Detection of an Influenza B Virus Strain with Reduced Susceptibility to Neuraminidase Inhibitor Drugs

LETTER

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.

Table 1.

Drug susceptibility and genotype of influenza B/Ontario/RV75-11/2010 virus^a

Virus strain	Passage no. of isolate or description	NA change	Oseltamivir		Zanamivir
			IC50 (nM)	Fold increaseb	IC50 (nM)	Fold increaseb
B/Ontario/RV75-11/2010	1	G109E	19.87 ± 0.41	7	23.11 ± 1.57	6
B/Ontario/RV75-11/2010	2	G109E	39.99 ± 5.99	13	69.29 ± 5.88	18
B/Ontario/RV535/2011	1	G109	3.60	1.2	6.37	1.7
Controls
B/Hong Kong/45/2005	Susceptible control	None (WTc)	3.00 ± 0.47	1	3.83 ± 0.34	1
B/Hong Kong/36/2005	Resistant control	R371K	633.33 ± 185.59		211.93 ± 89.38

^aThe susceptibility to oseltamivir and zanamivir was determined by a chemiluminescence neuraminidase inhibition assay, using the NA-Star kit (Applied Biosystems Inc.). Neuraminidase (NA) inhibition was assayed with viruses standardized to equivalent NA enzyme activity and incubated with NA inhibitor (NAI) at concentrations of 0.0316 nM to 1,000 nM. The 50% inhibitory concentration (IC₅₀) was calculated by plotting the percentage of inhibition of NA activity against the inhibitor concentration, using GraphPad PRISM 4 software for curve fitting.

^bFold increase in the IC₅₀ compared to the values for the wild-type control B/Hong Kong/36/2005 for oseltamivir and zanamivir.

^cWT, wild type.

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.