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Find video protocols related to scientific articles indexed in Pubmed.
Overview of the 3rd isirv-Antiviral Group Conference - advances in clinical management.
Influenza Other Respir Viruses
PUBLISHED: 10-14-2014
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This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv-Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted.
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Integrating influenza antigenic dynamics with molecular evolution.
Elife
PUBLISHED: 02-06-2014
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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: http://dx.doi.org/10.7554/eLife.01914.001.
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Changes in the hemagglutinin of H5N1 viruses during human infection--influence on receptor binding.
Virology
PUBLISHED: 06-21-2013
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As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.
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Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain.
Virol. J.
PUBLISHED: 04-09-2013
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Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case.
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Design of inhibitors of influenza virus membrane fusion: synthesis, structure-activity relationship and in vitro antiviral activity of a novel indole series.
Antiviral Res.
PUBLISHED: 02-04-2013
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The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure-activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.
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Improving influenza vaccine virus selection: report of a WHO informal consultation held at WHO headquarters, Geneva, Switzerland, 14-16 June 2010.
Influenza Other Respir Viruses
PUBLISHED: 08-08-2011
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• For almost 60 years, the WHO Global Influenza Surveillance and Response System (GISRS) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. The GISRS has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • The expanded and enhanced role of the GISRS following the adoption of the International Health Regulations (2005), recognition of the continuing threat posed by avian H5N1 and the aftermath of the 2009 H1N1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. In addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy-making community of the complexity of influenza vaccine virus selection. • The selection process is highly coordinated and involves continual year-round integration of virological data and epidemiological information by National Influenza Centres (NICs), thorough antigenic and genetic characterization of viruses by WHO Collaborating Centres (WHOCCs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by WHO Essential Regulatory Laboratories (ERLs). • Ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. However, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non-antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • Effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza A viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • Training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of NICs and other laboratories. This has resulted in a significant increase in the volume of information reported to WHO on the spread, intensity and impact of influenza. In addition, initiatives such as the WHO Shipment Fund Project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. It will be important to sustain and build upon the gains made in these and other areas. • Although the haemagglutination inhibition (HAI) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays - for example based upon advanced recombinant DNA and protein technologies - may be more adaptable to automation. Other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • Microneutralization assays provide an important adjunct to the HAI assay in virus antigenic characterization. Improvements in the use and potential automation of such assays should facilitate large-scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • Standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well-characterized banks of age-stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • Advances in high-throughput genetic sequencing coupled with advanced bioinformatics tools, together with more X-ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • Complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. However, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. The application of simpler non-mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • The adoption of alternative vaccine technologies - such as live-attenuated, quadrivalent or non-HA-based vaccines - has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. Recent collaboration between the GISRS and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. WHO will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • The WHO GISRS and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. WHO will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations.
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High throughput virus plaque quantitation using a flatbed scanner.
J. Virol. Methods
PUBLISHED: 03-30-2011
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The plaque assay is a standard technique for measuring influenza virus infectivity and inhibition of virus replication. Counting plaque numbers and quantifying virus infection of cells in multiwell plates quickly, accurately and automatically remain a challenge. Visual inspection relies upon experience, is subjective, often time consuming, and has less reproducibility than automated methods. In this paper, a simple, high throughput imaging-based alternative is proposed which uses a flatbed scanner and image processing software to quantify the infected cell population and plaque formation. Quantitation results were evaluated with reference to visual counting and achieved better than 80% agreement. The method was shown to be particularly advantageous in titration of the number of plaques and infected cells when influenza viruses produce a heterogeneous population of small plaques. It was also shown to be insensitive to the densities of plaques in determination of neutralization titres and IC(50)s of drug susceptibility. In comparison to other available techniques, this approach is cost-effective, relatively accurate, and readily available.
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Combinatorial effect of two framework mutations (E119V and I222L) in the neuraminidase active site of H3N2 influenza virus on resistance to oseltamivir.
Antimicrob. Agents Chemother.
PUBLISHED: 03-21-2011
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Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.
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Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles.
Virology
PUBLISHED: 01-14-2011
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Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.
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Charting the host adaptation of influenza viruses.
Mol. Biol. Evol.
PUBLISHED: 11-25-2010
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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.
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[Impact of avian influenza virus H5N1 neuraminidase mutations on the activity of neuraminidase and the sensibility to neuraminidase inhibitors].
Zhonghua Yi Xue Za Zhi
PUBLISHED: 10-29-2010
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To study the impact of avian influenza virus H5N1 neuraminidase mutations I117V, I314V and I117V + I314V on the sensibility of neuraminidase inhibitors (NAIs) and the activity of neuraminidase (NA).
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Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus.
J. Virol.
PUBLISHED: 09-08-2010
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Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of ?2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.
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Amantadine resistance in relation to the evolution of influenza A(H3N2) viruses in Iran.
Antiviral Res.
PUBLISHED: 05-23-2010
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The aminoadamantanes, amantadine and rimantadine, have been used to prevent and treat influenza A virus infections for many years. Several reports have shown an increased level of resistance to these drugs, particularly among influenza A(H3N2) subtype viruses, during recent years. We observed an increase in amantadine resistance, due to a Ser31Asn mutation in the M2 channel protein, among A(H3N2) viruses circulating in Iran during 2005-2007. Sequence analyses of the haemagglutinin and neuraminidase genes as well as the M gene of these viruses revealed that the emergence of resistance was in general consistent with the progressive worldwide evolution of H3N2 viruses.
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Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?
J. Virol.
PUBLISHED: 04-21-2010
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Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the "151 mutant," the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding.
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Interaction of aminoadamantane derivatives with the influenza A virus M2 channel-docking using a pore blocking model.
Bioorg. Med. Chem. Lett.
PUBLISHED: 04-08-2010
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Interaction of aminoadamantanes with the influenza A virus M2 proton channel was analyzed by docking simulations of a series of synthetic aminoadamantane derivatives, of differing binding affinity, into the crystal structure of the transmembrane (M2TM) pore. The pore blocking model tested in the gas phase describes qualitatively the changes on the relative binding affinities of the compounds (although a series of highly hydrophobic ligands which seem to have little capacity for different specific interactions with their receptor). The docking calculations predicted poses in which the adamantane ring is surrounded mainly by the alkyl side chains of Val27 or Ala30 and the ligands amino group is generally hydrogen bonded with hydroxyls of Ser31 or carbonyls of Val27 or carbonyls of Ala30, the former (Ser31) being the most stable and most frequently observed. The binding of the ligand is a compromise between hydrogen bonding ability, which is elevated by a primary amino group, and apolar interactions, which are increased by the ability of the lipophilic moiety to adequately fill a hydrophobic pocket within the M2TM pore. A delicate balance of these hydrophobic contributions is required for optimal interaction.
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Circulation of human influenza viruses and emergence of Oseltamivir-resistant A(H1N1) viruses in Cameroon, Central Africa.
BMC Infect. Dis.
PUBLISHED: 03-08-2010
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While influenza surveillance has increased in most developing countries in the last few years, little influenza surveillance has been carried out in sub-Saharan Africa and no information is available in Central Africa. The objective of this study was to assess the prevalence of influenza viruses circulating in Yaounde, Cameroon and determine their antigenic and genetic characteristics.
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Epidemiological, antigenic and genetic characteristics of seasonal influenza A(H1N1), A(H3N2) and B influenza viruses: basis for the WHO recommendation on the composition of influenza vaccines for use in the 2009-2010 Northern Hemisphere season.
Vaccine
PUBLISHED: 11-13-2009
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Influenza vaccines form an important component of the global response against infections and subsequent illness caused in man by influenza viruses. Twice a year, in February and September, the World Health Organisation through its Global Influenza Surveillance Network (GISN), recommends appropriate influenza viruses to be included in the seasonal influenza vaccine for the upcoming Northern and Southern Hemisphere winters. This recommendation is based on the latest data generated from many sources and the availability of viruses that are suitable for vaccine manufacture. This article gives a summary of the data and background to the recommendations for the 2009-2010 Northern Hemisphere influenza vaccine formulation.
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Using non-homogeneous models of nucleotide substitution to identify host shift events: application to the origin of the 1918 Spanish influenza pandemic virus.
J. Mol. Evol.
PUBLISHED: 06-05-2009
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Nonhomogeneous Markov models of nucleotide substitution have received scant attention. Here we explore the possibility of using nonhomogeneous models to identify host shift nodes along phylogenetic trees of pathogens evolving in different hosts. It has been noticed that influenza viruses show marked differences in nucleotide composition in human and avian hosts. We take advantage of this fact to identify the host shift event that led to the 1918 Spanish influenza. This disease killed over 50 million people worldwide, ranking it as the deadliest pandemic in recorded history. Our model suggests that the eight RNA segments which eventually became the 1918 viral genome were introduced into a mammalian host around 1882-1913. The viruses later diverged into the classical swine and human H1N1 influenza lineages around 1913-1915. The last common ancestor of human strains dates from February 1917 to April 1918. Because pigs are more readily infected with avian influenza viruses than humans, it would seem that they were the original recipient of the virus. This would suggest that the virus was introduced into humans sometime between 1913 and 1918.
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Comparison of the activities of BM2 protein and its H19 and W23 mutants of influenza B virus with activities of M2 protein and its H37 and W41 mutants of influenza A virus.
Arch. Virol.
PUBLISHED: 06-01-2009
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Co-expression of the BM2 protein with pH-sensitive HA reduces the conversion of HA to its low-pH conformation during transport to the cell surface in the same way as human M2 proteins. BM2 protein is capable of increasing vesicular pH by as much as 0.4 pH units. Mutation analysis showed that replacement of H19 in BM2 protein by A and L resulted in loss of activity, while M2, with the mutation H37A, remained active, but its severe toxicity was intolerable for cells. Whereas substitution of L or A for W23 abolished detectable activity of the BM2 channel, substitution of L for W41 in the M2 protein resulted in a functional ion channel but with reduced activity. W41 was not essential for functional activity of the M2 protein. Our results show some differences in the nature of the interaction of the histidine and tryptophan in the transmembrane domains of BM2 and M2 ion channels.
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Identifying changes in selective constraints: host shifts in influenza.
PLoS Comput. Biol.
PUBLISHED: 05-27-2009
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The natural reservoir of Influenza A is waterfowl. Normally, waterfowl viruses are not adapted to infect and spread in the human population. Sometimes, through reassortment or through whole host shift events, genetic material from waterfowl viruses is introduced into the human population causing worldwide pandemics. Identifying which mutations allow viruses from avian origin to spread successfully in the human population is of great importance in predicting and controlling influenza pandemics. Here we describe a novel approach to identify such mutations. We use a sitewise non-homogeneous phylogenetic model that explicitly takes into account differences in the equilibrium frequencies of amino acids in different hosts and locations. We identify 172 amino acid sites with strong support and 518 sites with moderate support of different selection constraints in human and avian viruses. The sites that we identify provide an invaluable resource to experimental virologists studying adaptation of avian flu viruses to the human host. Identification of the sequence changes necessary for host shifts would help us predict the pandemic potential of various strains. The method is of broad applicability to investigating changes in selective constraints when the timing of the changes is known.
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Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007-08 season.
Emerging Infect. Dis.
PUBLISHED: 04-01-2009
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In Europe, the 2007-08 winter season was dominated by influenza virus A (H1N1) circulation through week 7, followed by influenza B virus from week 8 onward. Oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y mutation in the neuraminidase emerged independently of drug use. By country, the proportion of ORVs ranged from 0% to 68%, with the highest proportion in Norway. The average weighted prevalence of ORVs across Europe increased gradually over time, from near 0 in week 40 of 2007 to 56% in week 19 of 2008 (mean 20%). Neuraminidase genes of ORVs possessing the H275Y substitution formed a homogeneous subgroup closely related to, but distinguishable from, those of oseltamivir-sensitive influenza viruses A (H1N1). Minor variants of ORVs emerged independently, indicating multiclonal ORVs. Overall, the clinical effect of ORVs in Europe, measured by influenza-like illness or acute respiratory infection, was unremarkable and consistent with normal seasonal activity.
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Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol.
Antiviral Res.
PUBLISHED: 02-14-2009
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The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.
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Stability and function of the influenza A virus M2 ion channel protein is determined by both extracellular and cytoplasmic domains.
Arch. Virol.
PUBLISHED: 02-04-2009
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A series of M2/NB chimeras were used to investigate the ion channel activity of the IAV M2 protein. Replacing the M2 cytoplasmic domain with the equivalent NB domain (AAB chimera) did not influence ion channel activity, while replacement of N-terminal domains (BAA and BAB chimeras) resulted in loss of activity. Extension of the M2 protein N-terminal domain resulted in full restoration of ion channel activity in BAA chimeras but only partial restoration in BAB. While not directly involved in ion channel activity, the N- and C-terminals of M2 are important for stabilization of the transmembrane domain structure.
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Efficient synthesis of highly active phospha-isosteres of the influenza neuraminidase inhibitor oseltamivir.
ChemMedChem
PUBLISHED: 01-22-2009
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With a Hunsdiecker-Barton iododecarboxylation strategy, we converted the carboxylate group of the oseltamivir precursor into exemplary phosphonate monoesters. In all cases, K(i) values towards influenza virus sialidase remained in the sub-nanomolar range. We have thus made valuable structural space available for the design of novel oseltamivir-based tools for influenza virus research.
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Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin.
Proc. Natl. Acad. Sci. U.S.A.
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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 (?2,3-linked sialic acid) to a preference for human receptors (?2,6-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.
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Sentinel surveillance for influenza in Senegal, 1996-2009.
J. Infect. Dis.
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Data on influenza in tropical and resource-limited countries are scarce. In this study we present results from 14 years of influenza surveillance in Senegal, one of the few tropical countries in Africa from which longitudinal data are available.
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H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis.
PLoS Pathog.
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Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.
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Rescue of a H3N2 influenza virus containing a deficient neuraminidase protein by a hemagglutinin with a low receptor-binding affinity.
PLoS ONE
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Influenza viruses possess at their surface two glycoproteins, the hemagglutinin and the neuraminidase, of which the antagonistic functions have to be well balanced for the virus to grow efficiently. Ferraris et al. isolated in 2003-2004 viruses lacking both a NA gene and protein (H3NA- viruses) (Ferraris O., 2006, Vaccine, 24(44-46):6656-9). In this study we showed that the hemagglutinins of two of the H3NA- viruses have reduced affinity for SA?2.6Gal receptors, between 49 and 128 times lower than that of the A/Moscow/10/99 (H3N2) virus and no detectable affinity for SA?2.3Gal receptors. We also showed that the low hemagglutinin affinity of the H3NA- viruses compensates for the lack of NA activity and allows the restoration of the growth of an A/Moscow/10/99 virus deficient in neuraminidase. These observations increase our understanding of H3NA- viruses in relation to the balance between the functional activities of the neuraminidase and hemagglutinin.
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