Research paperSubstitutions at H134 and in the 430-loop region in influenza B neuraminidases can confer reduced susceptibility to multiple neuraminidase inhibitors
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).
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Cited by (1)
Characterization of influenza B viruses with reduced susceptibility to influenza neuraminidase inhibitors
2022, Antiviral ResearchCitation 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.