Detection of antiviral resistance and genetic lineage markers in influenza B virus neuraminidase using pyrosequencing

We report here the design of a pyrosequencing approach for the detection of molecular markers of resistance to the neuraminidase inhibitors zanamivir and oseltamivir in influenza viruses of type B. Primers were designed to analyze the sequences at eight amino acid positions E119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering) in the neuraminidase (NA) which have been previously found to be associated with resistance or reduced susceptibility to oseltamivir and/or zanamivir in the NA inhibition assay. In addition, the designed primers could be utilized to the distinguish between the NAs of influenza B viruses from the two major lineages (Victoria and Yamagata) that have co-circulated globally in recent years, thus providing a valuable tool for virus strain surveillance.