2 distinct influenza B lineages: B/Victoria/2/87-like (B/Vic) and B/Yamagata/16/88-like (B/Yam) co-circulate in the human population since 2008, however, a B/Vic deletion variant (B/Vic-del) with 2 amino acid deletions at positions 162 and 163 in the haemagglutinin (HA) emerged in the US in 2016. The B/Vic-del viruses are antigenically distinct from the wild-type B/Vic lineage viruses. While real-time RT-PCR (rRT-PCR) assays can detect and differentiate between B/Yam and B/Vic, none can identify the B/Vic-del viruses. Thus we aim to develop a rapid and robust assay based on pyrosequencing to distinguish amongst the 3 influenza B-HA groups.
A B-HA pyrosequencing assay was developed to detect and differentiate currently circulating influenza B viruses belonging to B/Yam, B/Vic and B/Vic-del groups. Specificity was validated using 56 influenza B-positive samples. Sensitivity was evaluated using 10-fold serial dilutions of RNAs from representative viruses of each B-HA group. These were compared to the rRT-PCR assay from CDC (Atlanta, USA).
We tested 106 original specimens and 90 isolates with both assays. The pyrosequencing assay successfully identified B/Yam, B/Vic and B/Vic-del viruses with 100% accuracy, but the CDC rRT-PCR could not differentiate the B/Vic-del viruses from the wild-type. Detection limit based on RNA copy numbers for the pyrosequencing assay was 10 copies for B/Yam and B/Vic, 100 copies for B/Vic-del viruses. In comparison, the CDC rRT-PCR assay required at least 100 copies to differentiate between B/Yam and B/Vic. Using this pyrosequencing assay, we detected the first B/Vic-del in Australia.
Overall, this pyrosequencing assay has high sensitivity and specificity in distinguishing B/Vic-del from wild-type B/Vic and B/Yam viruses in a rapid, high throughput single reaction. The test will help laboratories performing influenza surveillance and provide important information for influenza vaccine updates over the next few years.