In JoVE (1)

Other Publications (40)

Articles by John W. McCauley in JoVE

Other articles by John W. McCauley on PubMed

Identification of the Glycosaminoglycan-binding Site on the Glycoprotein E(rns) of Bovine Viral Diarrhoea Virus by Site-directed Mutagenesis

The Journal of General Virology. Sep, 2002  |  Pubmed ID: 12185268

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein E(rns) interacts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant E(rns) was undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of E(rns) was essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in E(rns) reduced the binding of E(rns) to immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal E(rns), E(rns) that was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with E(rns). It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of E(rns) constitutes a GAG-binding site.

Inhibition of Beta Interferon Transcription by Noncytopathogenic Bovine Viral Diarrhea Virus is Through an Interferon Regulatory Factor 3-dependent Mechanism

Journal of Virology. Sep, 2002  |  Pubmed ID: 12186882

The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-alpha/beta or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-beta induction pathway have been analyzed. The transcription factor NF-kappa B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-kappa B by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

Influenza Type A in Humans, Mammals and Birds: Determinants of Virus Virulence, Host-range and Interspecies Transmission

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Jul, 2003  |  Pubmed ID: 12815721

The virulence of a virus is determined by its ability to adversely affect the host cell, host organism or population of host organisms. Influenza A viruses have been responsible for four pandemics of severe human respiratory disease this century. Avian species harbour a large reservoir of influenza virus strains, which can contribute genes to potential new pandemic human strains. The fundamental importance of understanding the role of each of these genes in determining virulence in birds and humans was dramatically emphasised by the recent direct transmission of avian influenza A viruses to humans, causing fatal infection but not community spread. An understanding of the factors involved in transmission between avian and mammalian species should assist in the development of better surveillance strategies for early recognition of influenza A virus strains having human pandemic potential, and possibly in the design of anti-viral strategies.

Differences in Membrane Association and Sub-cellular Distribution Between NS2-3 and NS3 of Bovine Viral Diarrhoea Virus

Virus Research. Nov, 2003  |  Pubmed ID: 14602200

The sub-cellular location and mechanism of membrane association of NS3 and NS2-3 polypeptides of bovine viral diarrhoea virus (BVDV) have been examined. Both NS3 and NS2-3 proteins were detected in post-nuclear membrane fractions but not in cytosolic fractions of BVDV infected cells; a proportion of NS3, but not NS2-3, could be dissociated from the membranes with 800 mM KCl or at pH 11. Following extraction with 1% Triton X-114, NS3 was predominantly present in the aqueous phase, but NS2-3 was only recovered in the detergent phase. Confocal microscopy showed that in BVDV infected cells, NS3 and/or NS2-3 co-localise with the endoplasmic reticulum (ER) protein, ERP60, but not Golgi or lysosomal proteins. Sub-cellular fractionation analysis demonstrated that NS2-3 was almost exclusively associated with the rough ER membrane but a significant proportion of NS3 was present in the smooth ER membrane fractions in addition to the rough ER membrane. These differences in the distribution of NS2-3 and NS3 on ER membranes in cells infected with cytopathogenic (CP) strains of BVDV were also observed using confocal microscopy and antibodies that are specific to either NS2 or NS3. This distinct distribution of NS3 and NS2-3 on the ER membrane has revealed a further difference between CP and non-cytopathogenic (NCP) strains of BVDV.

Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-stranded RNA

Journal of Virology. Jan, 2004  |  Pubmed ID: 14671095

Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-kappaB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.

Differential Activation of Interferon Regulatory Factors-3 and -7 by Non-cytopathogenic and Cytopathogenic Bovine Viral Diarrhoea Virus

Veterinary Immunology and Immunopathology. Aug, 2004  |  Pubmed ID: 15207451

Non-cytopathogenic bovine viral diarrhoea virus (ncpBVDV) has previously been shown to inhibit the function of interferon regulatory factor-3 in cultured cells [J. Virol. 76 (2002) 8979]. In this study, we show that, like ncpBVDV, when cells were previously exposed to cytopathogenic BVDV (cpBVDV) the appearance of an IRF-3-DNA complex from nuclear extracts that can be induced by heterologous virus infection was not observed. Infection of cells with ncpBVDV or cpBVDV resulted in neither the translocation of IRF-7 from the cytoplasm to the nucleus of infected cells, nor an inhibition of its nuclear translocation in cells super-infected by Semliki Forest Virus. We conclude that cpBVDV and ncpBVDV both share the ability to inhibit the full function of IRF-3 but neither stimulate or block the nuclear uptake of IRF-7.

The NPro Product of Bovine Viral Diarrhea Virus Inhibits DNA Binding by Interferon Regulatory Factor 3 and Targets It for Proteasomal Degradation

Journal of Virology. Dec, 2006  |  Pubmed ID: 16971436

Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of interferon regulatory factor 3 (IRF-3), a transcription factor essential for interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I interferon induction or the targeting of IRF-3 for degradation.

Studies of an Influenza A Virus Temperature-sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions

Journal of Virology. Jan, 2009  |  Pubmed ID: 18987140

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

PloS One. Jun, 2009  |  Pubmed ID: 19517011

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Within-host Variation of Avian Influenza Viruses

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Sep, 2009  |  Pubmed ID: 19687042

The emergence and spread of H5N1 avian influenza viruses from Asia through to Europe and Africa pose a significant animal disease problem and have raised concerns that the virus may pose a pandemic threat to humans. The epizootological factors that have influenced the wide distribution of the virus are complex, and the variety of viruses currently circulating reflects these factors. Sequence analysis of the virus genes sheds light on the H5N1 virus evolution during its emergence and spread, but the degree of virus variation at the level of an individual infected bird has been described in only a few studies. Here, we describe some results of a study in which turkeys, ducks and chickens were infected with either one of two H5N1 or one of three H7N1 viruses, and the degree of sequence variation within an individual infected avian host was examined. We developed 'deep amplicon' sequence analysis for this work, and the methods and results provide a background framework for application to disease outbreaks in the field.

The Relationship Between Mutation Frequency and Replication Strategy in Positive-sense Single-stranded RNA Viruses

Proceedings. Biological Sciences. Mar, 2010  |  Pubmed ID: 19906671

For positive-sense single-stranded RNA virus genomes, there is a trade-off between the mutually exclusive tasks of transcription, translation and encapsidation. The replication strategy that maximizes the intracellular growth rate of the virus requires iterative genome transcription from positive to negative, and back to positive sense. However, RNA viruses experience high mutation rates, and the proportion of genomes with lethal mutations increases with the number of replication cycles. Thus, intracellular mutant frequency will depend on the replication strategy. Introducing apparently realistic mutation rates into a model of viral replication demonstrates that strategies that maximize viral growth rate could result in an average of 26 mutations per genome by the time plausible numbers of positive strands have been generated, and that virus viability could be as low as 0.1 per cent. At high mutation rates or when a high proportion of mutations are deleterious, the optimal strategy shifts towards synthesizing more negative strands per positive strand, and in extremis towards a 'stamping-machine' replication mode where all the encapsidated genomes come from only two transcriptional steps. We conclude that if viral mutation rates are as high as current estimates suggest, either mutation frequency must be considerably higher than generally anticipated and the proportion of viable viruses produced extremely small, or replication strategies cannot be optimized to maximize viral growth rate. Mechanistic models linking mutation frequency to replication mechanisms coupled with data generated through new deep-sequencing technologies could play an important role in improving the estimates of viral mutation rate.

Dynamics of Influenza Virus Infection and Pathology

Journal of Virology. Apr, 2010  |  Pubmed ID: 20130053

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon ("cytokine storms"), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.

Intra- and Interhost Evolutionary Dynamics of Equine Influenza Virus

Journal of Virology. Jul, 2010  |  Pubmed ID: 20444896

Determining the evolutionary basis of cross-species transmission and immune evasion is key to understanding the mechanisms that control the emergence of either new viruses or novel antigenic variants with pandemic potential. The hemagglutinin glycoprotein of influenza A viruses is a critical host range determinant and a major target of neutralizing antibodies. Equine influenza virus (EIV) is a significant pathogen of the horse that causes periodical outbreaks of disease even in populations with high vaccination coverage. EIV has also jumped the species barrier and emerged as a novel respiratory pathogen in dogs, canine influenza virus. We studied the dynamics of equine influenza virus evolution in horses at the intrahost level and how this evolutionary process is affected by interhost transmission in a natural setting. To this end, we performed clonal sequencing of the hemagglutinin 1 gene derived from individual animals at different times postinfection. Our results show that despite the population consensus sequence remaining invariant, genetically distinct subpopulations persist during the course of infection and are also transmitted, with some variants likely to change antigenicity. We also detected a natural case of mixed infection in an animal infected during an outbreak of equine influenza, raising the possibility of reassortment between different strains of virus. In sum, our data suggest that transmission bottlenecks may not be as narrow as originally perceived and that the genetic diversity required to adapt to new host species may be partially present in the donor host and potentially transmitted to the recipient host.

Selection of Variant Viruses During Replication and Transmission of H7N1 Viruses in Chickens and Turkeys

Virology. Nov, 2012  |  Pubmed ID: 22944111

The influence of different glycosylation patterns of the haemagglutinin glycoprotein of H7N1 avian influenza viruses on virus replication in vivo was examined. Experimental infection of chickens and turkeys was carried out with H7N1 avian influenza viruses with alternative sites of glycosylation in the haemagglutinin and infected birds were sampled daily by swabbing the buccal and cloacal cavities. cDNAs of the HA1 coding region of the HA gene were prepared from the swabs and cloned into plasmids. Sequencing multiple plasmids made from individual swabs taken over the period of virus shedding showed that viruses with specific patterns of glycosylation near the receptor binding site were stable when birds were infected with a single variant, but when presented with a mixed population of viruses encoding differing patterns of glycosylation a specific variant was rapidly selected in the infected host.

Quantifying Transmission of Highly Pathogenic and Low Pathogenicity H7N1 Avian Influenza in Turkeys

PloS One. 2012  |  Pubmed ID: 23028760

Outbreaks of avian influenza in poultry can be devastating, yet many of the basic epidemiological parameters have not been accurately characterised. In 1999-2000 in Northern Italy, outbreaks of H7N1 low pathogenicity avian influenza virus (LPAI) were followed by the emergence of H7N1 highly pathogenic avian influenza virus (HPAI). This study investigates the transmission dynamics in turkeys of representative HPAI and LPAI H7N1 virus strains from this outbreak in an experimental setting, allowing direct comparison of the two strains. The fitted transmission rates for the two strains are similar: 2.04 (1.5-2.7) per day for HPAI, 2.01 (1.6-2.5) per day for LPAI. However, the mean infectious period is far shorter for HPAI (1.47 (1.3-1.7) days) than for LPAI (7.65 (7.0-8.3) days), due to the rapid death of infected turkeys. Hence the basic reproductive ratio, [Formula: see text] is significantly lower for HPAI (3.01 (2.2-4.0)) than for LPAI (15.3 (11.8-19.7)). The comparison of transmission rates and [Formula: see text] are critically important in relation to understanding how HPAI might emerge from LPAI. Two competing hypotheses for how transmission rates vary with population size are tested by fitting competing models to experiments with differing numbers of turkeys. A model with frequency-dependent transmission gives a significantly better fit to experimental data than density-dependent transmission. This has important implications for extrapolating experimental results from relatively small numbers of birds to the commercial poultry flock size, and for how control, including vaccination, might scale with flock size.

Evolution of the Receptor Binding Properties of the Influenza A(H3N2) Hemagglutinin

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2012  |  Pubmed ID: 23236176

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.

Receptor Binding by a Ferret-transmissible H5 Avian Influenza Virus

Nature. May, 2013  |  Pubmed ID: 23615615

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.

The Influenza Virus Protein PB1-F2 Interacts with IKKβ and Modulates NF-κB Signalling

PloS One. 2013  |  Pubmed ID: 23704945

PB1-F2, a protein encoded by a second open reading frame of the influenza virus RNA segment 2, has emerged as a modulator of lung inflammatory responses but the molecular mechanisms underlying this are only poorly understood. Here we show that PB1-F2 inhibits the activation of NF-κB dependent signalling pathways in luciferase reporter assays. PB1-F2 proteins from four different viruses interact with IKKβ in yeast two-hybrid assays and by co-immunoprecipitation. PB1-F2 expression did not inhibit IKKβ kinase activity or NF-κB translocation into the nucleus, but NF-κB binding to DNA was severely impaired in PB1-F2 transfected cells as assessed by Electrophoretic Mobility Shift Assay. Neither the N-terminal 57 amino acid truncated forms nor the C-terminus of PB1-F2 were able to inhibit NF-κB dependent signalling, indicating that the full length protein is necessary for the inhibition.

Receptor Binding by an H7N9 Influenza Virus from Humans

Nature. Jul, 2013  |  Pubmed ID: 23787694

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.

Glyconanoparticles for the Plasmonic Detection and Discrimination Between Human and Avian Influenza Virus

Organic & Biomolecular Chemistry. Nov, 2013  |  Pubmed ID: 24057694

A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent α2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25:75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.

Infectivity and Transmissibility of H9N2 Avian Influenza Virus in Chickens and Wild Terrestrial Birds

Veterinary Research. Oct, 2013  |  Pubmed ID: 24134616

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.

Development of a Surveillance Scheme for Equine Influenza in the UK and Characterisation of Viruses Isolated in Europe, Dubai and the USA from 2010-2012

Veterinary Microbiology. Mar, 2014  |  Pubmed ID: 24480583

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2.

Virus Pathotype and Deep Sequencing of the HA Gene of a Low Pathogenicity H7N1 Avian Influenza Virus Causing Mortality in Turkeys

PloS One. 2014  |  Pubmed ID: 24489838

Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

Integrating Influenza Antigenic Dynamics with Molecular Evolution

ELife. 2014  |  Pubmed ID: 24497547

Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. DOI:

A Novel I221L Substitution in Neuraminidase Confers High-level Resistance to Oseltamivir in Influenza B Viruses

The Journal of Infectious Diseases. Oct, 2014  |  Pubmed ID: 24795482

Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.

Receptor Binding by H10 Influenza Viruses

Nature. Jul, 2014  |  Pubmed ID: 24870229

H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.

Enhanced Human Receptor Binding by H5 Haemagglutinins

Virology. May, 2014  |  Pubmed ID: 24889237

Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between Asn-186 and Gln-226. In contrast, a double mutant, Δ133/Ile155Thr, isolated in Egypt has greater avidity for human receptor while retaining wild-type avidity for avian receptor. Despite these increases in human receptor binding, none of the mutants prefers human receptor, unlike aerosol transmissible H5N1 viruses. Nevertheless, mutants with high avidity for both human and avian receptors may be intermediates in the evolution of H5N1 viruses that could infect both humans and poultry.

Infection and Pathogenesis of Canine, Equine, and Human Influenza Viruses in Canine Tracheas

Journal of Virology. Aug, 2014  |  Pubmed ID: 24899186

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity.

Receptor Binding Properties of the Influenza Virus Hemagglutinin As a Determinant of Host Range

Current Topics in Microbiology and Immunology. 2014  |  Pubmed ID: 25078920

Host cell attachment by influenza A viruses is mediated by the hemagglutinin glycoprotein (HA), and the recognition of specific types of sialic acid -containing glycan receptors constitutes one of the major determinants of viral host range and transmission properties. Structural studies have elucidated some of the viral determinants involved in receptor recognition of avian-like and human-like receptors for various subtypes of influenza A viruses, and these provide clues relating to the mechanisms by which viruses evolve to adapt to human hosts. We discuss structural aspects of receptor binding by influenza HA, as well as the biological implications of functional interplay involving HA binding, NA sialidase functions, the effects of antigenic drift, and the inhibitory properties of natural glycans present on mucosal surfaces.

Human Infection with Highly Pathogenic A(H7N7) Avian Influenza Virus, Italy, 2013

Emerging Infectious Diseases. Oct, 2014  |  Pubmed ID: 25271444

During an influenza A(H7N7) virus outbreak among poultry in Italy during August-September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.

The Possible Impact of Vaccination for Seasonal Influenza on Emergence of Pandemic Influenza Via Reassortment

PloS One. 2014  |  Pubmed ID: 25494180

One pathway through which pandemic influenza strains might emerge is reassortment from coinfection of different influenza A viruses. Seasonal influenza vaccines are designed to target the circulating strains, which intuitively decreases the prevalence of coinfection and the chance of pandemic emergence due to reassortment. However, individual-based analyses on 2009 pandemic influenza show that the previous seasonal vaccination may increase the risk of pandemic A(H1N1) pdm09 infection. In view of pandemic influenza preparedness, it is essential to understand the overall effect of seasonal vaccination on pandemic emergence via reassortment.

Biophysical Measurement of the Balance of Influenza a Hemagglutinin and Neuraminidase Activities

The Journal of Biological Chemistry. Mar, 2015  |  Pubmed ID: 25586179

The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.

Focused Antibody Response to Influenza Linked to Antigenic Drift

The Journal of Clinical Investigation. Jul, 2015  |  Pubmed ID: 26011643

The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.

Global Circulation Patterns of Seasonal Influenza Viruses Vary with Antigenic Drift

Nature. Jul, 2015  |  Pubmed ID: 26053121

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

Optimization of a Micro-neutralisation Assay and Its Application in Antigenic Characterisation of Influenza Viruses

Influenza and Other Respiratory Viruses. Jun, 2015  |  Pubmed ID: 26073976

The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, additional to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate interpretation of HI results.

Improving the Representativeness of Influenza Viruses Shared Within the WHO Global Influenza Surveillance and Response System

Influenza and Other Respiratory Viruses. Mar, 2016  |  Pubmed ID: 26589132

Sharing influenza viruses within the WHO Global Influenza Surveillance and Response System is crucial for monitoring evolution of influenza viruses.

Effects of Egg-adaptation on Receptor-binding and Antigenic Properties of Recent Influenza A (H3N2) Vaccine Viruses

The Journal of General Virology. Jun, 2016  |  Pubmed ID: 26974849

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

PLoS Pathogens. Apr, 2016  |  Pubmed ID: 27057693

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997-2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.

Antigenic Characterization of Influenza Viruses Produced Using Synthetic DNA and Novel Backbones

Vaccine. Jul, 2016  |  Pubmed ID: 27219338

The global system for manufacturing seasonal influenza vaccines has been developed to respond to the natural evolution of influenza viruses, but the problem of antigenic mismatch continues to be a challenge in certain years. In some years, mismatches arise naturally due to the antigenic drift of circulating viruses after vaccine strain selection has already been made. In other years, antigenic differences between the vaccine virus and circulating viruses are introduced as part of the current system, which relies on the use of egg-adapted isolates as a starting material for candidate vaccine viruses (CVVs). Improving the current process for making vaccine viruses can provide great value. We have previously established a synthetic approach for rapidly generating influenza viruses in a vaccine-approved Madin Darby canine kidney (MDCK) cell line using novel, high-growth backbones that increase virus rescue efficiency and antigen yield. This technology also has the potential to produce viruses that maintain antigenic similarity to the intended reference viruses, depending on the hemagglutinin (HA) and neuraminidase (NA) sequences used for gene synthesis. To demonstrate this utility, we generated a panel of synthetic viruses using HA and NA sequences from recent isolates and showed by hemagglutination inhibition (HI) tests that all synthetic viruses were antigenically-like their conventional egg- or cell-propagated reference strains and there was no impact of the novel backbones on antigenicity. This synthetic approach can be used for the efficient production of CVVs that may be more representative of circulating viruses and may be used for both egg- and cell-based vaccine manufacturing platforms. When combined with mammalian cell culture technology for antigen production, synthetic viruses generated using HA and NA sequences from a non-egg-adapted prototype can help to reduce the potential impact of antigenic differences between vaccine virus and circulating viruses on vaccine effectiveness.

Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity

Journal of Virology. Oct, 2016  |  Pubmed ID: 27489273

Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses.

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