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

Abstract

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.