Abstract
BackgroundEffective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Methods and Principal FindingsAnalysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Conclusion/SignificanceThus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.
Highlights
Influenza viruses cause serious global economic and public health burdens
The recent outbreak of highly pathogenic H5N1 avian influenza virus (HPAI), which originated in Southeast Asia and has since spread globally, has resulted in 166 deaths (272 confirmed human cases) as of February 6, 2007
We have developed a method based on broad-range reverse transcription PCR (RT-PCR) followed by electrospray ionization mass spectrometry (RT-PCR/ ESI-MS) for rapid and accurate detection of influenza virus, subspecies characterization, and early identification of genetic changes in circulating viruses
Summary
Influenza viruses cause serious global economic and public health burdens. Annual influenza epidemics resulted in more than 30,000 deaths a year in the United States during 1990–1999[1,2]. Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide subspecies identification and infer influenza virus H and N subtypes. Rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance
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