title: Identification of virulence determinants in influenza viruses.
abstract: To date there is no rapid method to screen for highly pathogenic avian influenza strains that may be indicators of future pandemics. We report here the first development of an oligonucleotide-based spectroscopic assay to rapidly and sensitively detect a N66S mutation in the gene coding for the PB1-F2 protein associated with increased virulence in highly pathogenic pandemic influenza viruses. 5'-Thiolated ssDNA oligonucleotides were employed as probes to capture RNA isolated from six influenza viruses, three having N66S mutations, two without the N66S mutation, and one deletion mutant not encoding the PB1-F2 protein. Hybridization was detected without amplification or labeling using the intrinsic surfaced-enhanced Raman spectrum of the DNA-RNA complex. Multivariate analysis identified target RNA binding from noncomplementary sequences with 100% sensitivity, 100% selectivity, and 100% correct classification in the test data set. These results establish that optical-based diagnostic methods are able to directly identify diagnostic indicators of virulence linked to highly pathogenic pandemic influenza viruses without amplification or labeling.
authors.affiliation: Department of Chemistry, University of Georgia , Athens, Georgia 30602 United States.
authors.forename: Pierre
authors.initials: P
authors.lastname: Negri
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Cheryl
authors.initials: C
authors.lastname: Jones
authors.forename: S Mark
authors.initials: SM
authors.lastname: Tompkins
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Tripp
authors.forename: Richard A
authors.initials: RA
authors.lastname: Dluhy
mesh_terms: D000818:Animals; D015342:DNA Probes / Q000235:genetics; D004277:DNA, Single-Stranded; D004285:Dogs; D017403:In Situ Hybridization / Q000295:instrumentation; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000472:pathogenicity; D053125:Influenza A Virus, H5N2 Subtype / Q000235:genetics / Q000472:pathogenicity; D009980:Influenza A virus / Q000235:genetics / Q000472:pathogenicity*; D016018:Least-Squares Analysis; D061985:Madin Darby Canine Kidney Cells / Q000821:virology; D008954:Models, Biological; D009154:Mutation; D043942:Nanotubes; D009841:Oligonucleotides / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D013059:Spectrum Analysis, Raman / Q000295:instrumentation / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C442816:PB1-F2 protein, Influenza A virus; D012367:RNA, Viral; D014764:Viral Proteins; D037521:Virulence Factors