H5N1亚型高致病性禽流感病毒A/Duck/FuJian/01/02株感染性克隆构建

摘 要：1999-2002年本试验室从我国南方健康鸭体内分离到21株H5N1亚型禽流感病毒，对其进行系统地生物学特性和遗传演化分析发现，其中A/Duck/FuJian/01/02(简DKFJ)和A/Duck/Guangxi/53/02(简DKGX) 属于同一基因型, 对鸡都呈高致病性，但对哺乳动物模型Balb/c小鼠的致病性明显不同: DKGX对小鼠呈低致病性（MLD50>106.5），DKGX株反向基因操作系统本试验室已经建立，而DKFJ对小鼠呈高致病性（MLD50<100.5）。为了研究这两株病毒对哺乳动物模型Balb/c小鼠致病性差异的分子机制，本研究构建了对DKFJ的8质粒反向基因操作系统，并通过细胞转染技术成功拯救了该病毒（R-DKFJ）。R-DKFJ在对哺乳动物模型Balb/c小鼠的致病性保持了与亲本野生毒（W-DKFJ, MLD50<100.5）一致的生物学特性，并且对小鼠呈全身感染，即106EID50鼻腔感染小鼠后第三天在脑、脾、肾、肺脏等脏器检测到病毒。DKFJ反向基因操作系统的成功建立为阐明H5N1亚型禽流感病毒对哺乳动物模型Balb/c小鼠的致病机制等方面奠定了基础。

Establishment of infectious clone of highly pathogenic H5N1avian influenza virus A/Duck/FuJian/01/02

Abstract: From 1999-2002, we had isolated 21 H5N1 subtype avian influenza virus in health ducks from South China, We analyzed the biological properties and the heredity evolution of the H5N1 viruses systematically found that A/Duck/FuJian/01/02(DKFJ) and A/Duck/Guangxi/53/02(DKGX) are genetically similar, which were highly pathogenic to chickens but have different pathogenicity obviously in the BALB/c mice that is a useful mammalian model. DKGX is low pathogenic in mice (MLD50>106.5EID50), the reverse genetics system of DKGX had established in our Laboratory, whereas A/duck/FuJi/01/02 (DKFJ) is highly pathogenic (MLD50<100.5EID50). With the purpose of study the Molecular mechanisms of A duck H5N1 subtype avian influenza Pathogenic difference in mice of the two virus, an eight-plasmid reverse genetics system was established of DKFJ and the reassortant virus was rescued from cell transfection. The rescued virus(R-DKFJ) maintained the same biological properties as the wild virus (W-DKFJ MLD50<100.5). Both of the viruses could be recovered from the brain and spleen and kidney and lung of the Balb/c mice 3 days after inoculated intranasally with 106EID50. The successful establishment of the reverse genetics system of DKFJ will play an important role in the studies of the molecular basis of the evolution and the relationship between the structure and function of H5N1 subtype avian influenza viruses.