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

Abstract

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.

Figure 1. RT-PCR/ESI-MS identified initial H1N1pdm strain as unusual virus with genome components of swine, human, and avian origin.

RNA from clinical specimens was prepared from one of the first cases of the 2009 pandemic and tested using the RT-PCR/ESI-MS assay. BC signatures did not match any of the known reference genomic signatures in the database. The signatures indicated a close relationship to swine, human and avian sequences. The gene segments targeted were PB1 (VIR2798), NP (VIR1266), M1 (1279), PA (1287), NS1 (2775), and NS2 (2777). The base counts determined for each target are highlighted in green; below them are the number of viral RNA copies and the SNP variations from the matching strain, respectively. ^a VIR2798 =  PB1; VIR1266 =  NP; 1279 =  M1; 1287 =  PA; 2775 =  NS1; 2777 =  NS2; ^b The tested sample had 4 different close matches in the reference database, depending on the six targets; ^c The closest matching virus, from reference the database, to the sample tested; ^d The numerators refer to # of targets identified. The denominators refer to the number of entries (# of genes) from the different viruses represented with BC signatures in the database; ^e The sample tested had matches to viruses from swine, human, and avian, but identification of origin was not correct for all genes (e.g. the NP had a closest match in a human H1N1 virus, while genome sequencing revealed the virus has NP related to North American triple reassortant swine H1N1).

Figure 2. The genomic print of H1N1 pdm viruses is highly conserved and distinct from other influenza viruses.

Thirteen (13) seasonal H1N1 and H3N2 viruses from various seasons (top half); 12 samples from the H1N1pdm viruses (including the two viruses of the first two cases of the 2009 pandemic, referred to as NHRC1 and 2) (bottom half in yellow highlight); and a North American swine of H1N1 subtype were analyzed. All 2009 H1N1 pdm viruses have the same genomic print. BC signatures of five of the six targets in the H1N1pdm virus (NP, M1, PA, NS1, and NS2) were different from the other subtypes. The BC signature determined for the PB1 (A39, G32, C24, T33) was identical to some human H3N2 viruses, as expected. The H1N1pdm viruses were also distinguishable from the triple reassortant swine H1N1 at all six targets (compare yellow and purple signature). Differences in color patterns within the same subtype either reflects different genetic groups (H3N2 and H1N1), or a single nucleotide polymorphism (2009 H1N1 pdm). The numbers preceded by letters in each box correspond to the base counts (number A, G, C, and T) determined from the amplicons of the respective target genes.

Figure 3. Spatial representation of BC signatures covered by the six primer pairs for Influenza A viruses characterization.

Black numerals refer to the individual primer pair numbers, and are followed by the designation of the segment from which they stem (between parentheses). For each primer pair, each sphere represents one strain of influenza circulating within the seasons 2007–2009, and is color coded by host and subtype: 1) light blue, human H1N1; 2) dark blue, swine triple reassortant H1N1; 3) light green, human H3N2; 4) dark green, swine triple reassortant H3N2; 5) and red, H1N1pdm. For clarity, ellipses regroup the most common subtypes in circulation. Each isolate is mapped in a three-dimensional space according to its amplicon BC signature using the designated primer pair.

Figure 4. RT-PCR/ESI-MS assay allows differentiation between the two major seasonal H1N1virus clades 2B and 2C.

Fourteen H1N1 viruses with previously known clades (based on phylogenetic data) were blindly tested and the results were analyzed against data base reference viruses: A/South Dakota/06/2007 and A/Colorado/UR06-0053/2007, of clades 2B and 2C, respectively. Nine of the viruses were identified as clade 2B and the remaining five as clade 2C, mainly based on the combination of BC signatures of PB1, M1, and PA (highlighted in yellow). The remaining 3 targets (NP, NS1, and NS2) had identical BC signatures in both groups of viruses (highlighted in light green). One or two SNPs were also detected in two viruses (light blue) but had no effect on clade identification. *The numbers preceded by letters in each box correspond to the base counts (number A, G, C, and T) determined from the amplicons of the respective target genes.

Figure 5. Detection of intra-subtype reassortment among seasonal H1N1 viruses using the RT-PCR/ESI-MS assy.

A set of 23 viruses with unusual drug resistance profiles (resistant to both oseltamivir and the adamantanes) were analyzed; fourteen (14) of them showed genomic prints identical to the prototype clade 2B virus, A/South Dakota/06/2007 (green highlight), based on all six targets, with the exception of three viruses that had single SNPs each in the PB1 amplicon (two) and the NP (one) (light purple). Sample 23 did not amplify with the PB1 target. Seven viruses had five BC signatures (from PB1, NP, PA, NS1, and NS2) matching the clade 2B virus, while their M1 BC signature was typical of clade 2C virus, A/Colorado/UR06-0053/2007 (light yellow). Two viruses had genomic prints uncharacteristic of either clade 2B or clade 2C (samples 1 and 2). The numbers preceded by letters in each box correspond to the base counts (number A, G, C, and T) determined from the amplicons of the respective target genes.