Influenza B Viruses with Mutation in the Neuraminidase Active Site, North Carolina, USA, 2010–11

Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.

Oseltamivir is 1 of 2 antiviral medications available for the treatment of infl uenza B virus infections. We describe and characterize a cluster of infl uenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir. I nfl uenza B viruses are responsible for sporadic seasonal infl uenza illness and can be associated with severe illness and death. In the United States, there are 2 classes of antiviral drugs licensed by the Food and Drug Administration for treatment of infl uenza infections. The adamantanes are ineffective against infl uenza B viruses, which limits the available antiviral options to 2 neuraminidase inhibitors (NAIs), inhaled zanamivir and oral oseltamivir. Infl uenza B viruses seem to have reduced susceptibility to NAIs compared with infl uenza A viruses on the basis of neuraminidase inhibition (NAI) assays (1,2). Furthermore, in clinical studies, changes conferring either resistance or reduced susceptibility to NAIs have been identifi ed in the neuraminidase (NA) of infl uenza B viruses isolated from patients after treatment (3)(4)(5)(6). Although the use of an antiviral agent can lead to the development of drug resistance, infl uenza B viruses with a reduced NAI susceptibility have also been recovered from patients with no history of exposure (5,(7)(8)(9)(10). It is therefore plausible that such mutations may be naturally occurring within the NA of infl uenza B viruses.

The Study
During routine infl uenza antiviral susceptibility surveillance, an infl uenza B virus, B/North Carolina/11/2010, with reduced susceptibility to oseltamivir and the investigational NAI peramivir was detected by using the fl uorescent NAI assay based on IC 50 values (amount of NAI required to inhibit 50% of viral NA activity). According to the current algorithm, viruses with elevated IC 50 values, when compared with a drug-susceptible control reference virus, are further investigated by using either conventional sequencing or pyrosequencing. Sequence analysis for the NA gene of B/North Carolina/11/2010 showed a novel substitution, present as a mixed population, of isoleucine (I) to valine (V) at position 221 (B NA numbering corresponds to 222 in N2 NA amino acid numbering). A substitution of I to threonine (T) at 221 has previously been associated with reduced susceptibility to NAIs in infl uenza B viruses (1,5,9). Moreover, reduced susceptibility to oseltamivir has been reported in viruses with variation at the corresponding residue (223, N1 NA numbering) in the pandemic (H1N1) 2009 virus (11,12) and in infl uenza A/H5N1 (13) and A/ H3N2 viruses (14).
Subsequent fl uorescent NAI testing of isolates recovered during surveillance showed a cluster of 14 infl uenza B viruses from North Carolina with elevated oseltamivir IC 50 values compared with reference wildtype infl uenza B, wild-type pandemic (H1N1) 2009, and wild-type A(H3N2) viruses; a similar trend was observed for peramivir IC 50 values (Table 1). When comparing the pandemic (H1N1) 2009 virus with the oseltamivirresistance conferring H275Y substitution and an infl uenza A (H3N2) virus with the oseltamivir-resistance conferring E119V substitution, the North Carolina B viruses showed intermediate susceptibility ( Table 1). The infl uenza B virus carrying the R152K substitution was resistant to all NAIs compared with the infl uenza B viruses with I221V (Table  1). In the chemiluminescent NAI assay, the oseltamivir IC 50 values for the I221V variants were greater than that for the E119V infl uenza A (H3N2) virus variant, which has been associated with oseltamivir resistance (Table 1) (15). Pyrosequencing analysis showed I221V as well as wild-type (I221) in the propagated viruses used in the NAI assays. The presence of wild-type variants is likely to reduce IC 50 values.
A total of 258 infl uenza B virus isolates from domestic and foreign laboratories submitted to the Centers for Disease Control and Prevention for routine surveillance were screened for the I221V substitution by using singlenucleotide polymorphism (SNP) pyrosequencing analysis (10). All viruses were wild type at this position, with the exception of the 14 viruses from North Carolina with reduced susceptibility in the NAI assay ( Table 2). All 14 viruses were collected from patients in North Carolina during November 2010 through February 2011.
Because some susceptibility-altering NA mutations have been shown to arise from virus propagation in tissue culture (15), pyrosequencing analysis at position 221 in available matching clinical specimens was performed to rule out cell culture selection. The I221V substitution was identifi ed in the 9 available matching clinical specimens (

Conclusions
Although the NA change I221V has been seen among the N1 NA subtype of infl uenza A viruses (1,13), such a change has not been reported in infl uenza B viruses. Amino acid 221 is known to be a highly conserved residue of the NA enzyme active site. To date, all infl uenza B viruses with the I221V substitution appear to be limited geographically; however, monitoring is ongoing. Although oseltamivir IC 50 values obtained with the infl uenza B viruses carrying the I221V substitution are similar to those seen with infl uenza A(H3N2) viruses carrying the oseltamivir-resistance conferring substitution E119V (Table 1)