scholarly journals In Vitro Selection and Characterization of Influenza A (A/N9) Virus Variants Resistant to a Novel Neuraminidase Inhibitor, A-315675

2002 ◽  
Vol 76 (11) ◽  
pp. 5380-5386 ◽  
Author(s):  
Akhteruzzaman Molla ◽  
Warren Kati ◽  
Robert Carrick ◽  
Kevin Steffy ◽  
Yan Shi ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Cross Resistance ◽  
Virus Infections ◽  
Wild Type ◽  
Oseltamivir Carboxylate ◽  
Ha Gene ◽  
Na Gene

ABSTRACT With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 μM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 μM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.

scholarly journals Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

2016 ◽  
Vol 90 (7) ◽  
pp. 3684-3693 ◽  
Author(s):  
Léa Meyer ◽  
Alix Sausset ◽  
Laura Sedano ◽  
Bruno Da Costa ◽  
Ronan Le Goffic ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

scholarly journals Effect of Oseltamivir Carboxylate Consumption on Emergence of Drug-Resistant H5N2 Avian Influenza Virus in Mallard Ducks

2013 ◽  
Vol 57 (5) ◽  
pp. 2171-2181 ◽  
Author(s):  
Jenna E. Achenbach ◽  
Richard A. Bowen
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Resistance Mutation ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Human Populations ◽  
Wild Type ◽  
Oseltamivir Carboxylate

ABSTRACTOseltamivir carboxylate (OC) has been detected in environmental waters at various levels during recent influenza seasons in humans, reflecting levels of usage and stability of this drug. In consideration of the role of waterfowl as hosts for influenza viruses that may contribute to human infections, we evaluated the effect of consumption of low doses of OC on development of oseltamivir-resistant influenza virus mutants in mallard ducks (Anas platyrhynchos) infected with two different low-pathogenic (LP) H5N2 avian influenza viruses (AIV). We detected development of virus variants carrying a known molecular marker of oseltamivir resistance (neuraminidase E119V) in 4 out of 6 mallards infected with A/Mallard/Minnesota/182742/1998 (H5N2) and exposed to 1,000 ng/liter OC. The mutation first appeared as a minor population on days 5 to 6 and was the dominant genotype on days 6 to 8. Oseltamivir-resistant mutations were not detected in virus from ducks not exposed to the drug or in ducks infected with a second strain of virus and similarly exposed to OC. Virus isolates carrying the E119V mutation displayedin vitroreplication kinetics similar to those of the wild-type virus, butin vivo, the E119V virus rapidly reverted back to wild type in the absence of OC, and only the wild-type parental strain was transmitted to contact ducks. These results indicate that consumption by wild waterfowl of OC in drinking water may promote selection of the E119V resistance mutation in some strains of H5N2 AIV that could contribute to viruses infecting human populations.

scholarly journals Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James D. Allen ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Virus Infections ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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 Identification of GS 4104 as an Orally Bioavailable Prodrug of the Influenza Virus Neuraminidase Inhibitor GS 4071

1998 ◽  
Vol 42 (3) ◽  
pp. 647-653 ◽  
Author(s):  
Weixing Li ◽  
Paul A. Escarpe ◽  
Eugene J. Eisenberg ◽  
Kenneth C. Cundy ◽  
Clive Sweet ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Ethyl Ester ◽  
Oral Bioavailability ◽  
Influenza A ◽  
Neuraminidase Inhibitor ◽  
Virus Infections ◽  
Neuraminidase Activity ◽  
Orally Bioavailable ◽  
Influenza Virus Neuraminidase

ABSTRACT GS 4071 is a potent carbocyclic transition-state analog inhibitor of influenza virus neuraminidase with activity against both influenza A and B viruses in vitro. GS 4116, the guanidino analog of GS 4071, is a 10-fold more potent inhibitor of influenza virus replication in tissue culture than GS 4071. In this study we determined the oral bioavailabilities of GS 4071, GS 4116, and their respective ethyl ester prodrugs in rats. Both parent compounds and the prodrug of the guanidino analog exhibited poor oral bioavailability (2 to 4%) and low peak concentrations in plasma (C maxs; C max<0.06 μg/ml). In contrast, GS 4104, the ethyl ester prodrug of GS 4071, exhibited good oral bioavailability (35%) as GS 4071 and high C maxs of GS 4071 (Cmax = 0.47 μg/ml) which are 150 times the concentration necessary to inhibit influenza virus neuraminidase activity by 90%. The bioavailability of GS 4104 as GS 4071 was also determined in mice (30%), ferrets (11%), and dogs (73%). The plasma of all four species exhibited high, sustained concentrations of GS 4071 such that at 12 h postdosing the concentrations of GS 4071 in plasma exceeded those necessary to inhibit influenza virus neuraminidase activity by 90%. These results demonstrate that GS 4104 is an orally bioavailable prodrug of GS 4071 in animals and that it has the potential to be an oral agent for the prevention and treatment of influenza A and B virus infections in humans.

scholarly journals The mechanism of resistance to favipiravir in influenza

2018 ◽  
Vol 115 (45) ◽  
pp. 11613-11618 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Aartjan J. W. te Velthuis ◽  
Robert A. Fletcher ◽  
Pinky Langat ◽  
Maria Zambon ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
H1n1 Influenza ◽  
Viral Fitness ◽  
Virus Infections ◽  
Virus Rna ◽  
Evolution Of Resistance ◽  
Emergence Of Resistance

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

scholarly journals Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

2004 ◽  
Vol 11 (1) ◽  
pp. 21-28 ◽  
Author(s):  
James J. McSharry ◽  
Ann C. McDonough ◽  
Betty A. Olson ◽  
George L. Drusano
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Virus Yield ◽  
Drug Resistant ◽  
Wild Type ◽  
Vitro Assay ◽  
Drug Concentrations ◽  
Clinical Resistance ◽  
Na Gene

ABSTRACT A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.

scholarly journals TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

2005 ◽  
Vol 79 (20) ◽  
pp. 12773-12782 ◽  
Author(s):  
Johan Vingerhoets ◽  
Hilde Azijn ◽  
Els Fransen ◽  
Inky De Baere ◽  
Liesbet Smeulders ◽  
...  
Keyword(s):  
Wild Type Virus ◽  
Cross Resistance ◽  
Resistant Virus ◽  
Wild Type ◽  
Genetic Barrier ◽  
Development Of Resistance ◽  
Selection Experiments ◽  
The Impact ◽  
Hiv 1

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

scholarly journals Protective Role of Combined Polyphenols and Micronutrients against Influenza A Virus and SARS-CoV-2 Infection In Vitro

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1721
Author(s):  
Marta De Angelis ◽  
David Della-Morte ◽  
Gabriele Buttinelli ◽  
Angela Di Martino ◽  
Francesca Pacifici ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Viral Proteins ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Protective Role ◽  
Virus Infections ◽  
Infected Cells ◽  
Respiratory Virus Infections

Polyphenols have been widely studied for their antiviral effect against respiratory virus infections. Among these, resveratrol (RV) has been demonstrated to inhibit influenza virus replication and more recently, it has been tested together with pterostilbene against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present work, we evaluated the antiviral activity of polydatin, an RV precursor, and a mixture of polyphenols and other micronutrients, named A5+, against influenza virus and SARS-CoV-2 infections. To this end, we infected Vero E6 cells and analyzed the replication of both respiratory viruses in terms of viral proteins synthesis and viral titration. We demonstrated that A5+ showed a higher efficacy in inhibiting both influenza virus and SARS-CoV-2 infections compared to polydatin treatment alone. Indeed, post infection treatment significantly decreased viral proteins expression and viral release, probably by interfering with any step of virus replicative cycle. Intriguingly, A5+ treatment strongly reduced IL-6 cytokine production in influenza virus-infected cells, suggesting its potential anti-inflammatory properties during the infection. Overall, these results demonstrate the synergic and innovative antiviral efficacy of A5+ mixture, although further studies are needed to clarify the mechanisms underlying its inhibitory effect.

scholarly journals In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

2010 ◽  
Vol 54 (8) ◽  
pp. 3442-3450 ◽  
Author(s):  
Ashley N. Brown ◽  
James J. McSharry ◽  
Qingmei Weng ◽  
Elizabeth M. Driebe ◽  
David M. Engelthaler ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Infection Model ◽  
Virus Infections ◽  
Drug Pressure

ABSTRACT One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

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