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
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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. / Henritzi, Dinah; Zhao, Na; Starick, Elke; Simon, Gaelle; Krog, Jesper S.; Larsen, Lars Erik; Reid, Scott M.; Brown, Ian H.; Chiapponi, Chiara; Foni, Emanuela; Wacheck, Silke; Schmid, Peter; Beer, Martin; Hoffmann, Bernd; Harder, Timm C.
I: Influenza and Other Respiratory Viruses, Bind 10, Nr. 6, 01.11.2016, s. 504-517.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - 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
AU - Henritzi, Dinah
AU - Zhao, Na
AU - Starick, Elke
AU - Simon, Gaelle
AU - Krog, Jesper S.
AU - Larsen, Lars Erik
AU - Reid, Scott M.
AU - Brown, Ian H.
AU - Chiapponi, Chiara
AU - Foni, Emanuela
AU - Wacheck, Silke
AU - Schmid, Peter
AU - Beer, Martin
AU - Hoffmann, Bernd
AU - Harder, Timm C.
PY - 2016/11/1
Y1 - 2016/11/1
N2 - Background: 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. Objectives: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. Methods: 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. Results: 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. Conclusions: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.
AB - Background: 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. Objectives: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. Methods: 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. Results: 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. Conclusions: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.
KW - influenza A virus
KW - multiplex RT-qPCR
KW - subtyping
KW - surveillance
KW - swine
KW - zoonosis
U2 - 10.1111/irv.12407
DO - 10.1111/irv.12407
M3 - Journal article
C2 - 27397600
AN - SCOPUS:84991107062
VL - 10
SP - 504
EP - 517
JO - Influenza and other Respiratory Viruses
JF - Influenza and other Respiratory Viruses
SN - 1750-2640
IS - 6
ER -
ID: 247395004