Host Range Of Influenza Virus Research Articles

Molecular modeling and phylogenetic analyses highlight the role of amino acid 347 of the N1 subtype neuraminidase in influenza virus host range and interspecies adaptation.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A.

The avian-origin influenza A virus polymerase is restricted in human cells. This restriction has been associated with species differences in host factor ANP32A. Avian ANP32A supports the activity of an avian-origin polymerase. However, the avian-origin polymerase is incompatible with human ANP32A. Avian ANP32A proteins harbor an additional 33 amino acids compared to human ANP32A proteins, which are crucial for their ability to support the avian-origin influenza virus polymerase. Here, we elucidate the interactions between ANP32A proteins and the influenza A virus polymerase using split luciferase complementation assays, coimmunoprecipitation, and in situ split Venus interaction assays. We show greater interaction of chicken ANP32A than human ANP32A with the viral polymerase and visualize these interactions in situ in the cell nucleus. We demonstrate that the 33 amino acids of chicken ANP32A and the PB2 627 domain of viral polymerase complex both contribute to this enhanced interaction. Finally, we show how these interactions are affected by the presence of viral RNA and the processivity of the polymerase, giving insights into the way that ANP32A might act during virus infection.IMPORTANCE Successful zoonotic transmission of influenza A virus into humans can lead to pandemics in an immunologically naive population. Host-encoded ANP32A proteins are required to support influenza A virus polymerase activity, and species differences in ANP32A can restrict the host range of influenza virus. Understanding how ANP32A proteins support the viral polymerase and how differences in ANP32A affect the ability of the polymerase to coopt these proteins will enhance our understanding of viral replication and species restriction as well as suggesting targeted antiviral approaches to treat influenza virus infection.

Novel Flu Viruses in Bats and Cattle: "Pushing the Envelope" of Influenza Infection.

Influenza viruses are among the major infectious disease threats of animal and human health. This review examines the recent discovery of novel influenza viruses in bats and cattle, the evolving complexity of influenza virus host range including the ability to cross species barriers and geographic boundaries, and implications to animal and human health.

Influenza Hemagglutinin Protein Stability, Activation, and Pandemic Risk.

For decades, hemagglutinin (HA) protein structure and its refolding mechanism have served as a paradigm for understanding protein-mediated membrane fusion. HA trimers are in a high-energy state and are functionally activated by low pH. Over the past decade, HA stability (or the pH at which irreversible conformational changes are triggered) has emerged as an important determinant in influenza virus host range, infectivity, transmissibility, and human pandemic potential. Here, we review HA protein structure, assays to measure its stability, measured HA stability values, residues and mutations that regulate its stability, the effect of HA stability on interspecies adaptation and transmissibility, and mechanistic insights into this process. Most importantly, HA stabilization appears to be necessary for adapting emerging influenza viruses to humans.

Novel approach for identification of influenza virus host range and zoonotic transmissible sequences by determination of host-related associative positions in viral genome segments.

BackgroundRecent (2013 and 2009) zoonotic transmission of avian or porcine influenza to humans highlights an increase in host range by evading species barriers. Gene reassortment or antigenic shift between viruses from two or more hosts can generate a new life-threatening virus when the new shuffled virus is no longer recognized by antibodies existing within human populations. There is no large scale study to help understand the underlying mechanisms of host transmission. Furthermore, there is no clear understanding of how different segments of the influenza genome contribute in the final determination of host range.MethodsTo obtain insight into the rules underpinning host range determination, various supervised machine learning algorithms were employed to mine reassortment changes in different viral segments in a range of hosts. Our multi-host dataset contained whole segments of 674 influenza strains organized into three host categories: avian, human, and swine. Some of the sequences were assigned to multiple hosts. In point of fact, the datasets are a form of multi-labeled dataset and we utilized a multi-label learning method to identify discriminative sequence sites. Then algorithms such as CBA, Ripper, and decision tree were applied to extract informative and descriptive association rules for each viral protein segment.ResultWe found informative rules in all segments that are common within the same host class but varied between different hosts. For example, for infection of an avian host, HA14V and NS1230S were the most important discriminative and combinatorial positions.ConclusionHost range identification is facilitated by high support combined rules in this study. Our major goal was to detect discriminative genomic positions that were able to identify multi host viruses, because such viruses are likely to cause pandemic or disastrous epidemics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3250-9) contains supplementary material, which is available to authorized users.

Novel Polymerase Gene Mutations for Human Adaptation in Clinical Isolates of Avian H5N1 Influenza Viruses.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

Virology: Host protein clips bird flu's wings in mammals.

The polymerase enzyme from avian influenza A viruses does not function well in human cells. The protein ANP32A has been identified as the cellular factor mediating a major component of this host restriction. See Letter p.101 Wendy Barclay and colleagues identify a previously unknown factor underlying the restricted host range of influenza virus. They show that the host protein ANP32A acts a species barrier to the function of avian virus polymerase in mammalian cells. Crucially, the mutation E627K in viral protein PB2, which allows ANP32 family proteins to support the avian virus polymerase, is known to be associated with increased virulence of avian viruses in mammals.

Characteristics of nucleocytoplasmic transport of H1N1 influenza A virus nuclear export protein.

The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference. Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.

Influenza A(H1N1)pdm09 Virus Infection in Giant Pandas, China

We confirmed infection with influenza A(H1N1)pdm09 in giant pandas in China during 2009 by using virus isolation and serologic analysis methods. This finding extends the host range of influenza viruses and indicates a need for increased surveillance for and control of influenza viruses among giant pandas.

Identification of Amino Acid Changes That May Have Been Critical for the Genesis of A(H7N9) Influenza Viruses

Novel influenza A viruses of the H7N9 subtype [A(H7N9)] emerged in the spring of 2013 in China and had infected 163 people as of 10 January 2014; 50 of them died of the severe respiratory infection caused by these viruses. Phylogenetic studies have indicated that the novel A(H7N9) viruses emerged from reassortment of H7, N9, and H9N2 viruses. Inspections of protein sequences from A(H7N9) viruses and their immediate predecessors revealed several amino acid changes in A(H7N9) viruses that may have facilitated transmission and replication in the novel host. Since mutations that occurred more ancestrally may also have contributed to the genesis of A(H7N9) viruses, we inferred historical evolutionary events leading to the novel viruses. We identified a number of amino acid changes on the evolutionary path to A(H7N9) viruses, including substitutions that may be associated with host range, replicative ability, and/or host responses to infection. The biological significance of these amino acid changes can be tested in future studies. The novel influenza A viruses of the H7N9 subtype [A(H7N9)], which first emerged in the spring of 2013, cause severe respiratory infections in humans. Here, we performed a comprehensive evolutionary analysis of the progenitors of A(H7N9) viruses to identify amino acid changes that may have been critical for the emergence of A(H7N9) viruses and their ability to infect humans. We provide a list of potentially important amino acid changes that can be tested for their significance for the influenza virus host range, replicative ability, and/or host responses to infection.

Egyptian H5N1 Influenza Viruses—Cause for Concern?

Highly pathogenic avian H5N1 influenza viruses are now enzootic in parts of Southeast Asia and the Middle East. Occasionally, these viruses transmit to humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred [1]–[4]. A major determinant of influenza virus host range is the viral hemagglutinin (HA) protein: avian virus HA binds preferentially to sialic acid linked to the penultimate galactose residue by an α2,3-linkage (Siaα2,3Gal) [5]–[7], as found for sialic acid–containing receptors of the epithelial cells in duck intestine [8], the site of avian influenza virus replication. By contrast, human virus HA has higher affinity for Siaα2,6Gal [5]–[7], the main sialyloligosaccharide on the epithelial cells of the human upper respiratory tract [9], [10].

Mouse-Adapted H9N2 Influenza A Virus PB2 Protein M147L and E627K Mutations Are Critical for High Virulence

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.

Influenza Virus Sequence Feature Variant Type Analysis: Evidence of a Role for NS1 in Influenza Virus Host Range Restriction

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.

The contribution of animal models to the understanding of the host range and virulence of influenza A viruses

Since ferrets were first used in 1933 during the initial isolation of influenza A viruses, animal models have been critical for influenza research. The following review discusses the contribution of mice, ferrets, and non-human primates to the study of influenza virus host range and pathogenicity.

DOMESTIC CATS AS HOSTS FOR HUMAN INFLUENZA A AND B VIRUSES IN BRAZIL

Studies on the host range of influenza viruses have been of great importance to prove the role of some animals that were already considered as unlikely links, in the virus transmission chain. This study aimed to investigate the circulation of the influenza virus in cats in Brazil. Domestic cats, assisted at the clinic of the Faculty of Veterinary Medicine at the University of Sao Paulo, were grouped according to gender and age (young and adult). Serum samples were collected and analyzed for antibodies to influenza A and B viruses by the haemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Twenty per cent of the cats aged between 6 and 20 years old responded with high antibody titers (≥ 640 HIU/25 µL) against human influenza A (H1N1) virus. Lower percentages of the animals in the same age group, 11% and 8%, presented the same high titers in response to human influenza A (H3N2) and B virus samples respectively. When the animals were classified by gender, 17 % of males and 8% of females showed a poor antibody response against the influenza A (H1N1) virus (titers of ≤ 20 HIU/25 µL). For the first time in Brazil, protective antibody titers against human influenza viruses revealed the presence of virus infection possibly transmitted to the domestic cats by man. In conclusion, our results show that domestic cats, like other mammals, may play a role in interspecies transmission and spread of the influenza virus. DOI: http://dx.doi.org/10.17525/vrr.v15i2.43

Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry

At present there is much interest in the cell tropism and host range of influenza viruses, especially those of the H5N1 subtype. We wished to develop a method that would enable investigation of attachment of infectious virus through the interaction of the hemagglutinin molecule and live mammalian and avian cells and the subsequent infection of these cells. To this end, influenza viruses of various HA subtypes were constructed that either carry the green fluorescent protein (GFP) instead of the neuraminidase protein, or that express GFP in the cytoplasm of infected cells. The HA genes were derived from influenza viruses A/PR/8/34 (H1N1), A/Netherlands/178/95 (H3N2) and A/Vietnam/1194/04 (H5N1). Using these pairs of viruses, attachment and post-attachment events in the virus replication cycle can be distinguished. In general, the expression of NeuAc(α2-3)Gal or NeuAc(α2-6)Gal receptors on the cells tested corresponded with the attachment of the viruses that were studied with respect to predicted receptor specificity. Virus attachment was not always predictive for efficient infection of the cells.

Differential Polymerase Activity in Avian and Mammalian Cells Determines Host Range of Influenza Virus

As recently shown, mutations in the polymerase genes causing increased polymerase activity in mammalian cells are responsible for the adaptation of the highly pathogenic avian influenza virus SC35 (H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad. Sci. USA 102:18590-18595, 2005). We have now compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant SC35M in avian and mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells was linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both host specificity and pathogenicity.

Qinghai-like H5N1 from Domestic Cats, Northern Iraq

Qinghai-like H5N1 from Domestic Cats, Northern Iraq

Influenza Virus Infection in Racing Greyhounds

To the Editor: Influenza is globally the most economically important respiratory disease in humans, pigs, horses, and fowl (1). Influenza virus is known for its continuous genetic and antigenic changes, which impedes effective influenza control (1,2). More importantly, emergence of a new subtype by genetic reassortment or interspecies transmission is of great concern for preventing influenza epidemics and pandemics (1). Recently, influenza outbreaks have occurred in species (feline and canine) that historically do not carry influenza virus (3,4), which alerted both regulatory and scientific communities to expansion of the host range of influenza virus. We report an outbreak of respiratory disease by influenza virus infection in Iowa racing greyhounds after influenza outbreaks in Florida in 2004. In mid-April, an influx of racing greyhounds into Iowa greyhound tracks resulted in outbreaks of respiratory disease within the track compounds. The disease was characterized by rapid onset of fever and cough, rapid respiration, and hemorrhagic nasal discharge. The illness rate was almost 100% in both racetrack compounds, although the death rate was <5%. Most affected dogs recovered, yet many died of hemorrhagic pneumonia. Therapeutic administration of broad-spectrum antimicrobial drugs reduced the severity of the disease but could not control it. Tissue samples from 4 animals that died of severe pneumonia were submitted to the Iowa State University Veterinary Diagnostic Laboratory. The animals represented 2 different racing tracks located in eastern and western Iowa. On gross examination, lungs exhibited extensive red to red-black discoloration with moderate to marked palpable firmness. Mild fibrinous pleuritis was also noted. Microscopically, lung sections were characterized by severe hemorrhagic interstitial to bronchointerstitial pneumonia. Patchy interstitial change with alveolar septal thickening, coagula of debris in alveoli, and associated atelectasis were evident. Focally extensive pyogranulomatous bronchointerstitial pneumonia with dilatation of airways by degenerate cells and debris was observed. Scattered vasculitis and vascular thrombi were apparent. Microbiologic testing for conventional viral and bacterial agents did not show any important pathogens except Streptococcus equi subsp. zooepidemicus from lung tissues of all animals examined. Two of the 4 lung samples were positive for influenza A virus by a real-time reverse transcription–polymerase chain reaction (RT-PCR) (5). Viral pneumonic lesions of both lungs were positive for immunohistochemistry (IHC) with monoclonal antibody specific for the nucleoprotein of influenza A virus (6) and with antigen-capturing enzyme-linked immunosorbent assay (Directigen Flu A, Becton-Dickinson, Sparks, MD, USA). Bronchioalveolar lavage samples from the 2 positive lungs were also positive by RT-PCR for influenza A virus. Virus isolation was attempted; the influenza virus in canine lungs was unexpected since no influenza virus infection in dogs had been reported, except a recent communication at a meeting of veterinary diagnosticians (4). A virus that was able to agglutinate rooster erythrocytes was isolated in Madin-Darby canine kidney cells from lung and bronchioalveolar lavage fluid of 1 of the 2 animals in which influenza virus was detected by IHC and RT-PCR. Isolates were determined by RT-PCR to be influenza A virus of H3 subtype. The US Department of Agriculture National Veterinary Services Laboratory (Ames, IA) subtyped the virus isolates (A/Canine/Iowa/13628/2005) as H3N8 by using hemagglutination-inhibition assay and neuraminidase-inhibition assay. Sequencing hemagglutinin (HA) and neuraminidase (NA) genes of both isolates showed 100% and 99.8% identity, respectively, between the 2 isolates. Phylogenetically, the HA gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ146419,term_id:76362990}}DQ146419) of the isolates was genetically close (96%–98% nucleotide homology) to the HA gene of recent H3N8 equine influenza viruses (7). The NA gene ({type:entrez-nucleotide,attrs:{text:DQ146420,term_id:76574407}}DQ146420) of the isolates also showed 96%–98% homology with the NA gene of recent H3N8 equine influenza viruses. Internal genes remain to be sequenced. In conclusion, recent outbreaks of hemorrhagic pneumonia and associated deaths in Iowa racing greyhounds were primarily due to infection by an H3N8 influenza virus genetically and antigenically similar to equine influenza viruses. This conclusion can be supported by a previous report of fatal hemorrhagic pneumonia by H3N8 virus infection in racing greyhounds in Florida (4). The fact that greyhounds in 2 different racetracks, which are in geographically remote sites in Iowa, simultaneously died of the disease without the involvement of sick horses suggests that the influenza virus isolate is likely a canine-adapted strain and able to perpetuate and spread among dogs. While influenza virus infection was likely responsible for the disease outbreaks, the contribution that S. zooepidemicus might have made to the disease and the severity of clinical manifestations remains to be further evaluated since the bacterium has been implicated in respiratory disease and septicemia-associated problems in many different animal species (8,9).