Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

PLOS ONE, May 2007

Background Effective 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 Findings Analysis 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/Significance Thus, 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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Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

et al (2007) Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry. PLoS ONE 2(5): e489. doi:10.1371/journal.pone.0000489 Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry Rangarajan Sampath 0 1 3 Kevin L. Russell 0 1 3 Christian Massire 0 1 3 Mark W. Eshoo 0 1 3 Vanessa Harpin 0 1 3 Lawrence B. Blyn 0 1 3 Rachael Melton 0 1 3 Cristina Ivy 0 1 3 Thuy Pennella 0 1 3 Feng Li 0 1 3 Harold Levene 0 1 3 Thomas A. Hall 0 1 3 Brian Libby 0 1 3 Nancy Fan 0 1 3 Demetrius J. Walcott 0 1 3 Raymond Ranken 0 1 3 Michael Pear 0 1 3 Amy Schink 0 1 3 Jose Gutierrez 0 1 3 Jared Drader 0 1 3 David Moore 0 1 3 David Metzgar 0 1 3 Lynda Addington 0 1 3 Richard Rothman 0 1 3 Charlotte A. Gaydos 0 1 3 Samuel Yang 0 1 3 Kirsten St. George 0 1 3 Meghan E. Fuschino 0 1 3 Amy B. Dean 0 1 3 David E. Stallknecht 0 1 3 Ginger Goekjian 0 1 3 Samuel Yingst 0 1 3 Marshall Monteville 0 1 3 Magdi D. Saad 0 1 3 Chris A. Whitehouse 0 1 3 Carson Baldwin 0 1 3 Karl H. Rudnick 0 1 3 Steven A. Hofstadler 0 1 3 Stanley M. Lemon 0 1 3 David J. Ecker 0 1 3 0 Funding: The RT-PCR/ESI-MS technology development was funded by DARPA, a division of the U.S. Department of Defense. Funding from the CDC and NIAID supported the influenza surveillance assay development and screening described here. The sponsors had no role in the design, implementation or conlusions of the assay 1 Academic Editor: Joel Montgomery, US Naval Medical Research Center De- tachment/Centers for Disease Control , United States of America 2 3, Cairo, Egypt, 7 United States Army Medical Research in Infectious Diseases , Ft. Detrick, Maryland , United States of America, 8 Science Applications International Corporation, San Diego, California, United States of America, 9 Institute for Human Infections and Immunity, University of Texas Medical Branch , Galveston, Texas , United States of America 3 1 Ibis Biosciences Inc., A Wholly Owned Subsidiary of Isis Pharmaceuticals, Carlsbad, California, United States of America, 2 Naval Respiratory Disease Laboratory, Naval Health Research Center, San Diego, California, United States of America, 3 Department of Emergency Medicine and Medicine, The Johns Hopkins Medical Institutions , Baltimore , Maryland, United States of America, 4 Wadsworth Center, New York State Department of Health , Albany , New York, United States of America, 5 College of Veterinary Medicine, The University of Georgia , Athens , Georgia , United States of America, 6 Naval Medical Research Unit Background. Effective 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 Findings. 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. 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/Significance. Thus, 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. - INTRODUCTION 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 19901999[1,2]. Periodic pandemics result in significantly higher death tolls. Emergence of new influenza A virus strains can be caused by antigenic shift, resulting from reassortment of gene segments, including H and/or N types[3,4], antigenic drift resulting from the continuing accumulation of mutations in the H and N genes[5], or a pathogenic virus jumping species and acquiring the ability to infect and be transmitted among humans, as i (...truncated)


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Rangarajan Sampath, Kevin L. Russell, Christian Massire, Mark W. Eshoo, Vanessa Harpin, Lawrence B. Blyn, Rachael Melton, Cristina Ivy, Thuy Pennella, Feng Li, Harold Levene, Thomas A. Hall, Brian Libby, Nancy Fan, Demetrius J. Walcott, Raymond Ranken, Michael Pear, Amy Schink, Jose Gutierrez, Jared Drader, David Moore, David Metzgar, Lynda Addington, Richard Rothman, Charlotte A. Gaydos, Samuel Yang, Kirsten St. George, Meghan E. Fuschino, Amy B. Dean, David E. Stallknecht, Ginger Goekjian, Samuel Yingst, Marshall Monteville, Magdi D. Saad, Chris A. Whitehouse, Carson Baldwin, Karl H. Rudnick, Steven A. Hofstadler, Stanley M. Lemon, David J. Ecker. Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry, PLOS ONE, 2007, Volume 2, Issue 5, DOI: 10.1371/journal.pone.0000489