Elsevier

Antiviral Research

Volume 182, October 2020, 104895
Antiviral Research

Research paper
Substitutions at H134 and in the 430-loop region in influenza B neuraminidases can confer reduced susceptibility to multiple neuraminidase inhibitors

https://doi.org/10.1016/j.antiviral.2020.104895Get rights and content

Highlights

  • Two influenza B viruses from surveillance samples contained some novel neuraminidase inhibitor resistant quasispecies.

  • Plaques with wild type and H134Y neuraminidases were isolated from both viruses and a novel H134R mutant from one virus.

  • Several substitutions were located in the 430-loop region of the neuraminidase, including H431R, T436P and W438R.

  • None of the substitutions was in the active site, but most led to reduced susceptibility to at least two inhibitors.

  • Substitutions would affect flexibility of the neuraminidase 150 and 430-loops, confirming their importance for influenza B.

Abstract

With the introduction of the influenza specific neuraminidase inhibitors (NAIs) in 1999, there were concerns about the emergence and spread of resistant viruses in the community setting. Surveillance and testing of community isolates for their susceptibility to the NAIs was initially carried out by the Neuraminidase Inhibitor Susceptibility Network (NISN) and has subsequently been taken on by the global WHO influenza network laboratories. During the NISN surveillance, we identified two Yamagata lineage influenza B viruses with amino acid substitutions of H134Y (B/Auckland/2/2001) or W438R (B/Yokohama/12/2005) which had slightly elevated IC50 values for zanamivir and/or oseltamivir, but not sufficiently to be characterized as mild outliers at the time. As it has now been well demonstrated that mixed populations can mask the true magnitude of resistance of a mutant, we re-examined both of these isolates by plaque purification to see if the true susceptibilities were being masked due to mixed populations. Results confirmed that the B/Auckland isolate contained both wild type and H134Y mutant populations, with mutant IC50 values > 250 nM for both oseltamivir and peramivir in the enzyme inhibition assay. The B/Yokohama isolate also contained both wild type and W438R mutant populations, the latter now demonstrating IC50 values > 400 nM for zanamivir, oseltamivir and peramivir. In addition, plaque purification of the B/Yokohama isolate identified viruses with other single neuraminidase substitutions H134Y, H134R, H431R, or T436P. H134R and H431R viruses had IC50 values > 400 nM and >250 nM respectively against all three NAIs. All changes conferred much greater resistance to peramivir than to zanamivir, and less to oseltamivir, and affected the kinetics of binding and dissociation of the NAIs. Most affected affinity (Km) for the MUNANA substrate, but some had decreased while others had increased affinity. Despite resistance in the enzyme assay, no reduced susceptibility was seen in plaque reduction assays in MDCK cells for any of the mutant viruses. None of these substitutions was in the active site. Modelling suggests that these substitutions affect the 150 and 430-loop regions described for influenza A NAs, suggesting they may also be important for substrate and inhibitor binding for influenza B NAs.

Introduction

With the introduction of the influenza specific neuraminidase inhibitors (NAIs) there were concerns about the emergence and spread of resistant viruses in the community setting. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established in 1999 to carry out surveillance and facilitate testing of the susceptibility of viruses from within the community. As part of this screening, we identified the B/Auckland/2/2001 (Monto et al., 2006) and B/Yokohama/12/2005 (Tashiro et al., 2009) viruses as having slightly elevated IC50 values in the enzyme inhibition assay, but not high enough to consider them statistically as mild outliers. There are two lineages of influenza Bs circulating, and comparison to B/Hong Kong/330/2001 (B/Victoria/2/1987 lineage) and B/Hong Kong/557/2000 (B/Yamagata/16/1988 lineage) (Shaw et al., 2002) sequences showed the NA of both viruses belonged to the B/Yamagata lineage. Sequence analysis revealed a W438R substitution in the neuraminidase (NA) of the B/Yokahama virus (Genbank JF733877, M Tashiro, 2011) and an H134Y in the B/Auckland virus NA (Genbank JF972552, M Zambon, 2011) (influenza B numbering). Neither of these changes was known to confer reduced NAI susceptibility at the time. There were six additional differences reported between the NAs of the B/Yokohama and B/Auckland viruses I49T, V63A, A68T, T125K, K186R and P244S.

The NA active site consists of an inner shell of eight highly conserved residues, which interact with the sialic acid substrate, (R118, D151, R152, R224, E276, R292, R371, Y406). In addition 11 highly conserved framework residues form an outer shell, (E119, R156, W178, S179, D198, I222, E227, H274, E277, N294, E425) (N2 numbering) (Burmeister et al., 1992; Colman et al., 1983, 1993). These do not contact the sialic acid, but play an important structural role. Most resistance to the NAIs in influenza B NAs occurs primarily due to substitutions in these conserved residues, e.g. E119, R152, D198, I222, H274, R292, N294 (N2 numbering) (Burnham et al., 2014; WHO, 2018) although some non-active site substitutions conferring high levels of resistance in influenza B NAs have also been reported e.g. H134N, G104E (B numbering) (Gubareva et al., 2017; WHO, 2018).

We previously reported that mixed populations complicated the apparent NAI susceptibility in other samples from NISN screening (Mohr et al., 2011; Hurt et al., 2006; McKimm-Breschkin et al., 2013), and mixed wild type and mutant populations masked the true level of resistance of an R292K substitution in an H7N9 isolate (Yen et al., 2013). We therefore re-examined these two influenza B stocks by plaque purification to determine if they also contained mixed populations with wild type viruses masking the real magnitude of resistance of these mutant viruses that could be of clinical concern if seen in a patient undergoing treatment with an NAI. Plaque purification identified both virus samples contained mixed wild type and mutant populations, including ones with the original substitutions, with much higher levels of resistance than detected in the original assays. In addition, other highly resistant viruses with novel mutations were isolated.

Section snippets

Cells and viruses

Madin Darby canine kidney cells (MDCK) were grown in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) with 7.5% fetal calf serum and antibiotics as previously described (McKimm-Breschkin et al., 2013). Medium for amplification of viruses was a 1:1 mix of minimal essential medium (MEM) and Leibovitz L15, supplemented with 1 μg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin (Worthington, USA). Viruses were plaqued in MDCK cells with an overlay of DMEM/F12

Characterization of plaques

As both ViroMed virus stocks contained mixed plaque morphologies, our hypothesis was that mixed plaque phenotypes may have obscured the true magnitude of resistance seen by ViroMed. We therefore carried out plaque to plaque purification, picking and amplifying several plaques at each passage for NAI testing. This was repeated until amplified progeny of each plaque type had the same morphology and NAI phenotype, confirming homogeneity. Final plaques were amplified once.

The ViroMed B/Auckland

Discussion

Two viruses B/Auckland/2/2001 and B/Yokahama/12/2005 were previously identified in surveillance of community samples, with slightly reduced NAI susceptibility, but not sufficiently reduced to classify them as resistant (Monto et al., 2006; Tashiro et al., 2009). Sequencing revealed an H134Y or W438R substitution respectively, neither of which was in the active site, nor was known to affect NAI sensitivity at that time. Deep sequencing of influenza viruses has become widespread, providing

Declaration of competing interest

None.

Acknowledgements

This work was partly funded through a grant from GlaxoSmithKline (UK). They had no role in interpretation of data or the preparation of the manuscript. The ACDP Facility is supported by the Australian Government Department of Agriculture and Water Resources and the National Collaborative Research Infrastructure Strategy (NCRIS).

References (41)

  • R.E. Amaro et al.

    Remarkable loop flexibility in avian influenza N1 and its implications for antiviral drug design

    J. Am. Chem. Soc.

    (2007)
  • T. Baranovich et al.

    Antiviral drug-resistant influenza B viruses carrying H134N substitution in neuraminidase, Laos, February 2016

    Emerg. Infect. Dis.

    (2017)
  • S. Barrett et al.

    Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses

    PloS One

    (2011)
  • W.P. Burmeister et al.

    The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid

    EMBO J.

    (1992)
  • A.J. Burnham et al.

    Fitness costs for Influenza B viruses carrying neuraminidase inhibitor-resistant substitutions: underscoring the importance of E119A and H274Y

    Antimicrob. Agents Chemother.

    (2014)
  • P.M. Colman et al.

    Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase

    J. Virol.

    (1993)
  • P.M. Colman et al.

    Structure of the catalytic and antigenic sites in influenza virus neuraminidase

    Nature

    (1983)
  • V. Escuret et al.

    A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses

    J. Infect. Dis.

    (2014)
  • R. Farrukee et al.

    Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility

    J. Antimicrob. Chemother.

    (2015)
  • E. Feng et al.

    Structure-based design and synthesis of C-1- and C-4-modified analogs of zanamivir as neuraminidase inhibitors

    J. Med. Chem.

    (2013)
  • Cited by (1)

    • Characterization of influenza B viruses with reduced susceptibility to influenza neuraminidase inhibitors

      2022, Antiviral Research
      Citation Excerpt :

      Interestingly, despite the increases in the IC50 for peramivir and zanamivir in RG-B/Brisbane/27/2016, the presence of arginine at 439 appeared to reduce the oseltamivir IC50 to 1.27 nM (versus 13.96 nM) (Table 2). This increased sensitivity of a virus to one NAI (when compared to the wild type virus) whilst reducing its sensitivity to other NAIs, has also recently reported in another study (Mohr et al., 2020). Of these five viruses, the only clinical sample that was available for sequencing was B/Wellington/111/2019, in which the NA A200T was also seen.

    1

    Present address: The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, 3052, Australia.

    2

    Present Address: Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, 792 Elizabeth Street, Parkville, Victoria, 3000 Australia.

    View full text