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The neuraminidase inhibitors (NAIs) oseltamivir and zanamivir have played an essential role in the prophylaxis and treatment of influenza. The residues forming the NA active sites are conserved among influenza A and B viruses (3). Conserved residues are in direct contact with the substrate or provide structural framework for the functional residues. There have been reports of in vivo resistance for influenza B viruses (4, 7). Here we report the isolation of a novel influenza B virus with reduced sensitivity to NAIs.
On 22 December 2010, an 87-year-old woman presented to a hospital in Ontario, Canada, with an influenza-like illness. Her symptoms began on 19 December 2010. She was admitted to the hospital and treated with oseltamivir for 5 days (75 mg twice daily), making an uneventful recovery. Influenza B virus was detected in a nasopharyngeal swab collected on 22 December 2010. The specimen was cultured in rhesus monkey kidney cells, and the isolate was designated B/Ontario/RV75-11/2010. The susceptibility of B/Ontario/RV75-11/2010 to NAIs was determined by a chemiluminescence neuraminidase inhibition assay. The 50% inhibitory concentrations (IC50s) for B/Ontario/RV75-11/2010 showed a 7- to 13-fold increase and a 6- to 18-fold increase compared to the values for the wild-type control B/Hong Kong/36/2005 for oseltamivir and zanamivir, respectively (Table 1). Specimen collection and drug treatment initiation occurred on the same day, indicating that the reduced sensitivity may have occurred naturally.
Sequencing of the NA gene showed a G109E substitution and a N340D substitution compared to the reference strain B/Brisbane/60/2008. The N340D substitution has been found in NAI-susceptible strains of influenza B virus circulating in Canada. In contrast, the G109E substitution is unique to B/Ontario/RV75-11/2010. To determine whether the G109E mutation was responsible for the reduced susceptibility to NAIs, we tested another Canadian isolate with an NA sequence identical to that of B/Ontario/RV75-11/2010 except for the G109E mutation. B/Ontario/RV535/2011 was susceptible to oseltamivir and zanamivir (Table 1). To our knowledge, this is the first report linking a change at residue 109 to reduced susceptibility to NAIs. The mechanism by which this change leads to reduced susceptibility to NAIs is unknown. Residue 109 is not one of the highly conserved residues that form the NA active site. However, it is located near residue R118 that interacts with sialic acid and E119 that provides structural framework for the active site (1). It has been reported that substitutions in NA at positions that confer resistance to NAIs may compromise enzyme function and result in reduced enzyme stability (5, 6), instability of the NA tetramer (2), or a change in the optimum pH for NA activity (5). Further research is needed to determine the mechanism by which the G109E substitution alters susceptibility to NAIs.
Since the patient recovered without complication, the clinical significance of the G109E substitution may be limited but remains to be determined. The recovery of influenza B virus with the new G109E substitution which affects susceptibility to two drugs available for treatment of influenza B virus infections highlights the importance of monitoring NAI susceptibility using functional assays.
This work was supported by the Ontario Ministry of Health, the Public Health Agency of Canada, and the Centers for Disease Control and Prevention. Jonathan B. Gubbay received funding from GlaxoSmithKline and Hoffman La Roche to study resistance in influenza viruses.
The findings and conclusions of the report are those of the authors and do not necessarily represent the views of the funding agency, the Centers for Disease Control and Prevention (CDC). We do not have a commercial or other association that might pose a conflict of interest.
Published ahead of print on 7 September 2011.
Nathalie Bastien, National Microbiology Laboratory
Public Health Agency of Canada
Winnipeg, Manitoba, Canada.
Jonathan B. Gubbay, Ontario Agency for Health Protection and Promotion
Public Health Laboratories
Toronto, Ontario, Canada.
David Richardson, William Osler Health System
Brampton, Ontario, Canada.
Larisa Gubareva, Influenza Division
National Center for Immunization and Respiratory Diseases
Centers for Disease Control and Prevention
Yan Li, National Microbiology Laboratory
Canadian Science Center for Human and Animal Health
1015 Arlington Street
Winnipeg, Manitoba, Canada R3E 3R2.