Electrochemical Assay to Detect Influenza Viruses and Measure Drug Susceptibility

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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Drug susceptibility surveillance of influenza viruses circulating in the United States in 2011-2012: application of the WHO antiviral working group criteria

Influenza and Other Respiratory Viruses ◽

10.1111/irv.12215 ◽

2013 ◽

Vol 8 (2) ◽

pp. 258-265 ◽

Cited By ~ 24

Author(s):

Margaret Okomo-Adhiambo ◽

Ha T. Nguyen ◽

Anwar Abd Elal ◽

Katrina Sleeman ◽

Alicia M. Fry ◽

...

Keyword(s):

United States ◽

Working Group ◽

The United States ◽

Drug Susceptibility ◽

Influenza Viruses

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Influenza Project in Myanmar

Journal of Disaster Research ◽

10.20965/jdr.2014.p0842 ◽

2014 ◽

Vol 9 (5) ◽

pp. 842-847

Author(s):

Reiko Saito ◽

◽

Yadanar Kyaw ◽

Yi Yi Myint ◽

Clyde Dapat ◽

...

Keyword(s):

Influenza A ◽

Drug Susceptibility ◽

Influenza Viruses ◽

World Health ◽

Influenza Surveillance ◽

Influenza B ◽

Laboratory Capacity ◽

Influenza A H1n1 ◽

Resistant Strains ◽

Health Organization

The epidemiological study of influenza in Southeast Asia is limited. We surveyed influenza in Myanmar from 2007 to 2013. Nasopharyngeal swabs were collected from patients in the two cities of Yangon and Nay Pyi Taw. Samples were screened using rapid influenza diagnostic kits and identified by virus isolation. Isolates were characterized by cyclingprobe-based real-time PCR, drug susceptibility assay, and sequencing. Samples collected numbered 5,173, from which 1,686 influenza viruses were isolated during the seven-year study period. Of these, 187 strains were of seasonal influenza A(H1N1), 274 of influenza A(H1N1)pdm09, 791 of influenza A(H3N2), and 434 of influenza B. Interestingly, two zanamivir and amantadine-resistant strains each were detected in 2007 and 2008. These rare dual-resistant strains had a Q136K mutation in the NA protein and S31N substitution in the M2 protein. Our collaboration raised the influenza surveillance laboratory capacity in Myanmar and led Yangon’s National Health Laboratory – one of the nation’s leading research institutes – to being designated a National Influenza Center by the World Health Organization.

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Plaque inhibition assay for drug susceptibility testing of influenza viruses.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.17.5.865 ◽

1980 ◽

Vol 17 (5) ◽

pp. 865-870 ◽

Cited By ~ 147

Author(s):

F G Hayden ◽

K M Cote ◽

R G Douglas

Keyword(s):

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Influenza Viruses ◽

Inhibition Assay

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Evaluation of the antiviral drug susceptibility of influenza viruses in Italy from 2004/05 to 2009/10 epidemics and from the recent 2009 pandemic

Antiviral Research ◽

10.1016/j.antiviral.2011.04.003 ◽

2011 ◽

Vol 90 (3) ◽

pp. 205-212 ◽

Cited By ~ 11

Author(s):

Simona Puzelli ◽

Marzia Facchini ◽

Angela Di Martino ◽

Concetta Fabiani ◽

Angie Lackenby ◽

...

Keyword(s):

Antiviral Drug ◽

Drug Susceptibility ◽

Influenza Viruses ◽

Antiviral Drug Susceptibility

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Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916825117 ◽

2020 ◽

Vol 117 (15) ◽

pp. 8593-8601 ◽

Cited By ~ 8

Author(s):

Jeremy C. Jones ◽

Philippe Noriel Q. Pascua ◽

Thomas P. Fabrizio ◽

Bindumadhav M. Marathe ◽

Patrick Seiler ◽

...

Keyword(s):

Influenza A ◽

Clinical Trial Data ◽

Drug Susceptibility ◽

Influenza Viruses ◽

Endonuclease Activity ◽

Influenza A Viruses ◽

Complex Activity ◽

Minimal Impact

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00334-09 ◽

2009 ◽

Vol 53 (10) ◽

pp. 4433-4440 ◽

Cited By ~ 74

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.

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