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J Clin Virol. 2013 Sep;58(1):94-9. doi: 10.1016/j.jcv.2013.04.018. Epub 2013 May 18.

The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses.

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WHO Collaborating Centre for Reference and Research on Influenza, 10 Wreckyn Street, North Melbourne, VIC 3051, Australia.



Influenza B viruses belong to two antigenically and genetically distinct lineages which co-circulate in varying proportions in many countries.


To develop simple, rapid, accurate and robust methods to detect and differentiate currently circulating B-lineage viruses in respiratory samples and virus isolates.


Haemagglutinin (HA) gene sequences from more than 6300 influenza B strains were analysed to identify signature sequences that could be used to distinguish between B-lineages and sublineages.


Pyrosequencing and a real time PCR assays were developed to detect the major B-lineages (B/Victoria/2/87 or B/Yamagata/16/88) and pyrosequencing for a unique mutation was used to further differentiate the B/Yamagata viruses into two currently co-circulating subgroups. More than 300 influenza virus-containing samples, including original specimens, cell and egg grown viruses, were tested with a 100% accuracy. Furthermore, when the same PCR primers were used in an rRT-PCR assay, the two lineages could be differentiated by their distinct ranges of melting temperature with an overall accuracy of 99% for 158 samples tested.


These new pyrosequencing and rRT-PCR methods have the potential to aid the rapid identification of influenza B-lineages for surveillance purposes and to increase the available data for bi-annual selection of viruses for updating influenza vaccines.


B-lineages; HA; Influenza B; MDCK; Madin-Darby canine kidney; NA; Pyrosequencing; Real-time RT-PCR; T(m); haemagglutinin; melting temperature; neuraminidase; rRT-PCR; real-time reverse transcriptase polymerase chain reaction

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