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

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

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

Abstract

BACKGROUND:

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

OBJECTIVE:

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

STUDY DESIGN:

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.

RESULTS:

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.

CONCLUSIONS:

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.

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