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PLoS One. 2016 Sep 22;11(9):e0163175. doi: 10.1371/journal.pone.0163175. eCollection 2016.

Sensitive Detection and Simultaneous Discrimination of Influenza A and B Viruses in Nasopharyngeal Swabs in a Single Assay Using Next-Generation Sequencing-Based Diagnostics.

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DETTD/OBRR/CBER, Food and Drug Administration, Silver Spring, MD, 20993, United States of America.
DVP/OVRR/CBER, Food and Drug Administration, Silver Spring, MD, 20993, United States of America.
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, 06520, United States of America.


Reassortment of 2009 (H1N1) pandemic influenza virus (pdH1N1) with other strains may produce more virulent and pathogenic forms, detection and their rapid characterization is critical. In this study, we reported a "one-size-fits-all" approach using a next-generation sequencing (NGS) detection platform to extensively identify influenza viral genomes for diagnosis and determination of novel virulence and drug resistance markers. A de novo module and other bioinformatics tools were used to generate contiguous sequence and identify influenza types/subtypes. Of 162 archived influenza-positive patient specimens, 161(99.4%) were positive for either influenza A or B viruses determined using the NGS assay. Among these, 135(83.3%) were A(H3N2), 14(8.6%) were A(pdH1N1), 2(1.2%) were A(H3N2) and A(pdH1N1) virus co-infections and 10(6.2%) were influenza B viruses. Of the influenza A viruses, 66.7% of A(H3N2) viruses tested had a E627K mutation in the PB2 protein, and 87.8% of the influenza A viruses contained the S31N mutation in the M2 protein. Further studies demonstrated that the NGS assay could achieve a high level of sensitivity and reveal adequate genetic information for final laboratory confirmation. The current diagnostic platform allows for simultaneous identification of a broad range of influenza viruses, monitoring emerging influenza strains with pandemic potential that facilitating diagnostics and antiviral treatment in the clinical setting and protection of the public health.

Conflict of interest statement

The authors have declared that no competing interests exist.

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