Development and evaluation of a conventional RT-PCR for differentiating emerging influenza B/Victoria lineage viruses with hemagglutinin amino acid deletion from B/Yamagata lineage viruses

Background: Recent influenza B/Victoria lineage viruses contain amino acid deletions at positions 162 to 164 of the haemagglutinin (HA) protein. These amino acid deletions have affected the detection of B/Victoria lineage viruses by the lineage-specific conventional reverse-transcription polymerase chain reaction (RT-PCR) that was recommended by World Health Organization (WHO). Objectives: We aimed to develop and evaluate a novel lineage-specific RT-PCR for rapid differentiation of the contemporary B/Victoria lineage from B/Yamagata lineage viruses. Study Design: Primers of our in-house RT-PCR were designed to avoid amino acid positions 162 to 164 and to target conserved regions of the HA gene that are specific for B/Victoria and B/Yamagata lineage viruses. Our in-house RT-PCR and WHO RT-PCR were evaluated using influenza B positive clinical specimens or virus culture isolates. Influenza B virus lineage was confirmed by Sanger sequencing. Results: A total of 105 clinical specimens or virus culture isolates were retrieved, including 83 with B/Victoria lineage and 22 with B/Yamagata lineage viruses. Our in-house RT-PCR correctly identified B/Victoria lineage viruses in all 83 samples, including 82 samples with double or triple amino acid deletion in the HA protein. Conversely, the WHO lineage-specific conventional RT-PCR failed to detect any of the 82 samples with HA amino acid deletions. For the 22 samples with B/Yamagata lineage viruses, both RT-PCR assays have correctly identified B/Yamagata lineage in all samples. Conclusions: Our novel lineage-specific RT-PCR has successfully detected all contemporary B/Victoria lineage viruses with amino acid deletions in HA. This protocol is especially useful for laboratories without the equipment for real-time PCR.