Heine Hans G, Foord Adam J, Wang Jianning, Valdeter Stacey, Walker Som, Morrissy Chris, Wong Frank Y K, Meehan Brian
CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia.
Virol J. 2015 Feb 8;12:18. doi: 10.1186/s12985-015-0250-3.
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.
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.
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.
高致病性禽流感（HPAI）H5变异病毒最近因H5血凝素（HA）基因与不同神经氨酸酶（NA）基因（包括NA1、NA2、NA5、NA6和NA8）重配而出现。这些病毒形成了一个新提出的HA 2.3.4.4分支（以前临时称为2.3.4.6分支），并与中国、韩国、老挝、日本和越南的家禽疾病暴发以及中国的一例人类死亡病例有关。人们真切担心这个新分支可能广泛传播，并且使用现有的诊断算法难以识别。
开发了基于荧光探针的逆转录定量聚合酶链反应（RT-qPCR）检测方法，以促进对亚洲出现的新型2.3.4.4分支H5N6亚型病毒的鉴定。检测方法针对用于分支鉴定的血凝素（HA）基因以及用于鉴定N6的NA基因。采用小沟结合剂（MGB）探针的HA检测方法能够检测并区分A/鸭/老挝/XBY004/2014（H5N6）和属于提议的2.3.4.4分支的相关甲型流感病毒（H5N6）分离株与其他H5高致病性禽流感病毒。此外，一种欧亚N6检测方法能够将N6与其他NA亚型区分开来。
使用分支特异性HA RT-qPCR检测方法检测到了代表提议的2.3.4.4分支的老挝甲型流感病毒（H5N6），并将其与其他H5N1分支的病毒区分开来，而N6-NA亚型则通过欧亚N6 RT-qPCR检测方法确定。这种分支特异性检测方法对于监测以及在无法随时进行测序的诊断实验室中将具有特别价值。
