Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

Burnham, Andrew J.; Armstrong, Jianling; Webster, Robert G.; Govorkova, Elena A.

doi:10.1128/jvi.02473-14

Cited by 22 publications

(23 citation statements)

References 77 publications

(80 reference statements)

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“…We show that the MUT-Y273 virus had equivalent NA activity and expression to the WT-H273 virus, but delayed growth in cell culture. Previous experiments using a rg-H273Y virus showed that relative to the rg-WT, the rg-H273Y virus had significantly higher NA activity and surface expression, and superior replication kinetics in a competitive mixtures experiment in normal human bronchial epithelial cells (14, 22, 23). The discrepancy in these fitness outcomes between our 2015 H273Y virus and a 1998 rg-H273Y virus may be due to differences in viral background (16 amino acid differences).…”

Section: Discussionmentioning

confidence: 98%

“…A number of different NA substitutions at conserved amino acid positions (e.g. E117, D197, I221 and H273) have previously been described to confer reduced inhibition by the NAIs in vitro (8, 12-21), but the impact of these substitutions on enzyme function, virus replication or transmissibility, has only been assessed in a limited number of studies (14, 22, 23). The fitness of influenza B viruses with either the H273Y or D197N NA substitution is of particular interest as a number of viruses with either substitution have been recently found in patients in community settings who, unlike hospitalized or immunocompromised patients, do not typically receive NAI treatment (8, 9, 17, 18, 24).…”

Section: Introductionmentioning

confidence: 99%

“…The effect of H273Y NA substitutions in influenza B viruses has been previously studied using reverse genetics (rg) in the B/Yamanashi/166/98 virus background (15, 22, 23). To date, few studies have reported the effect of the H273Y or the D197N NA substitution in contemporary viruses, which is important because it has been shown that the fitness consequences of resistance-conferring mutations can vary due to the genetic background of the NA (33, 34).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

Farrukee

Alex

McCaw

et al. 2018

Preprint

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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 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 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 analyse within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had similar NA enzyme function relative to 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 variant with 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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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

Farrukee

Alex

McCaw

et al. 2018

Preprint

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“…Application of antiviral therapies is another approach to increase our readiness for pandemic outbreaks. Antivirals such as oseltamivir, which target viral processes, have shown utility, but drug resistant viruses can emerge [4]. Antivirals that target host processes important for the virus have potential to circumvent the development of resistance, and targeting of host processes has shown therapeutic promise for other viruses [5].…”

Section: Introductionmentioning

confidence: 99%

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication

Rupp

Locatelli

Grieser

et al. 2017

Virol J

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BackgroundThe essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication.MethodsInfluenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference.ResultsExogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell.ConclusionsThese results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.

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“…Novel IBV antivirals are needed. There are only two approved antivirals (oseltamivir and zanamivir) for treatment of pediatric influenza virus infections (26,27). Additionally, as with IAV, antiviral resistance is present in IBV isolates (28)(29)(30).…”

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confidence: 99%

Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies

Fulton

Palese

Heaton

2015

J Virol

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cInfluenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the development of both a replication-competent fluorescent influenza B reporter virus and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies. Influenza B virus (IBV) is a common human pathogen that causes yearly epidemics and significant disease in the pediatric population (1-4). Despite the importance of this virus, far less is known about IBV relative to influenza A virus (IAV). Research into IAV has been greatly enhanced by several replication-competent reporter viruses (5-17). These reporter viruses have been crucial for identifying host factors, understanding basic viral pathogenesis, screening for antiviral compounds, and characterizing broadly reactive antibodies (6)(7)(8)(9)(10)(12)(13)(14)(15)(16)(17)(18)(19). Lack of viruses encoding reporters has delayed similar progress for IBV.Development of a fluorescent influenza B reporter virus. We have previously published a description of an IAV reporter virus that contains the Gaussia luciferase gene directly behind the PB2 open reading frame (ORF) in segment 1 of A/Puerto Rico/8/1934 virus (PR8) (7). A 2A proteolytic cleavage site inserted between the PB2 ORF and the Gaussia luciferase gene allows for the cotranslational separation of the two proteins (20).Utilizing a similar approach, an influenza virus codon-optimized ORF of mNeonGreen (mNeon), a gene encoding a bright monomeric fluorescent protein (21), was cloned behind each of the three polymerase segments of the B/Yamagata/16/ 1988 (Ya88) virus (Fig. 1A). The reporter segments were rescued individually utilizing standard protocols (22-24) to generate the PB1 mNeon, PB2 mNeon, and PA mNeon viruses. Madin-Darby canine kidney (MDCK) cells were infected at a multiplicity of infection (MOI) of 0.5 for 18 h with the recombinant wild-type (rWT) Ya88 or one of the three reporter viruses (with all experiments carried out at 33°C). Tosyl phenylalanyl chloromethyl ketone (TPCK) trypsin, which is required for spread of viral progeny, was excluded from the infection medium. The cells were imaged or subjected to flow cytometry after being labeled with Hoechst stain (Thermo Scientific) or LIVE/DEAD stain (Life Technologies), respectively. By epifluorescence microscopy, the PB1 mNeon and PB2 mNeon viruses were bright and easily observed by 18 h postinfection, but the PA mNeon virus was less detectable (Fig. 1B to E). Flow cytometry (BD LSRIIA) recapitulated the microscopy results (Fig. 1F to I). Quantification of the flow cytometry data with FlowJo indicated that both the PB1 mNeon and PB2 mNeon viruses yielded the brightest signals ( Fig. 1J and K), and the PB1 mNeon virus was chosen for further study.The PB1 mNeon virus can rapidly monitor the growth of IBV in vi...

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Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

Cited by 22 publications

References 77 publications

Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication

Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies

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