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Robert G Webster - One of the best experts on this subject based on the ideXlab platform.
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unique determinants of Neuraminidase Inhibitor resistance among n3 n7 and n9 avian influenza viruses
Journal of Virology, 2015Co-Authors: Robert G Webster, Minsuk Song, Bindumadhav M Marathe, Gyanendra Kumar, Sooksan Wong, Adam Rubrum, Mark Zanin, Young Ki Choi, Elena A GovorkovaAbstract:Human infections with avian influenza viruses are a serious public health concern. The Neuraminidase (NA) Inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically. IMPORTANCE The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to Neuraminidase (NA) Inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.
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susceptibility of avian influenza viruses of the n6 subtype to the Neuraminidase Inhibitor oseltamivir
Antiviral Research, 2012Co-Authors: Terri D Stoner, Elena A Govorkova, Scott Krauss, Jasmine Turner, Patrick Seiler, Nicholas J Negovetich, David E Stallknecht, Sharon Frase, Robert G WebsterAbstract:Abstract Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 Neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA Inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC50 values ranging from 50.0 nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P ⩽ 0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC50, 30.0 and 40.0 nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.
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fitness of Neuraminidase Inhibitor resistant influenza a viruses
Current Opinion in Virology, 2011Co-Authors: Tatiana Baranovich, Robert G Webster, Elena A GovorkovaAbstract:Antiviral drugs currently approved by the United States Food and Drug Administration to control influenza infection are M2-ion channel blockers (ie, amantadine and rimantadine) and Neuraminidase (NA) Inhibitors [(NAI), ie, zanamivir and oseltamivir). Both classes of drugs target specific influenza A virus proteins and interfere with either viral uncoating inside the cell (M2-blockers) or the release of influenza virions from infected cell (NAI) [1]. The NAIs are effective against all 9 NA subtypes of influenza A viruses and 2 lineages of influenza B viruses; adamantanes are only effective against influenza A viruses. The incidence of naturally occurring amantadine-resistant variants has increased dramatically since 2003 and thus limited our options to NAIs. Although NAIs were assumed to be less prone to select resistant influenza viruses, NAI-resistant variants exist [2–4]. Clinically derived drug-resistant viruses have mutations that are NA subtype-specific and differ with the NAI used [5]. The most frequently observed mutations in NAI-resistant variants of influenza A viruses of the N1 NA subtype are H274Y and N294S (N2 numbering here and throughout the text); those of N2 NA subtype most frequently harbor R292K and E119V, and influenza B viruses most frequently harbor R152K and D198N. Although amino acid substitutions at other positions at the catalytic or framework NA residues of influenza A viruses also reduce NAI susceptibility [5], the contribution of these substitutions in clinic is uncertain.
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intramuscularly administered Neuraminidase Inhibitor peramivir is effective against lethal h5n1 influenza virus in mice
Antiviral Research, 2008Co-Authors: David A Boltz, Robert G Webster, Natalia A Ilyushina, Shane C Arnold, Sudhakar Y Babu, Elena A GovorkovaAbstract:Abstract The replication efficiency and multi-organ dissemination of some influenza A (H5N1) viruses requires a rapid re-evaluation of the available antiviral strategies. We assessed five regimens of the Neuraminidase (NA) Inhibitor peramivir in mice inoculated with H5N1 virus. The regimens differed by: (1) frequency of administration on first day (once vs twice); (2) duration of administration (1 day vs 8 days); (3) route of administration (intramuscular [IM] injection alone or followed by oral administration). In all regimens, BALB/c mice were administered 30 mg/kg peramivir IM 1 h after lethal challenge with 5 MLD50 of A/Vietnam/1203/04 (H5N1) influenza virus. When given only on the day of inoculation, a single IM injection produced a 33% survival rate, which increased to 55% with two injections. Eight-day regimens significantly increased survival; two IM injections followed by seven daily IM injections was the most effective regimen (100% survival; inhibition of replication in lungs and brain). When this 8-day regimen began at 24 h after inoculation, 78% of mice survived; 56% survived when treatment began at 48 hours. Anti-HA antibody titer differed with the peramivir regimen and corresponded to the severity of disease. Overall, our results demonstrate that IM administration of peramivir is effective in promoting the survival of mice infected with systemically replicating H5N1 virus.
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importance of Neuraminidase active site residues to the Neuraminidase Inhibitor resistance of influenza viruses
Journal of Virology, 2006Co-Authors: Erich Hoffmann, Arnold S Monto, Robert G Webster, G L Taylor, Christoph Scholtissek, Elena A GovorkovaAbstract: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.
Elena A Govorkova - One of the best experts on this subject based on the ideXlab platform.
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unique determinants of Neuraminidase Inhibitor resistance among n3 n7 and n9 avian influenza viruses
Journal of Virology, 2015Co-Authors: Robert G Webster, Minsuk Song, Bindumadhav M Marathe, Gyanendra Kumar, Sooksan Wong, Adam Rubrum, Mark Zanin, Young Ki Choi, Elena A GovorkovaAbstract:Human infections with avian influenza viruses are a serious public health concern. The Neuraminidase (NA) Inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically. IMPORTANCE The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to Neuraminidase (NA) Inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.
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susceptibility of avian influenza viruses of the n6 subtype to the Neuraminidase Inhibitor oseltamivir
Antiviral Research, 2012Co-Authors: Terri D Stoner, Elena A Govorkova, Scott Krauss, Jasmine Turner, Patrick Seiler, Nicholas J Negovetich, David E Stallknecht, Sharon Frase, Robert G WebsterAbstract:Abstract Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 Neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA Inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC50 values ranging from 50.0 nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P ⩽ 0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC50, 30.0 and 40.0 nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.
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fitness of Neuraminidase Inhibitor resistant influenza a viruses
Current Opinion in Virology, 2011Co-Authors: Tatiana Baranovich, Robert G Webster, Elena A GovorkovaAbstract:Antiviral drugs currently approved by the United States Food and Drug Administration to control influenza infection are M2-ion channel blockers (ie, amantadine and rimantadine) and Neuraminidase (NA) Inhibitors [(NAI), ie, zanamivir and oseltamivir). Both classes of drugs target specific influenza A virus proteins and interfere with either viral uncoating inside the cell (M2-blockers) or the release of influenza virions from infected cell (NAI) [1]. The NAIs are effective against all 9 NA subtypes of influenza A viruses and 2 lineages of influenza B viruses; adamantanes are only effective against influenza A viruses. The incidence of naturally occurring amantadine-resistant variants has increased dramatically since 2003 and thus limited our options to NAIs. Although NAIs were assumed to be less prone to select resistant influenza viruses, NAI-resistant variants exist [2–4]. Clinically derived drug-resistant viruses have mutations that are NA subtype-specific and differ with the NAI used [5]. The most frequently observed mutations in NAI-resistant variants of influenza A viruses of the N1 NA subtype are H274Y and N294S (N2 numbering here and throughout the text); those of N2 NA subtype most frequently harbor R292K and E119V, and influenza B viruses most frequently harbor R152K and D198N. Although amino acid substitutions at other positions at the catalytic or framework NA residues of influenza A viruses also reduce NAI susceptibility [5], the contribution of these substitutions in clinic is uncertain.
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intramuscularly administered Neuraminidase Inhibitor peramivir is effective against lethal h5n1 influenza virus in mice
Antiviral Research, 2008Co-Authors: David A Boltz, Robert G Webster, Natalia A Ilyushina, Shane C Arnold, Sudhakar Y Babu, Elena A GovorkovaAbstract:Abstract The replication efficiency and multi-organ dissemination of some influenza A (H5N1) viruses requires a rapid re-evaluation of the available antiviral strategies. We assessed five regimens of the Neuraminidase (NA) Inhibitor peramivir in mice inoculated with H5N1 virus. The regimens differed by: (1) frequency of administration on first day (once vs twice); (2) duration of administration (1 day vs 8 days); (3) route of administration (intramuscular [IM] injection alone or followed by oral administration). In all regimens, BALB/c mice were administered 30 mg/kg peramivir IM 1 h after lethal challenge with 5 MLD50 of A/Vietnam/1203/04 (H5N1) influenza virus. When given only on the day of inoculation, a single IM injection produced a 33% survival rate, which increased to 55% with two injections. Eight-day regimens significantly increased survival; two IM injections followed by seven daily IM injections was the most effective regimen (100% survival; inhibition of replication in lungs and brain). When this 8-day regimen began at 24 h after inoculation, 78% of mice survived; 56% survived when treatment began at 48 hours. Anti-HA antibody titer differed with the peramivir regimen and corresponded to the severity of disease. Overall, our results demonstrate that IM administration of peramivir is effective in promoting the survival of mice infected with systemically replicating H5N1 virus.
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importance of Neuraminidase active site residues to the Neuraminidase Inhibitor resistance of influenza viruses
Journal of Virology, 2006Co-Authors: Erich Hoffmann, Arnold S Monto, Robert G Webster, G L Taylor, Christoph Scholtissek, Elena A GovorkovaAbstract: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.
Frederick G Hayden - One of the best experts on this subject based on the ideXlab platform.
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surveillance for Neuraminidase Inhibitor resistant influenza viruses in japan 1996 2007
Antiviral Therapy, 2009Co-Authors: Masato Tashiro, Jennifer L Mckimmbreschkin, Takehiko Saito, Alexander Klimov, Catherine A Macken, Maria Zambon, Frederick G HaydenAbstract:Background: High usage of the Neuraminidase Inhibitor (NAI) oseltamivir in Japan since 2003 led the Neuraminidase Inhibitor Susceptibility Network to assess the susceptibility of community isolates of influenza viruses to oseltamivir and zanamivir. Methods: Isolates were tested by the enzyme inhibition assay and by Neuraminidase (NA) sequence analysis. Results: Among 1,141 A(H3N2) viruses and 171 type B viruses collected in Japan during the 2003-2004 season, 3 (0.3%) A(H3N2) isolates showed high 50% Inhibitory concentrations (IC 50 ) to oseltamivir. Each possessed a known resistance NA mutation at R292K or E119V. During the 2004-2005 season, no resistance was found among 567 influenza A(H3N2) or 60 A(H1N1) isolates, but 1 of 58 influenza B isolates had an NAI resistance mutation (D197N). Sequence analysis found that 4 (3%) of 132 A(H1N1) viruses from 2005-2006 had known NA resistance mutations (all H274Y), but no additional resistant isolates were detected from that or the subsequent 2006-2007 season. Concurrent testing of a selection of 500 influenza B viruses from 2000 to 2006 showed significant variations between seasons in both oseltamivir and zanamivir IC 50 values, but no persistent increases over this period. Conclusions: Our findings suggested possible low-level transmission of resistant variants from oseltamivir-treated patients in several seasons in Japan but no sustained reductions in NAI susceptibility or consistently increased frequency of detecting resistant variants for any strain or subtype, despite high levels of drug use. In particular, although oseltamivir-resistant A(H1N1) viruses with the H274Y mutation spread globally in 2007-2008, we found little evidence for increasing levels of resistant A(H1N1) variants in Japan in preceding years.
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Neuraminidase Inhibitor susceptibility network position statement antiviral resistance in influenza a h5n1 viruses
Antiviral Therapy, 2005Co-Authors: Frederick G Hayden, Masato Tashiro, Jennifer L Mckimmbreschkin, Alexander Klimov, Catherine A Macken, Arnold S Monto, Alan W Hampson, Robert G Webster, Michele Amyard, Maria ZambonAbstract:The emerging epidemic of H5N1 avian influenza virus with spillover into the human population in Asia has provoked intense concern globally about the potential of these particularly pathogenic viruses to evolve with the capacity for human-to-human transmission with a consequent pandemic. The availability of antiviral drugs with activity against influenza A viruses and the recognition of drug-resistant variants to these drugs prompted the following report by a select group of the global experts - members of the Neuraminidase Inhibitor Susceptibility Network - on the best use of the available drugs, both for prophylaxis and treatment. The editors of Antiviral Therapy are pleased to be able to provide this document in an expeditious manner.
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efficacy and safety of the oral Neuraminidase Inhibitor oseltamivir in treating acute influenza
JAMA, 2000Co-Authors: John J Treanor, Frederick G Hayden, Peter S Vrooman, Rick Barbarash, Robert Bettis, Dennis Riff, Sudeep Singh, Nelson Kinnersley, Penelope Ward, Roger G MillsAbstract:ContextPrevious studies have shown oseltamivir, a Neuraminidase Inhibitor, to be effective in preventing influenza and treating experimental influenza.ObjectiveTo evaluate the efficacy and safety of oseltamivir in the treatment of naturally acquired influenza infection.DesignRandomized, placebo-controlled, double-blind study conducted January through March 1998.SettingSixty primary care and university health centers throughout the United States.ParticipantsA total of 629 healthy nonimmunized adults aged 18 to 65 years with febrile respiratory illness of no more than 36 hours' duration with temperature of 38°C or more plus at least 1 respiratory symptom and 1 constitutional symptom.InterventionsIndividuals were randomized to 1 of 3 treatment groups with identical appearing pills: oral oseltamivir phosphate, 75 mg twice daily (n = 211) or 150 mg (n = 209) twice daily, or placebo (n = 209).Main Outcome MeasuresDuration and severity of illness in individuals infected with influenza.ResultsTwo individuals withdrew before receiving medication and were excluded from further analyses. A total of 374 individuals (59.6%) were infected with influenza. Their duration of illness was reduced by more than 30% with both oseltamivir, 75 mg twice daily (median, 71.5 hours; P<.001), and oseltamivir, 150 mg twice daily (median, 69.9 hours; P = .006), compared with placebo (median, 103.3 hours). Severity of illness was reduced by 38% (median score, 597 score-hours; P<.001) with oseltamivir, 75 mg twice daily, and by 35% (median score, 626 score-hours; P<.001) with oseltamivir, 150 mg twice daily, vs placebo (median score, 963 score-hours). Oseltamivir treatment reduced the duration of fever and oseltamivir recipients returned to usual activities 2 to 3 days earlier than placebo recipients (P≤.05). Secondary complications such as bronchitis and sinusitis occurred in 15% of placebo recipients compared with 7% of combined oseltamivir recipients (P = .03). Among all 629 subjects, oseltamivir reduced illness duration (76.3 hours and 74.3 hours for 75 mg and 150 mg, respectively, vs 97.0 hours for placebo; P = .004 for both comparisons) and illness severity (686 score-hours and 629 score-hours for 75 mg and 150 mg, respectively, vs 887 score-hours for placebo; P<.001 for both comparisons). Nausea and vomiting occurred more frequently in both oseltamivir groups (combined, 18.0% and 14.1%, respectively; P = .002) than in the placebo group (7.4% and 3.4%; P<.001).ConclusionsOur data suggest that oral oseltamivir treatment reduces the duration and severity of acute influenza in healthy adults and may decrease the incidence of secondary complications.
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use of the selective oral Neuraminidase Inhibitor oseltamivir to prevent influenza
The New England Journal of Medicine, 1999Co-Authors: Frederick G Hayden, Penelope Ward, Robert L Atmar, Margo Schilling, Casey T Johnson, Donald Poretz, David Paar, Les Huson, Roger G MillsAbstract:Background Safe and effective antiviral agents are needed to prevent infection with influenza A and B viruses. Oseltamivir (GS4104), which can be administered orally, is the prodrug of GS4071, a potent and selective Inhibitor of influenzavirus Neuraminidases. We studied the use of oseltamivir for long-term prophylaxis against influenza in two placebo-controlled, double-blind trials at different U.S. sites during the winter of 1997–1998. Methods We randomly assigned 1559 healthy, nonimmunized adults 18 to 65 years old to receive either oral oseltamivir (75 mg given once or twice daily, for a total daily dose of 75 or 150 mg) or placebo for six weeks during a peak period of local influenzavirus activity. The primary end point with respect to efficacy was laboratory-confirmed influenza-like illness (defined as a temperature of at least 37.2°C accompanied by at least one respiratory and at least one systemic symptom). Results In the two studies combined, the risk of influenza among subjects assigned to either...
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use of the oral Neuraminidase Inhibitor oseltamivir in experimental human influenza randomized controlled trials for prevention and treatment
JAMA, 1999Co-Authors: Frederick G Hayden, John J Treanor, Robert F Betts, Monica Lobo, Nelson Kinnersley, Penelope Ward, Roger G Mills, Scott R Fritz, Madeline Miller, Stephen E StrausAbstract:ContextInfluenza virus Neuraminidase is thought to be essential for virus replication in humans; however, to date, available Neuraminidase Inhibitors are limited to zanamivir, which is topically administered.ObjectiveTo determine the safety, tolerability, and antiviral activity of oral Neuraminidase Inhibitor oseltamivir (GS4104/Ro64-0796) for prevention and the early treatment of influenza in experimentally infected humans.DesignTwo randomized, double-blind, placebo-controlled trials conducted between June and July 1997.SettingIndividual hotel rooms; 2 large US university medical schools.ParticipantsA total of 117 healthy adult volunteers (aged 18-40 years; median age, 21 years) who were susceptible (hemagglutination-inhibition antibody titer ≤1:8).InterventionsAll subjects were inoculated intranasally with influenza A/Texas/36/91(H1N1) virus. For the prophylaxis study, oral oseltamivir (100 mg once daily [n=12], 100 mg twice daily [n=12], or matching placebo [n=13], starting 26 hours before virus inoculation) was administered. For the treatment study, the same drug was given (20 mg, 100 mg, or 200 mg twice daily, 200 mg once daily, or matching placebo [n=16], in each group starting 28 hours after inoculation). All regimens were continued for 5 days.Main Outcome MeasuresComparing placebo groups with pooled treatment groups, for prophylaxis, outcomes included frequency of infection and viral shedding; for treatment, viral shedding in titers.ResultsIn the prophylaxis study, 8 (67%) of 12 placebo and 8 (38%) of 21 oseltamivir recipients became infected (P=.16; efficacy, 61%); 6 (50%) placebo compared with 0 oseltamivir recipients shed virus (P<.001; efficacy, 100%), and 33% of placebo but no oseltamivir recipient had infection-related respiratory illness (P<.01). Among infected subjects in the treatment study (n=69), the viral titer area under the curve of the combined oseltamivir groups (n=56) was lower (median [interquartile range {IQR}], 80 [23-151] vs 273 [79-306] log10 tissue culture-infective doses50 per milliliter×hour; P=.02) than the placebo group (n=13), and the median (IQR) duration of viral shedding with therapy was reduced from 107 (83-131) to 58 (35-59) hours (P=.003). Oseltamivir treatment also reduced symptom scores (median [IQR] score-hours, 225 [97-349] vs 400 [189-645]; P=.05), and nasal proinflammatory cytokine levels. Transient mild to moderate nausea after dosing was observed in 15 (17%) of 88 oseltamivir and 2 (7%) of 29 placebo recipients (95% confidence interval for difference, −11% to 68%), which was largely prevented by ingestion with food.ConclusionsIn these trials, prophylaxis and early treatment with oral oseltamivir were both associated with significant antiviral and clinical effects in experimental human influenza.
Aeron C Hurt - One of the best experts on this subject based on the ideXlab platform.
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Neuraminidase Inhibitor resistance in influenza a clinical perspective
Current Opinion in Infectious Diseases, 2018Co-Authors: Aeron C HurtAbstract:Purpose of reviewNeuraminidase Inhibitors (NAIs), including oseltamivir, zanamivir, and peramivir, is the main class of antiviral available for clinical use. As such, development of resistance toward these agents is of great clinical and public health concern.Recent findingsAt present, NAI resistanc
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characterization of influenza b virus variants with reduced Neuraminidase Inhibitor susceptibility
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Rubaiyea Farrukee, Alexander E Zarebski, James M Mccaw, Jesse D Bloom, Patrick C Reading, Aeron C HurtAbstract:Treatment options for influenza B virus infections are limited to Neuraminidase Inhibitors (NAIs), which block the Neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo. The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.
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Global update on the susceptibility of human influenza viruses to Neuraminidase Inhibitors and status of novel antivirals, 2016-2017.
Antiviral Research, 2018Co-Authors: Angie Lackenby, Aeron C Hurt, Terry G. Besselaar, Rod S. Daniels, Vicki Gregory, Larisa V. Gubareva, Weijuan Huang, Sook-kwan LeangAbstract:Abstract A total of 13672 viruses, collected by World Health Organization recognised National Influenza Centres between May 2016 and May 2017, were assessed for Neuraminidase Inhibitor susceptibility by four WHO Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance Epidemiology and Control of Influenza. The 50% Inhibitory concentration (IC50) was determined for oseltamivir and zanamivir for all viruses, and for peramivir and laninamivir in a subset (n = 8457). Of the viruses tested, 94% were obtained from the Western Pacific, Americas and European WHO regions, while limited viruses were available from the Eastern Mediterranean, African and South East Asian regions. Reduced inhibition (RI) by one or more Neuraminidase Inhibitor was exhibited by 0.2% of viruses tested (n = 32). The frequency of viruses with RI has remained low since this global analysis began (2015/16: 0.8%, 2014/15: 0.5%; 2013/14: 1.9%; 2012/13: 0.6%) but 2016/17 has the lowest frequency observed to date. Analysis of 13581 Neuraminidase sequences retrieved from public databases, of which 5243 sequences were from viruses not included in the phenotypic analyses, identified 58 further viruses (29 without phenotypic analyses) with amino acid substitutions associated with RI by at least one Neuraminidase Inhibitor. Bringing the total proportion to 0.5% (90/18915). This 2016/17 analysis demonstrates that Neuraminidase Inhibitors remain suitable for treatment and prophylaxis of influenza virus infections, but continued monitoring is important. An expansion of surveillance testing is paramount since several novel influenza antivirals are in late stage clinical trials with some resistance already having been identified.
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in vitro generation of Neuraminidase Inhibitor resistance in a h5n1 influenza viruses
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Aeron C Hurt, Jessica K Holien, Ian G BarrAbstract: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.
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influenza viruses with reduced sensitivity to the Neuraminidase Inhibitor drugs in untreated young children
Communicable diseases intelligence quarterly report, 2008Co-Authors: Aeron C Hurt, Ian G BarrAbstract:The Neuraminidase Inhibitors are a class of antiviral drugs used for both the prophylaxis and treatment of influenza infections. Clinical trials of these Inhibitors detected a low level of resistant viruses from treated individuals, although a higher frequency was detected in children (5%-6%) compared to adults (1%-4%). In addition, there have been some previous reports of NA Inhibitor resistant viruses being isolated from untreated individuals. Here we report on the NA Inhibitor sensitivity of over 1 000 influenza isolates collected through the World Health Organization (WHO) global influenza surveillance program. Of the total number of viruses analysed, only 2 (0.2%) strains (an A(H1N1) strain and an influenza B strain) were considered to have a significant reduction in sensitivity to at least one of the Neuraminidase Inhibitor drugs. Interestingly, both of these strains were isolated from untreated patients in the youngest age cohort (less than 2 years). Although the influenza B strain is unlikely to be clinically resistant, the A(H1N1) virus contained the same His274Tyr Neuraminidase mutation that has been observed in resistant mutants following oseltamivir treatment. Given these results it may be important to enhance Neuraminidase Inhibitor susceptibility testing of viruses from patients in the less than two years cohort. (author abstract)
Maria Zambon - One of the best experts on this subject based on the ideXlab platform.
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Neuraminidase Inhibitor resistance after oseltamivir treatment of acute influenza a and b in children
Clinical Infectious Diseases, 2009Co-Authors: Iain Stephenson, Angie Lackenby, Jane Democratis, Karl G Nicholson, Teresa Mcnally, James R Smith, Manish Pareek, J Ellis, Alison Bermingham, Maria ZambonAbstract:Background. Oseltamivir, a specific influenza Neuraminidase Inhibitor, is an effective treatment for seasonal influenza. Emergence of drug-resistant influenza viruses after treatment has been reported, particularly in children in Japan, where the dosing schedule is different from that used throughout the rest of the world. We investigated the emergence of drug-resistant infection in children treated with a tiered weight-based dosing regimen.Methods. We analyzed sequential clinical nasopharyngeal samples, obtained before and after tiered weight-based oseltamivir therapy, from children with acute influenza during 2005–2007. We isolated viruses, tested for drug resistance with use of a fluorescence-based Neuraminidase inhibition assay, performed Neuraminidase gene sequencing, and determined quantitative viral loads.Results. Sixty-four children (34 with influenza A subtype H3N2, 11 with influenza A subtype H1N1, and 19 with influenza B virus) aged 1–12 years (median age, 3 years, 1 month) were enrolled. By days 4–7 after initiation of treatment, of 64 samples tested, 47 (73.4%) and 26 (40.6%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. By days 8–12 after initiation of treatment, of 53 samples tested, 18 (33.9%) and 1 (1.8%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. We found no statistically significant differences in the reduction of viral shedding or time to clearance of virus between viral subtypes. Antiviral-resistant viruses were recovered from 3 (27.3%) of 11 children with influenza A subtype H1N1, 1 (2.9%) of 34 children with influenza A subtype H3N2, and 0 (0%) of 19 children with influenza B virus, all of whom were treated with oseltamivir (P=.004) There was no evidence of prolonged illness in children infected with drug-resistant virus.Conclusions. Drug resistance emerges at a higher rate in influenza A subtype H1N1 virus than in influenza A subtype H3N2 or influenza B virus after tiered weight-based oseltamivir therapy. Virological surveillance for patterns of drug resistance is essential for determination of antiviral treatment strategies and for composition of pandemic preparedness stockpiles.
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surveillance for Neuraminidase Inhibitor resistant influenza viruses in japan 1996 2007
Antiviral Therapy, 2009Co-Authors: Masato Tashiro, Jennifer L Mckimmbreschkin, Takehiko Saito, Alexander Klimov, Catherine A Macken, Maria Zambon, Frederick G HaydenAbstract:Background: High usage of the Neuraminidase Inhibitor (NAI) oseltamivir in Japan since 2003 led the Neuraminidase Inhibitor Susceptibility Network to assess the susceptibility of community isolates of influenza viruses to oseltamivir and zanamivir. Methods: Isolates were tested by the enzyme inhibition assay and by Neuraminidase (NA) sequence analysis. Results: Among 1,141 A(H3N2) viruses and 171 type B viruses collected in Japan during the 2003-2004 season, 3 (0.3%) A(H3N2) isolates showed high 50% Inhibitory concentrations (IC 50 ) to oseltamivir. Each possessed a known resistance NA mutation at R292K or E119V. During the 2004-2005 season, no resistance was found among 567 influenza A(H3N2) or 60 A(H1N1) isolates, but 1 of 58 influenza B isolates had an NAI resistance mutation (D197N). Sequence analysis found that 4 (3%) of 132 A(H1N1) viruses from 2005-2006 had known NA resistance mutations (all H274Y), but no additional resistant isolates were detected from that or the subsequent 2006-2007 season. Concurrent testing of a selection of 500 influenza B viruses from 2000 to 2006 showed significant variations between seasons in both oseltamivir and zanamivir IC 50 values, but no persistent increases over this period. Conclusions: Our findings suggested possible low-level transmission of resistant variants from oseltamivir-treated patients in several seasons in Japan but no sustained reductions in NAI susceptibility or consistently increased frequency of detecting resistant variants for any strain or subtype, despite high levels of drug use. In particular, although oseltamivir-resistant A(H1N1) viruses with the H274Y mutation spread globally in 2007-2008, we found little evidence for increasing levels of resistant A(H1N1) variants in Japan in preceding years.
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rapid quantitation of Neuraminidase Inhibitor drug resistance in influenza virus quasispecies
Antiviral Therapy, 2008Co-Authors: Angie Lackenby, Jane Democratis, Marilda M Siqueira, Maria ZambonAbstract:Background: Emerging resistance of influenza viruses to Neuraminidase Inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to complete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clinically meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community. Methods: We have developed a pyrosequencing methodology to detect and quantitate influenza Neuraminidase Inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms associated with drug resistance in the Neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities. Results: Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide information capable of informing individual patient or outbreak management. Conclusions: Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness.
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Neuraminidase Inhibitor susceptibility network position statement antiviral resistance in influenza a h5n1 viruses
Antiviral Therapy, 2005Co-Authors: Frederick G Hayden, Masato Tashiro, Jennifer L Mckimmbreschkin, Alexander Klimov, Catherine A Macken, Arnold S Monto, Alan W Hampson, Robert G Webster, Michele Amyard, Maria ZambonAbstract:The emerging epidemic of H5N1 avian influenza virus with spillover into the human population in Asia has provoked intense concern globally about the potential of these particularly pathogenic viruses to evolve with the capacity for human-to-human transmission with a consequent pandemic. The availability of antiviral drugs with activity against influenza A viruses and the recognition of drug-resistant variants to these drugs prompted the following report by a select group of the global experts - members of the Neuraminidase Inhibitor Susceptibility Network - on the best use of the available drugs, both for prophylaxis and treatment. The editors of Antiviral Therapy are pleased to be able to provide this document in an expeditious manner.