Development of a Real-Time RT-PCR Assay for Detecting H9 Subtype Avian Influenza Viruses

Abstract

H9N2 subtype avian influenza virus (AIV) is widely distributed in poultry in Asia, the Middle East, and Africa. If they were introduced into poultry in Japan, the rapid diagnosis of AIVs would be difficult because the current high pathogenicity avian influenza (HPAI) diagnosis system in Japan primarily targets H5 and H7 subtypes and could not identify H9 subtype AIVs. In this study, we compared five real-time RT-PCR methods under the same conditions using in vitro-transcribed RNAs from H9N2 AIV strains (n＝24) belonging to the Y439, G1, and Y280 lineages. The Saito method showed the highest detection efficiency, while reduced sensitivity was observed for one strain in the G1 lineage, possibly due to sequence mismatches within the forward primer binding region. To increase G1 strain detection efficiency, we redesigned the forward primers incorporating degenerate bases. The method using the redesigned primers successfully restored high sensitivity across all strains in three lineages and improved the overall robustness of detection. We also confirmed there was no cross-reactivity of the method using the redesigned primer with AIVs of other subtypes and live vaccine strains of other pathogens. Taken together, these results demonstrated that the method developed in this study provides a way to specifically and sensitively detect H9N2 AIVs, and it is expected to complement the AIV diagnosis system in Japan.