Abstract
BackgroundVariant 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.FindingsFluorescent 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.ConclusionsLaos 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.
Highlights
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
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 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay
Recent HPAI H5 virus variants of different NA subtypes have been implicated in disease outbreaks in poultry in China (H5N1, H5N2, H5N5, H5N6, H5N8), South Korea (H5N8), Japan (H5N8), Laos (H5N8) and Vietnam (H5N1, H5N6) and an H5N6 subtype virus caused a human fatality in China [1-3]
Summary
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. Recent HPAI H5 virus variants of different NA subtypes have been implicated in disease outbreaks in poultry in China (H5N1, H5N2, H5N5, H5N6, H5N8) , South Korea (H5N8), Japan (H5N8), Laos (H5N8) and Vietnam (H5N1, H5N6) and an H5N6 subtype virus caused a human fatality in China [1-3]. These variants have previously been referred to as “clade 2.3.4.6” viruses and originate from H5N1 clade 2.3.4 viruses [4,5]. The WHO/OIE/FAO H5N1 Evolution Working Group recently designated that these viruses fall into a Molecular identification of this new clade currently requires conventional RT-PCR amplification of the HA gene followed by nucleotide sequence determination. The clade specific HA probes contained the codon for amino acid substitution 273H that has been
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