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Ahead of Print -Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses - Volume 21, Number 3—March 2015 - Emerging Infectious Disease journal - CDC

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Ahead of Print -Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses - Volume 21, Number 3—March 2015 - Emerging Infectious Disease journal - CDC

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Volume 21, Number 3—March 2015


Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses

Jiangqin ZhaoComments to Author , Viswanath Ragupathy, Jikun Liu, Xue Wang, Sai Vikram Vemula, Haja Sittana El Mubarak, Zhiping Ye, Marie L. Landry, and Indira HewlettComments to Author 
Author affiliations: Food and Drug Administration, Silver Spring, Maryland, USA (J. Zhao, V. Ragupathy, J. Liu, X. Wang, S.V. Vemula, H.S. El Mubarak, Z. Ye, I. Hewlett)Yale University School of Medicine, New Haven, Connecticut, USA (M.L. Landry)


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.
Influenza A virus consists of 8 negative, single-stranded RNA segments encoding 11 proteins: polymerase basic 1 and 2 (PB1 and PB2); polymerase acidic (PA); hemagglutinin (HA); nucleoprotein (NP); neuraminidase (NA); matrix (M1/2); and nonstructural (NS1/2). Influenza A viruses are classified into 18 HA subtypes (H1–H18) and 11 NA subtypes (N1–N11), determined on the basis of the antigenic differences in the surface glycoproteins HA and NA (14). All known HA subtypes of influenza A virus are found in aquatic birds, and some, including H1, H2, H3, H5, H7, and H9, have been reported to infect humans (1,57). Direct transmission of avian influenza A virus subtypes H5N1, H7N2, H7N3, H7N7, H9N2, and H10N7 from domestic poultry to humans has been reported (813).
In early 2009, a novel swine-origin virus, designated influenza A(H1N1)pdm09 (pH1N1), emerged in Mexico and spread rapidly around the world, causing a global influenza pandemic (14,15). This virus was generated by multiple reassortment events over 10 years (16,17) and continued to circulate in humans after the initial pandemic period, replacing the previously circulating seasonal H1N1 viruses. Influenza A(H3N2) variant virus (H3N2v) isolated from humans in the United States in 2011 was also generated through reassortment originating from swine, avian, and human viruses, including the M gene from pH1N1 virus (18,19). More recently, a novel avian-origin influenza A(H7N9) virus capable of poultry-to-human transmission was identified in China (7;http://www.who.int/influenza/human_animal_interface/influenza_h7n9/140225_H7N9RA_for_web_20140306FM.pdf). Diagnosis of infection with this virus is difficult because infection does not kill infected poultry, but the virus may post a substantial risk for a human pandemic because of a lack of immunity in the general population (7). As these viruses demonstrate, reassortment of pH1N1 virus with other circulating seasonal strains can produce virulent variants that can be transmitted to and among humans and that could emerge as a future pandemic strain (15,20,21). Therefore, it is critical to determine whether transmitted viruses have pandemic potential in humans during the influenza season.
Multiple influenza strains are usually prevalent during an influenza season. Increasing global travel results in rapid spread of novel influenza viruses from one geographic region to another (13,22). Current approaches for screening and characterizing novel influenza viruses require many steps and multiple assays. A single test has not been available for simultaneous identification of newly emerging strains from known or unknown subtypes of influenza viruses and the characterization of unique virulence factors or putative antiviral resistance markers.
We previously described a method for detection of avian influenza A(H5N1) and swine-origin pH1N1 viruses that used a nanotechnology-based, PCR-free, whole-genome microarray assay (nanomicroarray) (23,24). In this article, we describe a new diagnostic platform for identification and characterization of subtypes of influenza A virus that uses nanomicroarray for screening and multiplex next-generation sequencing (NGS) for laboratory confirmation. We demonstrate that this platform enables accurate and simultaneous identification of multiple subtypes in a single sample. We used this platform to evaluate clinical nasopharyngeal swab specimens from patients with influenza-like illness that had tested positive for influenza virus to determine influenza virus subtype.

Thumbnail of 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 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...
Dr Zhao is a virologist and reviewer at the Food and Drug Administration, Silver Spring, Maryland. His research interests include new technologies and tools for diagnosis of infectious viral pathogens.


We are thankful to FDA Center for Biologics Evaluation and Research core facility staff for help with some NGS assays and oligosynthesis.
This work was funded through the FDA Center for Biologics Evaluation and Research intramural and Medical Countermeasures Initiative funds.
The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.


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Technical Appendix

Suggested citation for this article: Zhao J, Ragupathy V, Liu J, Wang X, Vemula SV, El Mubarak HS, et al. Nanomicroarray and multiplex next-generation sequencing for simultaneous identification and characterization of influenza viruses. Emerg Infect Dis [Internet]. 2015 Mar [date cited].http://dx.doi.org/10.3201/eid2103.141169
DOI: 10.3201/eid2103.141169

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