| 1. |
- Orozovic, Goran, et al.
(författare)
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Detection of Oseltamivir-and Zanamivir-Resistant Influenza A (H12N3) Virus from Wild Mallards in Sweden
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Resistance to neuraminidase inhibitors (NAIs) is a growing problem in battle against influenza A virus. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the virus. To date, no low-pathogenic avian influenza (LPAI) virus that is resistant to NAIs has been detected. The aim of this study is to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC)- or zanamivir (ZA)-related resistance mutations. In this work, 22 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The R118K mutation was the most recurrent, as it was observed in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA observed in the N1 and N2 groups of NAs. Furthermore, both negative controls (NC) in the N6 and one NC in the N9 subtype were less sensitive to ZA than were genotypically related mutants of the respective subtypes. One H12N3 strain (80087 isolate) was cross-resistant, with an IC50 of >104 nM. This virus carried D151K and R156K mutations that were not associated with NAI resistance. Analyses of the 3D structure of NA indicated that the NAI-resistant phenotype that retained NA catalytic activity was likely to be due to the D151K mutation. However, the residues occupying positions 149, 150 (part of the 150 loop) and 153 may have contributed to this resistance. Other possible factors included the presence of the 150 cavity, the nature of the substrate (anchored or unanchored) and the interaction of the ligand with the active site. These results demonstrate that NAI-resistant viruses exist in LPAI and highlight the importance of monitoring influenza A viruses in wild birds, as these viruses can be transmitted to humans and can thus become part of a human-adapted influenza virus with pandemic potential.
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| 2. |
- Orozovic, Goran, 1965-, et al.
(författare)
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Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir).
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| 3. |
- Orozovic, Goran, et al.
(författare)
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Oseltamivir- and Zanamivir-Resistance Related Mutations in Influenza A Viruses Isolated from Wild Mallards in Sweden Studied by a Colorimetric Neuraminidase Inhibition Assay
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Resistance to neuraminidase inhibitors (NAIs) is a growing problem in the battle against influenza A viruses. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To date, no low-pathogenic avian influenza (LPAI) virus functionally resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC)- or zanamivir (ZA)-related resistance mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The R118K mutation was the most frequently observed; it was seen in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA previously observed in the N1 and N2 groups of NAs. Furthermore, both negative controls (NCs) in the N6 and one NC in the N9 subtype were less sensitive to ZA than were genotypically related mutants of the respective subtypes. The presence of OC- and ZA-related mutations in the NA of viruses isolated from wild birds did not result in a NAI-resistant phenotype in this study. The R118K and R152K mutations seemed to somewhat increase the sensitivity to both NAIs compared to WT viruses which supports the thought that the mutations were a result of natural NA variation and not induced by NAI residuals in the environment. Although not resulting in a NAI resistant phenotype, the finding of the mutations D151N and I222V shows that mutations with the potential to enhance NAI resistance exist in the pool of LPAI viruses which stresses the need for surveillance of NAI resistance in wild birds.
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| 4. |
- Orozovic, Goran, et al.
(författare)
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Study of oseltamivir and zanamivir resistance-related mutations in influenza viruses isolated from wild mallards in Sweden
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:2, s. e89306-
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to neuraminidase inhibitors (NAIs) is a growing problem in battle against influenza A virus. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To our knowledge, no low-pathogenic avian influenza (LPAI) virus resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC) or zanamivir (ZA) resistance-related mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The results of assay showed no NAIs-resistant phenotype for any of the viruses. The R118K mutation was the most recurrent, as it was observed in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA observed in the N1 and N2 groups of NAs. Furthermore, both wild types (WTs) in the N6 and one WT in the N9 subtype were less sensitive to ZA than were genotypically related mutants with R152K and R118K change in the respective subtypes. This may indicate that these and probably even other NAIs resistance-related mutations found in our virus collection were not induced by NAIs residuals in the environment and that the impact of such mutations in an avian influenza could be dependent on subtype, strain and host species.
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| 5. |
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| 6. |
- Nicholls, Ian A., et al.
(författare)
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Synthetic Neuraminidases : Nanostructured Materials for Environmental Monitoring
- 2011
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Ingår i: Ecohealth, vol. 7, Supplement 1. - : Springer. ; , s. S97-S97
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Konferensbidrag (refereegranskat)abstract
- The risks to society associated with the spread of new strains of influenza with human pathogenicity, or with impact on agricultureare significant. Our capacity to challenge the threat of the virus is dependent upon our ability to develop new vaccines, and upon ouraccess to effective virus-targeted small molecule pharmaceuticals. The current primary small molecule weapons oseltamivir(Tamiflu) and zanamivir (Relenza) currently form our last line of defence against this virus. More recently, the identification ofstrains resistant to (in particular) drugs targeting neuraminidase has awoken serious concern. Equally as worrying is the clearevidence of the presence of these substances in the World’s water systems which has now come forth. Collectively, this makes thedevelopment of techniques giving us better insight into the virus and antiviral agents a priority. Robust methods for the rapid andsensitive determination of these substances are required, especially as the monitoring methods should be able to withstand therigours of environments not normally conducive to biomacromolecules (temperature, toxic substances etc) e.g. antibodies.Advanced materials fulfilling these requirements can be obtained by Molecular Imprinting, which is a technique forproducing highly selective synthetic receptors for biochemical and chemical structures in synthetic polymers. The polymerscontain nano-structured cavities that are of complementary functional and structural character to predetermined target.The technique entails the judicious selection of a monomer or monomer mixture with chemical functionality comple-mentary to that of the imprint species (template). The complementary interacting functionalities (reversible covalent ornon-covalent) form predictable solution structures, which after polymerisation in the presence of a suitable cross linkingagent and removal of the template lead to the defining of recognition sites of complementary steric and functionaltopography to the template molecule. These sites give selective recognition of the template. Furthermore, by analogy tocatalytic antibody production, using transition state analogues as templates yields synthetic enzymes.Synthetic polymers with neuraminidase-like behaviour have been designed through the screening of candidate polymersystems using a combination of molecular dynamics and NMR studies. The characterisation of the resulting materials hasdemonstrated systems with selectivity for the targeted antiviral agents. Our studies illustrate the potential of these uniquenanostructured materials for the monitoring of these antiviral agents in the environment, which is an important aspect inefforts aimed at limiting the development of resistant strains, and as a tool for policy makers.
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| 7. |
- Shoravi, Siamak, et al.
(författare)
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Towards Synthetic Neuraminidases
- 2010
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Konferensbidrag (refereegranskat)abstract
- Influenza endemics and pandemics have been a menace to humanity through the ages and pose ominous threats and with dire consequences for humanity [1]. A better understanding of the virus, the epidemiology of the disease, and its structure and function is essential for creating new therapies and even for better understanding resistance to existing treatments. At the molecular level the two capside proteins Neuraminidase and Hemagglutinin are engaged in interactions with host cell surface sialic acid residues, and are critical for contagion and budding off of the virus into and from the cell, and have been the targets for drug development strategies [2]. The rise of strains of the virus resistant to drugs targeting Neuraminidase make it crucial to develop techniques for better understanding of the virus and hence design of better antiviral agents [3].In this study a combination of molecular dynamics (MD) and NMR spectroscopy [4-7] is been utilised in order to screen some 60 polymers for candidate systems [8] for use in the preparation of synthetic polymer neauraminidase mimics. Progress in the design, synthesis and evaluation of these materials shall be presented.References1. Webster et al., Microbiol. Rev., 56, 152-175 (1992).2. von Itzstein et al., Nature, 263, 418-423 (1993).3. Lindberg et al., Chemosphere, 57, 1479-1488. (2004).4. Svensson et al., J. Chromatogr. A, 1024, 39-44 (2004).5. O’Mahony et al., Analyst, 23, 1147-1115 (2007).6. Karlsson et al., J. Am. Chem. Soc., 131, 13297-13304 (2009).7. Nicholls et al., Biosens. Bioelectron., 25, 553-557 (2009).8. Shoravi et al., unpublished results (2010).
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