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

full-text ►

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

Volume 21, Number 3—March 2015

Research

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

On This Page

• Materials and Methods
• Results
• Discussion
• Suggested Citation

Figures

• Figure 1
• Figure 2
• Figure 3
• Figure 4

Tables

• Table 1
• Table 2

Technical Appendicies

• Technical Appendix

Downloads

• RIS[TXT - 2 KB]

Jiangqin Zhao , Viswanath Ragupathy, Jikun Liu, Xue Wang, Sai Vikram Vemula, Haja Sittana El Mubarak, Zhiping Ye, Marie L. Landry, and Indira Hewlett 

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)

Suggested citation for this article

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.

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 (1–4). 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 (1,5–7). Direct transmission of avian influenza A virus subtypes H5N1, H7N2, H7N3, H7N7, H9N2, and H10N7 from domestic poultry to humans has been reported (8–13).

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.

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.

Acknowledgments

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.

References

1. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev. 1992;56:152–79.PubMed
2. Röhm C, Zhou N, Suss J, Mackenzie J, Webster RG. Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza A subtypes. Virology. 1996;217:508–16. DOIPubMed
3. Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol. 2005;79:2814–22. DOIPubMed
4. Tong S, Zhu X, Li Y, New world bats harbor diverse influenza A viruses. PLoS Pathog. 2013;9:e1003657. DOIPubMed
5. Horimoto T, Kawaoka Y. Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev. 2001;14:129–49. DOIPubMed
6. Hilleman MR. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine. 2002;20:3068–87. DOIPubMed
7. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med. 2013;368:1888–97.DOIPubMed
8. Arzey GG, Kirkland PD, Arzey KE, Frost M, Maywood P, Conaty S, Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia.Emerg Infect Dis. 2012;18:814–6. DOIPubMed
9. Cheng VC, Chan JF, Wen X, Wu WL, Que TL, Chen H, Infection of immunocompromised patients by avian H9N2 influenza A virus. J Infect.2011;62:394–9. DOIPubMed
10. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet. 2004;363:587–93. DOIPubMed
11. Ostrowsky B, Huang A, Terry W, Anton D, Brunagel B, Traynor L, Low pathogenic avian influenza A (H7N2) virus infection in immunocompromised adult, New York, USA, 2003. Emerg Infect Dis. 2012;18:1128–31. DOIPubMed
12. Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, Human illness from avian influenza H7N3, British Columbia. Emerg Infect Dis.2004;10:2196–9. DOIPubMed
13. Pabbaraju K, Tellier R, Wong S, Li Y, Bastien N, Tang JW, Full-genome analysis of avian influenza A(H5N1) virus from a human, North America, 2013.Emerg Infect Dis. 2014;20:887–91. DOIPubMed
14. Gallaher WR. Towards a sane and rational approach to management of influenza H1N1 2009. Virol J. 2009;6:51. DOIPubMed
15. Vijaykrishna D, Poon LL, Zhu HC, Ma SK, Li OT, Cheung CL, Reassortment of pandemic H1N1/2009 influenza A virus in swine. Science.2010;328:1529. DOIPubMed
16. Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. 2009;459:1122–5. DOIPubMed
17. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science. 2009;325:197–201. DOIPubMed
18. Lindstrom S, Garten R, Balish A, Shu B, Emery S, Berman L, Human infections with novel reassortant influenza A(H3N2)v viruses, United States, 2011. Emerg Infect Dis. 2012;18:834–7. DOIPubMed
19. Nelson MI, Vincent AL, Kitikoon P, Holmes EC, Gramer MR. Evolution of novel reassortant A/H3N2 influenza viruses in North American swine and humans, 2009–2011. J Virol. 2012;86:8872–8. DOIPubMed
20. Kitikoon P, Vincent AL, Gauger PC, Schlink SN, Bayles DO, Gramer MR, Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010–2011. J Virol. 2012;86:6804–14. DOIPubMed
21. Webby RJ, Webster RG. Emergence of influenza A viruses. Philos Trans R Soc Lond B Biol Sci. 2001;356:1817–28. DOIPubMed
22. Chang SY, Lin PH, Tsai JC, Hung CC, Chang SC. The first case of H7N9 influenza in Taiwan. Lancet. 2013;381:1621. DOIPubMed
23. Zhao J, Wang X, Ragupathy V, Zhang P, Tang W, Ye Z, Rapid detection and differentiation of swine-origin influenza a virus (H1N1/2009) from other seasonal influenza A viruses. Viruses. 2012;4:3012–9. DOIPubMed
24. Zhao J, Tang S, Storhoff J, Marla S, Bao YP, Wang X, Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay. BMC Biotechnol. 2010;10:74. DOIPubMed
25. Landry ML, Ferguson D. Cytospin-enhanced immunofluorescence and impact of sample quality on detection of novel swine origin (H1N1) influenza virus. J Clin Microbiol. 2010;48:957–9. DOIPubMed
26. Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses. J Virol. 2009;83:10309–13. DOIPubMed
27. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol.2001;146:2275–89. DOIPubMed
28. Treangen TJ, Salzberg SL. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet.2012;13:36–46.PubMed
29. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol.2007;24:1596–9. DOIPubMed
30. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet. 2013;381:1916–25. DOIPubMed
31. Yurovsky A, Moret BM. FluReF, an automated flu virus reassortment finder based on phylogenetic trees. BMC Genomics. 2011;12(Suppl 2):S3.DOIPubMed
32. Rabadan R, Levine AJ, Krasnitz M. Non-random reassortment in human influenza A viruses. Influenza Other Respir Viruses. 2008;2:9–22.
33. Lun AT, Wong JW, Downard KM. FluShuffle and FluResort: new algorithms to identify reassorted strains of the influenza virus by mass spectrometry. BMC Bioinformatics. 2012;13:208. DOIPubMed
34. Jonges M, Meijer A, Fouchier RA, Koch G, Li J, Pan JC, Guiding outbreak management by the use of influenza A(H7Nx) virus sequence analysis. Euro Surveill. 2013;18:20460 .PubMed
35. Munster VJ, de Wit E, van Riel D, Beyer WE, Rimmelzwaan GF, Osterhaus AD, The molecular basis of the pathogenicity of the Dutch highly pathogenic human influenza A H7N7 viruses. J Infect Dis. 2007;196:258–65. DOIPubMed
36. Mok CK, Lee HH, Lestra M, Nicholls JM, Chan MC, Sia SF, Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts. J Virol. 2014;88:3568–76. DOIPubMed
37. Hatta M, Gao P, Halfmann P, Kawaoka Y. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science. 2001;293:1840–2.DOIPubMed
38. Zaraket H, Saito R, Suzuki Y, Suzuki Y, Caperig-Dapat I, Dapat C, Genomic events contributing to the high prevalence of amantadine-resistant influenza A/H3N2. Antivir Ther. 2010;15:307–19. DOIPubMed
39. Liu Q, Lu L, Sun Z, Chen GW, Wen Y, Jiang S. Genomic signature and protein sequence analysis of a novel influenza A (H7N9) virus that causes an outbreak in humans in China. Microbes Infect. 2013;15:432–9. DOIPubMed
40. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterization of viral genome. Lancet. 2013;381:1916–25. DOIPubMed

Figures

• 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...
• 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...
• 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,...

Tables

• Table 1. Detection of influenza A viruses in nasopharyngeal swab samples collected from naturally infected patients, Connecticut, USA, 2012–13 influenza season
• Table 2. Summary of results from NGS data analysis for influenza A(H3N2) and A(H5N1) viruses obtained from the Centers for Disease Control and Prevention

Technical Appendix

• Technical Appendix. Detailed methods for the design of capture and intermediate oligonucleotides, testing of viral and clinical samples, reverse transcription PCR, and nanomicroarray printing and testing of samples.  596 KB

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