Adaptation Of Influenza Viruses Research Articles

Identification of Influenza A Virus PB2 Residues Involved in Enhanced Polymerase Activity and Virus Growth in Mammalian Cells at Low Temperatures.

Mutations in the polymerase genes are known to play a major role in avian influenza virus adaptation to mammalian hosts. Despite having avian origin PA and PB2, the 2009 pandemic H1N1 virus (pH1N1) can replicate well in mammalian respiratory tracts, suggesting that these proteins have acquired mutations for efficient growth in humans. We have previously shown that PA from the pH1N1 virus A/California/04/09 (Cal) strongly enhances activity of an otherwise avian polymerase complex derived from A/chicken/Nanchang/3-120/01 (Nan) in mammalian cells. However, this enhancement was observed at 37°C but not at the lower temperature of 34°C. An additional introduction of Cal PB2 enhanced activity at 34°C, suggesting the presence of unidentified residues in Cal PB2 that are required for efficient growth at low temperature. Here, we sought to determine the key PB2 residues which confer enhanced polymerase activity and virus growth in human cells at low temperature. Using a reporter gene assay, we identified novel mutations, PB2 V661A and V683T/A684S, which are involved in enhanced Cal polymerase activity at low temperature. The PB2 T271A mutation, which we previously reported, also contributed to enhanced activity. The growth of recombinant Cal containing PB2 with Nan residues 271T/661V/683V/684A was strongly reduced in human cells compared to wild-type virus at low temperature. Among the four residues, 271A and 684S are conserved in human and pH1N1 viruses but not in avian viruses, suggesting an important role in mammalian adaptation of pH1N1 virus. The PB2 protein plays a key role in the host adaptation, cold sensitivity, and pathogenesis of influenza A virus. Despite containing an avian origin PB2 lacking the mammalian adaptive mutations 627K or 701N, pH1N1 influenza virus strains can replicate efficiently in the low temperature upper respiratory tract of mammals, suggesting the presence of unknown mutations in the pH1N1 PB2 protein responsible for its low temperature adaptation. Here, in addition to PB2 271A, which has been shown to increase polymerase activity, we identified novel PB2 residues 661A and 683T/684S in pH1N1 which confer enhanced polymerase activity and virus growth in mammalian cells especially at low temperature. Our findings suggest that the presence of these PB2 residues contributes to efficient replication of the pH1N1 virus in the upper respiratory tract, which resulted in efficient human-to-human transmission of this virus.

Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence.

The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.

Mammalian adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck.

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

Role of receptor binding specificity in influenza A virus transmission and pathogenesis

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.

Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses.

Glycosylation Site Alteration in the Evolution of Influenza A (H1N1) Viruses

Influenza virus typically alters protein glycosylation in order to escape immune pressure from hosts and hence to facilitate survival in different host environments. In this study, the patterns and conservation of glycosylation sites on HA and NA of influenza A/H1N1 viruses isolated from various hosts at different time periods were systematically analyzed, by employing a new strategy combining genome-based glycosylation site prediction and 3D modeling of glycoprotein structures, for elucidation of the modes and laws of glycosylation site alteration in the evolution of influenza A/H1N1 viruses. The results showed that influenza H1N1 viruses underwent different alterations of protein glycosylation in different hosts. Two alternative modes of glycosylation site alteration were involved in the evolution of human influenza virus: One was an increase in glycosylation site numbers, which mainly occurred with high frequency in the early stages of evolution. The other was a change in the positional conversion of the glycosylation sites, which was the dominating mode with relatively low frequency in the later evolutionary stages. The mechanisms and possibly biological functions of glycosylation site alteration for the evolution of influenza A/H1N1 viruses were also discussed. Importantly, the significant role of positional alteration of glycosylation sites in the host adaptation of influenza virus was elucidated. Although the results still need to be supported by experimental data, the information here may provide some constructive suggestions for research into the glycosylation of influenza viruses as well as even the design of surveillance and the production of viral vaccines.

Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice

In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host.

Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus

Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission.

Charting the Host Adaptation of Influenza Viruses

Four influenza pandemics have struck the human population during the last 100 years causing substantial morbidity and mortality. The pandemics were caused by the introduction of a new virus into the human population from an avian or swine host or through the mixing of virus segments from an animal host with a human virus to create a new reassortant subtype virus. Understanding which changes have contributed to the adaptation of the virus to the human host is essential in assessing the pandemic potential of current and future animal viruses. Here, we develop a measure of the level of adaptation of a given virus strain to a particular host. We show that adaptation to the human host has been gradual with a timescale of decades and that none of the virus proteins have yet achieved full adaptation to the selective constraints. When the measure is applied to historical data, our results indicate that the 1918 influenza virus had undergone a period of preadaptation prior to the 1918 pandemic. Yet, ancestral reconstruction of the avian virus that founded the classical swine and 1918 human influenza lineages shows no evidence that this virus was exceptionally preadapted to humans. These results indicate that adaptation to humans occurred following the initial host shift from birds to mammals, including a significant amount prior to 1918. The 2009 pandemic virus seems to have undergone preadaptation to human-like selective constraints during its period of circulation in swine. Ancestral reconstruction along the human virus tree indicates that mutations that have increased the adaptation of the virus have occurred preferentially along the trunk of the tree. The method should be helpful in assessing the potential of current viruses to found future epidemics or pandemics.

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential.

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.

Molecular adaptation of an H7N3 wild duck influenza virus following experimental multiple passages in quail and turkey

To investigate the molecular adaptation of influenza viruses during natural interspecies transmission, we performed a phenotypic and genotypic analysis of a low-pathogenic duck H7N3 influenza virus after experimental passages in turkey and quail. Results obtained showed differences in the HA receptor-binding and in NA enzyme activities in viruses recovered after passages in quail, compared to those obtained from passages in turkey. Sequencing of the HA, NA and genes of internal proteins of the viruses obtained from quail and turkey, identified several amino acid substitutions in comparison with the progenitor virus. Of note, in the quail-adapted viruses the emergence of a 23-amino acid deletion in the stalk of the NA and the introduction of a glycosylation site in the HA were a reminiscence of changes typically observed in nature confirming a potential role of the quail in the adaptation of wild birds viruses to domestic poultry.

A 27-Amino-Acid Deletion in the Neuraminidase Stalk Supports Replication of an Avian H2N2 Influenza A Virus in the Respiratory Tract of Chickens

The events and mechanisms that lead to interspecies transmission of, and host adaptation to, influenza A virus are unknown; however, both surface and internal proteins have been implicated. Our previous report highlighted the role that Japanese quail play as an intermediate host, expanding the host range of a mallard H2N2 virus, A/mallard/Potsdam/178-4/83 (H2N2), through viral adaptation. This quail-adapted virus supported transmission in quail and increased its host range to replicate and be transmitted efficiently in chickens. Here we report that of the six amino acid changes in the quail-adapted virus, a single change in the hemagglutinin (HA) was crucial for transmission in quail, while the changes in the polymerase genes favored replication at lower temperatures than those for the wild-type mallard virus. Reverse genetic analysis indicated that all adaptive mutations were necessary for transmission in chickens, further implicating quail in extending this virus to terrestrial poultry. Adaptation of the quail-adapted virus in chickens resulted in the alteration of viral tropism from intestinal shedding to shedding and transmission via the respiratory tract. Sequence analysis indicated that this chicken-adapted virus maintained all quail-adaptive mutations, as well as an additional change in the HA and, most notably, a 27-amino-acid deletion in the stalk region of neuraminidase (NA), a genotypic marker of influenza virus adaptation to chickens. This stalk deletion was shown to be responsible for the change in virus tropism from the intestine to the respiratory tract.

Mechanism of Glycan Receptor Recognition and Specificity Switch for Avian, Swine and Human Adapted Influenza Virus Hemagglutinins: A Molecular Dynamics Perspective

Hemagglutinins (HA's) from duck, swine and human influenza viruses have previously been shown to prefer avian and human glycan receptor analogues with distinct topological profiles, pentasaccharides LSTa (α-2,3 linkage) and LSTc (α-2,6 linkage), in comparative molecular dynamics studies. Based upon detailed analyses of the dynamic motions of the receptor binding domains (RBD's) and interaction energy profiles with individual glycan residues, we have identified approximately 30 residue positions and secondary structural elements (SSE's) in the RBD that present distinct profiles with the receptor analogues. Glycan binding constrained the conformational space sampling by the HA. Electrostatic steering appeared to play a key role in glycan binding specificity. The complex dynamic behaviors of the major SSE and trimeric interfaces with or without bound glycans suggested that networks of interactions might account for species specificity in these low affinity and high avidity (multivalent) interactions between different HA and glycans. Contact frequency, energetic decomposition and H-bond analyses revealed species-specific differences in HA-glycan interaction profiles, not readily discernable from crystal structures alone. Interaction energy profiles indicated that mutation events at the set of residues such as 145, 156, 158 and 222 would favor human or avian receptor analogues, often through interactions with distal asialo-residues. These results correlate well with existing experimental evidence, and suggest new opportunities for simulation-based vaccine and drug development.

Mechanism of glycan receptor recognition and specificity switch for avian, swine, and human adapted influenza virus hemagglutinins: a molecular dynamics perspective.

Hemagglutinins (HA’s) from duck, swine, and human influenza viruses have previously been shown to prefer avian and human glycan receptor analogues with distinct topological profiles, pentasaccharides LSTa (α-2,3 linkage) and LSTc (α-2,6 linkage), in comparative molecular dynamics studies. On the basis of detailed analyses of the dynamic motions of the receptor binding domains (RBDs) and interaction energy profiles with individual glycan residues, we have identified ∼30 residue positions in the RBD that present distinct profiles with the receptor analogues. Glycan binding constrained the conformational space sampling by the HA. Electrostatic steering appeared to play a key role in glycan binding specificity. The complex dynamic behaviors of the major SSE and trimeric interfaces with or without bound glycans suggested that networks of interactions might account for species specificity in these low affinity and high avidity (multivalent) interactions between different HA and glycans. Contact frequency, energetic decomposition, and H-bond analyses revealed species-specific differences in HA−glycan interaction profiles, not readily discernible from crystal structures alone. Interaction energy profiles indicated that mutation events at the set of residues such as 145, 156, 158, and 222 would favor human or avian receptor analogues, often through interactions with distal asialo-residues. These results correlate well with existing experimental evidence, and suggest new opportunities for simulation-based vaccine and drug development.

Illuminating the switch in influenza viruses

Integrating data from glycan microarrays and X-ray structures sheds light on human adaptation of influenza viruses.

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.

Antigenic alteration of influenza B virus associated with loss of a glycosylation site due to host-cell adaptation.

Effects of host-cell adaptation of the hemagglutinin (HA) protein were evaluated by the analyses of four pairs of recent influenza B field isolates, each pair consisting of an Madin Darby canine kidney (MDCK)- and an embryonated chicken egg-derived isolates from the same clinical specimen. Among the isolates examined, all of the MDCK-derived isolates retained glycosylation site at amino acid 197 on the HA1 molecule, whereas three egg-derived isolates lost it. Antigenic difference in the HA molecule between an MDCK- and an egg-derived isolates of three of these pairs was demonstrated to be associated with the glycosylation 197. Replication of the MDCK-derived isolates was suppressed in eggs, suggesting that the presence of the glycosylation 197 was disadvantageous to replication in eggs. Virus-binding affinity assay revealed that the loss of carbohydrate chain did not significantly alter the preferential recognition of sialic acid linkage. Immunogenicity and vaccine efficacy of an MDCK- and an egg-derived clones of B/Akita/27/2001, the former retained the glycosylation 197 and the latter lost it, were compared in a hamster model. When formalin-inactivated whole virion vaccines prepared from the paired isolates were administered into hamsters, no significant difference between them was observed in protective ability against challenges by the homologous and heterologous clones. Implication of the egg adaptation of influenza virus to antigenic surveillance of the field isolates as well as the selection of vaccine strains, and possibility of the involvement of the viral protein(s) other than the HA in the egg adaptation were discussed.

Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens.

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

Influenza Virus Infection is not Affected by Serum Amyloid P Component

Binding of serum amyloid P component (SAP) to its ligands, including bacteria, chromatin and amyloid fibrils, protects them from degradation, is anti-opsonic and anti-immunogenic. SAP thereby enhances the virulence of pathogenic bacteria to which it binds. However SAP also contributes to host resistance against bacteria to which it does not bind. Human SAP has been reported to bind to the influenza virus and inhibit viral invasion of cells in tissue culture. We therefore investigated a possible role of SAP in either host resistance or viral virulence during influenza infection in vivo. The clinical course of mouse adapted influenza virus infection, the host antibody response, and viral replication, were compared in wild type mice, mice with targeted deletion of the SAP gene, and mice transgenic for human SAP. The effects of reconstitution of SAP deficient mice with pure human SAP, and of a drug that specifically blocks SAP binding in vivo, were also studied. Binding of mouse and human SAP to immobilized influenza virus was compared. The presence, absence, or availability for binding of SAP in vivo had no significant or consistent effect on the course or outcome of influenza infection, or on either viral replication or the anti-viral antibody response. Mouse SAP bound much less avidly than human SAP to influenza virus. In marked contrast to the dramatic effects of SAP deficiency on host resistance to different bacterial infections, mouse SAP apparently plays no significant role during infection of mice with influenza virus. Human SAP binds much more avidly than mouse SAP to the virus, but also had no effect on any of the parameters measured and is therefore unlikely to be involved in human influenza infection.

A mouse model of dual infection with influenza virus and Streptococcus pneumoniae

Background: A lethal synergism exists between influenza virus and Streptococcus pneumoniae accounting for excess mortality during influenza epidemics. A small animal model of dual infection with these organisms would be useful for study of pathogenic mechanisms underlying this interaction. Methods: Groups of mice were infected with either mouse adapted influenza virus A/Puerto Rico/8/34 (H1N1), S. pneumoniae strain D39, both simultaneously, or pneumococcus following influenza virus. Weight loss, as a measure of morbidity, and mortality were followed. Blood cultures were collected 24 h after infection. Results: Mice infected simultaneously with both influenza virus and pneumococcus exhibited gradual weight loss and mortality commensurate with expectations for an additive process. In contrast, mice infected with pneumococcus 7 days following infection with influenza virus uniformly died in less than 24 h and were highly bacteremic. Discussion: A mouse model of sequential infection with influenza virus and S. pneumoniae has been developed. Mice infected with pneumococcus seven days after infection with influenza virus exhibit a synergistic lethality caused by overwhelming sepsis. This model will be useful for study of the mechanisms involved in pathogenic interactions between influenza virus and pneumococcus.