Nanopore sequencing reveals novel targets for the diagnosis and surveillance of human and avian influenza A virus

Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for IAV RT-PCR assay using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5′ and 3′ ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well-matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed similar or lower limit of detection than M gene RT-PCR, and achieved 100% sensitivity and specificity in the detection of A(H1N1) and A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV M gene RT-PCR conversion in sequentially-collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates identification of novel diagnostic targets.