Genomic Signature-Based Identification of Influenza A Viruses Using RT-PCR/Electro-Spray Ionization Mass Spectrometry (ESI-MS) Technology

BACKGROUND: The 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 FINDINGS: Here, 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/SIGNIFICANCE: The 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.