Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.

Varough M Deyde,Heather Matthews,Rebecca J Garten,Matthew S Reed,Pavel Svoboda,Alexander I Klimov,Rangarajan Sampath,Jan Pohl,Christopher A Myers,Patrick J Blair,Christian Massire,Larisa V Gubareva

doi:10.1371/journal.pone.0013293

Abstract

BackgroundThe emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza “core” genes. Combination of the BC signatures represents a “genomic print” of an influenza A virus.Methodology/Principal FindingsHere, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with “core” genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between “core” genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir.Conclusions/SignificanceThe RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.

Highlights

Influenza A viruses are important respiratory pathogens that cause annual epidemics and occasional pandemics
Based on our previous experience with the assay using seasonal influenza viruses, and its reported performance in analysis of influenza A(H3N2) [25], the main goals of this study were to investigate: (A) whether the RT-PCR/ElectroSpray Ionization Mass Spectrometry (ESI-MS) assay can be applied for identification of the H1N1 pandemic influenza A virus (H1N1pdm) viruses; (B) whether this method could be helpful in the analysis of A(H3N2) virus lineages from the 2006-07 to 2008-09 seasons; (C) whether the assay could identify and distinguish between the major lineages of seasonal H1N1 viruses (2B and 2C clades), including the dual oseltamivir and adamantane-resistant viruses; and lastly (D) whether wild type viruses could be readily distinguished from the live attenuated vaccines (LAIV) using this assay
The criterion used for future identification of the H1N1pdm viruses was based on the detection of base composition (BC) signatures identical to those of A/California/04/2009 in at least five of the six targets in the ‘‘core’’ genes

Summary

Introduction

Influenza A viruses are important respiratory pathogens that cause annual epidemics and occasional pandemics. Influenza A virus evolution is considered to be predominantly driven by host-mediated selective pressure that leads to amino acid replacements at key antigenic sites in the HA, a mechanism known as antigenic drift [4,5]. Another mechanism of influenza virus evolution, antigenic shift, which occurs at a lower frequency, involves the replacement of HA and/or NA with new antigenic virus subtypes that have not circulated in humans viruses for a long time [6]. Combination of the BC signatures represents a ‘‘genomic print’’ of an influenza A virus

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Conclusion