Nanomicroarray and multiplex next-generation sequencing for simultaneous identification and characterization of influenza viruses

Abstract

Conventional methods for detection and discrimination of influenza viruses are time consuming and labor intensive. We developed a diagnostic platform for simultaneous identification and characterization of influenza viruses that uses a combination of nanomicroarray for screening and multiplex next-generation sequencing (NGS) assays for laboratory confirmation. The nanomicroarray was developed to target hemagglutinin, neuraminidase, and matrix genes to identify influenza A and B viruses. PCR amplicons synthesized by using an adapted universal primer for all 8 gene segments of 9 influenza A subtypes were detected in the nanomicroarray and confirmed by the NGS assays. This platform can simultaneously detect and differentiate multiple influenza A subtypes in a single sample. Use of these methods as part of a new diagnostic algorithm for detection and confirmation of influenza infections may provide ongoing public health benefits by assisting with future epidemiologic studies and improving preparedness for potential influenza pandemics.

Figure 1

Nanomicroarray layout design for testing of samples for influenza A and B viruses. The microarray internal positive control capture is listed in Technical Appendix Table 1. The negative control is the printing buffer. M, matrix protein.

Figure 2

Portion of the microarray images for DNA oligonucleotides of influenza viruses after hybridization with PCR products. Lighter shades represent greater silver intensities for each gene. Typical nanomicroarray silver staining images represent the hits for specific types or subtypes indicated. The positive controls of influenza A and B (left panels) use PCR products amplified by pair-specific primes for matrix gene.

Figure 3

Phylogenetic analysis of the matrix (M) gene sequences obtained from nasopharyngeal swab samples from patients who had received a diagnosis of influenza in Connecticut, USA, during the 2012–13 influenza season (see Table 1). Analysis was performed by using the neighbor-joining module in MEGA (29) with the Kimura 2-parameter method. The reference subtypes were fetched from the Influenza Research Database (http://www.fludb.org) and used to construct the tree. Bootstrap values >70% are shown. The M genes identified in this study are indicated by black circles; reference M genes are indicated by black squares for influenza A(H3N2)v and black triangles for pandemic influenza A(H1N1) 2009 (pH1N1) virus. Scale bar indicates 2% genetic distance.

Figure 4

Diagnostic algorithm for identification of an unknown risk for influenza by using nanomicroarray and next-generation sequencing (NGS) assays. To determine the virus type for a suspected influenza virus infection, viral RNA is extracted from a patient sample and initially analyzed in nanomicroarray assay for screening and determining the influenza A and B viruses (1). Once a novel, emerging, or co-infected influenza A and B virus is found, universal reverse transcription PCR (RT-PCR) is performed to generate whole-genome mega-amplicons (2), which can then be retested on the nanomicroarray assay to confirm the initial finding (3) or sent to the central laboratory performing the NGS assay and data analysis for final sequence confirmation (4).