scholarly journals In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

2009 ◽  
Vol 53 (10) ◽  
pp. 4433-4440 ◽  
Author(s):  
Aeron C. Hurt ◽  
Jessica K. Holien ◽  
Ian G. Barr
Keyword(s):  
Reverse Genetics ◽  
Selective Pressure ◽  
Neuraminidase Inhibitor ◽  
Drug Susceptibility ◽  
Influenza Viruses ◽  
Wild Type ◽  
H5n1 Influenza ◽  
Different Strains ◽  
Inhibitor Resistance

ABSTRACT To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.

scholarly journals Importance of Neuraminidase Active-Site Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses

2006 ◽  
Vol 80 (17) ◽  
pp. 8787-8795 ◽  
Author(s):  
Hui-Ling Yen ◽  
Erich Hoffmann ◽  
Garry Taylor ◽  
Christoph Scholtissek ◽  
Arnold S. Monto ◽  
...  
Keyword(s):  
Active Site ◽  
Influenza A ◽  
Selective Pressure ◽  
Neuraminidase Inhibitor ◽  
Influenza Viruses ◽  
Active Site Residues ◽  
Conserved Residues ◽  
Enzyme Active Site ◽  
Inhibitor Resistance

ABSTRACT Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (∼10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.

scholarly journals The pH of Activation of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity and Transmissibility in Ducks

2009 ◽  
Vol 84 (3) ◽  
pp. 1527-1535 ◽  
Author(s):  
Mark L. Reed ◽  
Olga A. Bridges ◽  
Patrick Seiler ◽  
Jeong-Ki Kim ◽  
Hui-Ling Yen ◽  
...  
Keyword(s):  
Weight Loss ◽  
Influenza Virus ◽  
Membrane Fusion ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
H5n1 Influenza ◽  
Ha Protein

ABSTRACT While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of +0.4 and −0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H241Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.

scholarly journals Neuraminidase Inhibitor-Resistant Recombinant A/Vietnam/1203/04 (H5N1) Influenza Viruses Retain Their Replication Efficiency and Pathogenicity In Vitro and In Vivo

2007 ◽  
Vol 81 (22) ◽  
pp. 12418-12426 ◽  
Author(s):  
Hui-Ling Yen ◽  
Natalia A. Ilyushina ◽  
Rachelle Salomon ◽  
Erich Hoffmann ◽  
Robert G. Webster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Pandemic ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
H5n1 Influenza Virus ◽  
Wild Type ◽  
Replication Efficiency ◽  
H5n1 Influenza

ABSTRACT Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC50] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC50 increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V max and Km ) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.

scholarly journals Early Control of H5N1 Influenza Virus Replication by the Type I Interferon Response in Mice

2009 ◽  
Vol 83 (11) ◽  
pp. 5825-5834 ◽  
Author(s):  
Kristy J. Szretter ◽  
Shivaprakash Gangappa ◽  
Jessica A. Belser ◽  
Hui Zeng ◽  
Hualan Chen ◽  
...  
Keyword(s):  
H5n1 Virus ◽  
Type I Interferon ◽  
Influenza Viruses ◽  
Interferon Response ◽  
Type I ◽  
Wild Type ◽  
H5n1 Influenza ◽  
Deficient Mice

ABSTRACT Widespread distribution of highly pathogenic avian H5N1 influenza viruses in domesticated and wild birds continues to pose a threat to public health, as interspecies transmission of virus has resulted in increasing numbers of human disease cases. Although the pathogenic mechanism(s) of H5N1 influenza viruses has not been fully elucidated, it has been suggested that the ability to evade host innate responses, such as the type I interferon response, may contribute to the virulence of these viruses in mammals. We investigated the role that type I interferons (alpha/beta interferon [IFN-α/β]) might play in H5N1 pathogenicity in vivo, by comparing the kinetics and outcomes of H5N1 virus infection in IFN-α/β receptor (IFN-α/βR)-deficient and SvEv129 wild-type mice using two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. IFN-α/βR-deficient mice experienced significantly more weight loss and more rapid time to death than did wild-type mice. HK/486 virus caused a systemic infection similar to that with HK/483 virus in IFN-α/βR-deficient mice, suggesting a role for IFN-α/β in controlling the systemic spread of this H5N1 virus. HK/483 virus replicated more efficiently than HK/486 virus both in vivo and in vitro. However, replication of both viruses was significantly reduced following pretreatment with IFN-α/β. These results suggest a role for the IFN-α/β response in the control of H5N1 virus replication both in vivo and in vitro, and as such it may provide some degree of protection to the host in the early stages of infection.

scholarly journals Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

1998 ◽  
Vol 42 (12) ◽  
pp. 3234-3241 ◽  
Author(s):  
Chun Y. Tai ◽  
Paul A. Escarpe ◽  
Robert W. Sidwell ◽  
Matthew A. Williams ◽  
Willard Lew ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Neuraminidase Inhibitor ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Human Influenza ◽  
Wild Type ◽  
Viral Virulence ◽  
High Level

ABSTRACT An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

scholarly journals Neuraminidase Inhibitor Sensitivity and Receptor-Binding Specificity of Cambodian Clade 1 Highly Pathogenic H5N1 Influenza Virus

2011 ◽  
Vol 55 (5) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Naughtin ◽  
J. C. Dyason ◽  
S. Mardy ◽  
S. Sorn ◽  
M. von Itzstein ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Binding Specificity ◽  
Neuraminidase Inhibitor ◽  
H5n1 Influenza Virus ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Receptor Binding Specificity ◽  
Pathogenic H5n1

ABSTRACTThe evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

scholarly journals PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2

2011 ◽  
Vol 92 (6) ◽  
pp. 1435-1444 ◽  
Author(s):  
Jing Li ◽  
Yongqiang Li ◽  
Yi Hu ◽  
Guohui Chang ◽  
Wei Sun ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Recombinant Virus ◽  
Influenza Viruses ◽  
H5n1 Influenza Virus ◽  
Wild Type ◽  
Small Plaque ◽  
Large Plaque ◽  
H5n1 Influenza ◽  
Virulence Attenuation ◽  
N Terminus

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

Factors Affecting Susceptibility of H5N1 Influenza Viruses to Neuraminidase Inhibitor Oseltamivir

2008 ◽  
Vol 78 (2) ◽  
pp. A18-A19
Author(s):  
Elena Govorkova ◽  
Natalia Ilyushina ◽  
Jennifer McClaren ◽  
Tri Naipospos ◽  
Neziha Yilmas ◽  
...  
Keyword(s):  
Neuraminidase Inhibitor ◽  
Influenza Viruses ◽  
Factors Affecting ◽  
H5n1 Influenza

scholarly journals Bioluminescence-Based Neuraminidase Inhibition Assay for Monitoring Influenza Virus Drug Susceptibility in Clinical Specimens

2013 ◽  
Vol 57 (11) ◽  
pp. 5209-5215 ◽  
Author(s):  
Henju Marjuki ◽  
Vasiliy P. Mishin ◽  
Katrina Sleeman ◽  
Margaret Okomo-Adhiambo ◽  
Tiffany G. Sheu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Point Of Care ◽  
Drug Susceptibility ◽  
Influenza Viruses ◽  
Clinical Specimens ◽  
Influenza Virus A ◽  
Oseltamivir Resistance ◽  
Resistance Markers ◽  
The Difference ◽  
Inhibitor Resistance

ABSTRACTThe QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n= 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n= 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.

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