Tsukamoto Kenji, Ashizawa Hisayoshi, Nakanishi Koji, Kaji Noriyuki, Suzuki Kotaro, Okamatsu Masatoshi, Yamaguchi Shigeo, Mase Masaji
Research Team for Zoonotic Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
J Clin Microbiol. 2008 Sep;46(9):3048-55. doi: 10.1128/JCM.02386-07. Epub 2008 Jul 2.
Serious concern about the worldwide transmission of the Asian H5N1 highly pathogenic (HP) avian influenza (AI) virus by migratory birds surrounds the importance of the AI global surveillance in wild aquatic birds and underscores the requirement for a reliable subtyping method of AI viruses. PCR is advantageous due to its simplicity, lower cross-reactivity, and unlimited reagent supply. Currently, the only available hemagglutinin (HA) subtyping primer set that can subtype H1 through H15 is not fully evaluated and, since it only targets HA1, is unavailable for molecular pathotyping of AI viruses. Our preliminary experiments found that these primer sets were cross-reactive and missed some recent AI viruses. In this study, we developed new primer sets against HA cleavage sites for subtyping H1 to H15 genes and for molecular pathotyping. Our primer sets were subtype specific and detected 99% of previously identified HA genes (115/116, 1949 to March 2006), and the correct amplifications of HA genes were confirmed by sequence analyses of all 115 PCR products. The primer sets successfully subtyped most of the recent AI viruses isolated in Japan (96% [101/105], October 2006 to March 2007). Taken together, our primer sets could efficiently detect HA genes (98% [216/221]) of both previously and recently identified HA genes or of both American (29/29) and Eurasian (187/192) lineages. All 38 H5 and 13 H7 viruses were molecularly pathotyped by sequencing analyses of the HA cleavage site. In contrast, despite efficient detection of previously identified strains (98% [114/116]), the published primer sets exhibited lower specificity and lower detection efficiency against recent AI viruses (80% [84 of 105]). These results indicate that our primers are useful not only for HA subtyping but also for molecular pathotyping of both previous and recent AI viruses. These advancements will enable general diagnostic laboratories to subtype AI viruses for the surveillance in wild aquatic birds.
候鸟传播亚洲H5N1高致病性禽流感（AI）病毒引发了全球范围内的严重关注，这凸显了对野生水鸟进行禽流感全球监测的重要性，并强调了需要一种可靠的禽流感病毒亚型分型方法。聚合酶链反应（PCR）因其操作简单、交叉反应性较低且试剂供应不受限制而具有优势。目前，唯一可用于H1至H15亚型分型的血凝素（HA）分型引物组尚未得到充分评估，并且由于其仅靶向HA1，无法用于禽流感病毒的分子致病型分析。我们的初步实验发现，这些引物组具有交叉反应性，并且遗漏了一些近期的禽流感病毒。在本研究中，我们开发了针对HA裂解位点的新引物组，用于H1至H15基因的亚型分型和分子致病型分析。我们的引物组具有亚型特异性，检测到了99%的先前鉴定的HA基因（115/116，1949年至2006年3月），并且通过对所有115个PCR产物的序列分析证实了HA基因的正确扩增。该引物组成功地对日本近期分离的大多数禽流感病毒进行了亚型分型（96% [101/105]，2006年10月至2007年3月）。综上所述，我们的引物组能够高效检测先前和近期鉴定的HA基因（98% [216/221]），或美洲（29/29）和欧亚（187/192）谱系的HA基因。通过对HA裂解位点的测序分析，对所有38株H5和13株H7病毒进行了分子致病型分析。相比之下，尽管已发表的引物组能够高效检测先前鉴定的毒株（98% [114/116]），但对近期的禽流感病毒表现出较低的特异性和检测效率（80% [105株中的84株]）。这些结果表明，我们的引物不仅可用于HA亚型分型，还可用于先前和近期禽流感病毒的分子致病型分析。这些进展将使普通诊断实验室能够对禽流感病毒进行亚型分型，以监测野生水鸟。
