmissible, human-infective H1N1 virus. We tested the virulence of the reassortants in
mice as a correlate for virulence in humans and tested transmissibility in guinea pigs, which have both avian and mammalian
types of airway receptor. Transmission studies showed that the H1N1 virus genes encoding acidic polymerase and nonstructural
protein made the H5N1 virus transmissible by respiratory droplet between guinea pigs without killing them. Further experiments
implicated other H1N1 genes in the enhancement of mammal-to-mammal transmission, including those that encode nucleoprotein,
neuraminidase, and matrix, as well as mutations in H5 HA that improve affinity for humanlike airway receptors. Hence, avian H5N1 subtype viruses do have the potential to acquire
mammalian transmissibility by reassortment in current agricultural scenarios.
Recent studies have identified several mutations in the hemagglutinin (HA) protein that allow the highly pathogenic avian
H5N1 influenza A virus to transmit between mammals by airborne route. Here, we determined the complex structures of wild-type
and mutant HAs derived from an Indonesia H5N1 virus bound to either avian or human receptor sialic acid analogs. A cis/trans
conformational change in the glycosidic linkage of the receptor analog was observed, which explains how the H5N1 virus alters
its receptor-binding preference. Furthermore, the mutant HA possessed low affinities for both avian and human receptors. Our
findings provide a structural and biophysical basis for the H5N1 adaptation to acquire human, but maintain avian, receptor-binding
properties.
Background:
Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China.
Methods:
Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patient's and the chicken's isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes.
Findings:
We identified four patients (mean age 56 years), all of whom had contact with poultry 3-8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2.
Interpretation:
Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness.
Funding:
Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.
The conformation is described of the sialylα(2 → 3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAcα(2 → 3) Galβ(1 → 4)GlcNAcβ(1 → N)Asn and NeuAcα-(2 → 6)Galβ(1 → 4)GlcNAcβ(1 → N)Asn is presented and for each glycosidic linkage, except for the α(2→6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the β(1 → 4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the α(2 → 3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the α(2 → 6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the α(2→6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAcα(2 → 6)Galβ(1 → 4)GlcNAcβ structural element. This provides an explanation for the shift effects induced by α(2 → 6) attachment of NeuAc to the N-acetyllactosamine unit.
Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.
Pandemic 2009 influenza A/H1N1 (H1N1v) is resistant to adamantanes, leaving neuraminidase inhibitors as the only therapeutic option. Other mutations are considered to be associated with virulence and clinical severity. However, out of the surveillance programs, few studies analyze the presence of resistance/virulent H1N1v variants in certain clinical circumstances.
To define the frequency and role of resistance and virulence mutations in a specific clinical circumstance-in patients with persistent infection by H1N1v.
Observational study of patients with persistent H1N1v infection admitted to our hospital.
NAI-resistance mutations were detected in 14.3% of cases with persistent infection (2/14), and in none of the non-persistent controls (0/15). These cases were initially infected with susceptible variants that acquired resistance at different time-points after therapy with oseltamivir (OTV). The first case (case 2) was an HIV-positive patient who rapidly acquired resistance 9 days after diagnosis (6 days on OTV) and whose infection resolved after standard OTV therapy. The second case (case 3) was a patient with chronic lymphocytic leukemia [corrected] and the longest viral persistence (59 days). The resistance mutation was detected in the specimen taken on day 37 after diagnosis (30 days on OTV). Once the resistance mutation was identified, OTV was substituted by zanamivir and the infection resolved. In addition to mutations encoding resistance, variants associated with virulence were also sought. The D225G mutation was not found in any case, whereas the D225E variant was identified in three persistent cases but also in two non-persistent ones. In one patient, the D225E substitution coincided with the H275 resistant mutation.
NAI-resistance mutations were detected, at rather different paces, in non-severe immunosuppressed cases with persistent infection by influenza A/H1N1v.