title: Rapid differentiation of mixed influenza A/H1N1 virus infections with seasonal and pandemic variants by multitemperature single-stranded conformational polymorphism analysis.
abstract: Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.
authors.affiliation: BioVectis, Pawinskiego 5a/D, 02-106 Warsaw, Poland. beata.pajak@biovectis.com
authors.forename: Beata
authors.initials: B
authors.lastname: Pajak
authors.forename: Ilona
authors.initials: I
authors.lastname: Stefanska
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Lepek
authors.forename: Stefan
authors.initials: S
authors.lastname: Donevski
authors.forename: Magdalena
authors.initials: M
authors.lastname: Romanowska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Szeliga
authors.forename: Lidia B
authors.initials: LB
authors.lastname: Brydak
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Szewczyk
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kucharczyk
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D005260:Female; D005838:Genotype; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D008297:Male; D008875:Middle Aged; D011044:Poland; D018807:Polymorphism, Single-Stranded Conformational; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D055815:Young Adult
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus