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

Abstract

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.

Fig 1. Study profile.

Enrollment of 162 patients aged 20 months to 89 years with symptoms of influenza-like illness during February 19, 2011 to March 8, 2013 from over twenty cities and towns in Connecticut, and submitted to the Clinical Virology Laboratory (CVL) at Yale New Haven Hospital, New Haven, Connecticut prior to detect by universal RT-PCR and NGS assays at the Laboratory of Molecular Virology (LMV) in FDA. A total of 381 genome sequences identified from 55 specimens including A(H3N2), A(pdH1N1), and Influenza B viruses were submitted into GenBank. DFA, direct fluorescent antigen test; RT-qPCR, quantitative RT-PCR; NGS, next-generation sequencing.

Fig 2

(A) Agarose gel illustrating the sensitivity of the universal RT-PCR assay. The picture shows PCR mega-amplicons in 2% agarose gel representing the whole-genome segments of A/Viet Nam/1203/2004(H5N1) virus in dilution series, and detection of viral RNA in a dilution of 10⁻⁷ (Table 1). (B) Illustrating average depth of coverage (DOC). (C) average breadth of coverage (BOC) in NGS characterization of the influenza genome.