title: Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.
abstract: A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood.
  
  
  
  Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis.
  
  
  
  New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples.
  
  
  
  A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping.
  
  
  
  These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany.
authors.forename: Dinah
authors.initials: D
authors.lastname: Henritzi
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany.
authors.forename: Na
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Starick
authors.affiliation: Anses, Ploufragan-Plouzané Laboratory, Swine Virology Immunology Unit, Ploufragan, France.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Simon
authors.affiliation: National Veterinary Institute; Technical University of Denmark (DTU), Frederiksberg C, Denmark.
authors.forename: Jesper S
authors.initials: JS
authors.lastname: Krog
authors.affiliation: National Veterinary Institute; Technical University of Denmark (DTU), Frederiksberg C, Denmark.
authors.forename: Lars Erik
authors.initials: LE
authors.lastname: Larsen
authors.affiliation: Department of Virology, Animal and Plant Health Agency-Weybridge, New Haw, Addlestone, Surrey, UK.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Reid
authors.affiliation: Department of Virology, Animal and Plant Health Agency-Weybridge, New Haw, Addlestone, Surrey, UK.
authors.forename: Ian H
authors.initials: IH
authors.lastname: Brown
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Parma, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Chiapponi
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Parma, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Foni
authors.affiliation: IDT Biologika GmbH, Dessau-Rosslau, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Wacheck
authors.affiliation: IDT Biologika GmbH, Dessau-Rosslau, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schmid
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institute (FLI), Greifswald-Insel Riems, Germany. timm.harder@fli.de.
authors.forename: Timm C
authors.initials: TC
authors.lastname: Harder
mesh_terms: D000818:Animals; D001717:Birds / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D062665:Epidemiological Monitoring / Q000662:veterinary; D005060:Europe / Q000453:epidemiology; D006388:Hemagglutinins / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000453:epidemiology / Q000635:transmission / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction; D009439:Neuraminidase / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology*
chemical_list: D017931:DNA Primers; D006388:Hemagglutinins; D009439:Neuraminidase
keywords: influenza A virus; multiplex RT-qPCR; subtyping; surveillance; swine; zoonosis