title: Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction.
abstract: BACKGROUND
  Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.4.4 (previously provisionally referred to as clade 2.3.4.6), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms.
  
  
  RESULTS
  Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 2.3.4.4 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 2.3.4.4 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes.
  
  
  CONCLUSIONS
  Laos influenza A(H5N6) virus representative of proposed clade 2.3.4.4, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Hans.Heine@csiro.au.
authors.forename: Hans G
authors.initials: HG
authors.lastname: Heine
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Adam.Foord@csiro.au.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Foord
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Jianning.Wang@csiro.au.
authors.forename: Jianning
authors.initials: J
authors.lastname: Wang
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Stacey.Valdeter@csiro.au.
authors.forename: Stacey
authors.initials: S
authors.lastname: Valdeter
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Som.Walker@csiro.au.
authors.forename: Som
authors.initials: S
authors.lastname: Walker
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Chris.Morrissy@csiro.au.
authors.forename: Chris
authors.initials: C
authors.lastname: Morrissy
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Frank.Wong@csiro.au.
authors.forename: Frank Y K
authors.initials: FY
authors.lastname: Wong
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. Brian.Meehan@csiro.au.
authors.forename: Brian
authors.initials: B
authors.lastname: Meehan
mesh_terms: D000818:Animals; D001208:Asia; D001717:Birds; D009980:Influenza A virus / Q000302:isolation & purification* / Q000472:pathogenicity*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011200:Poultry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*