【作者】 钟功勋；

【导师】 陈化兰；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 H5N1亚型禽流感病毒是重要的人兽共患病病原。1997年香港首次发生H5N1亚型高致病性禽流感病毒感染并致人死亡事件,目前H5N1亚型禽流感病毒已在15个国家感染近500人,致死率近60%,给公共卫生安全带来了极大挑战。病毒如何跨越种间屏障感染人类以及其对人类的高致病力决定因素,是流感病毒领域研究的重大课题。根据HA基因的不同,H5N1亚型禽流感病毒被划分为不同进化分支(clade),其中clade 2.3.4的病毒在中国和东南亚多个国家的家禽和感染病人中发现,是地理分布范围较为广泛的一个分支。我国分离的clade 2.3.4的病毒NA和内部基因具有较大差异,形成多个基因型。本研究分析了我国分离的16株clade 2.3.4病毒对哺乳动物(BALB/c小鼠模型)的感染和致病力,发现它们对小鼠表现为高、中、低不同致病型。在上述研究基础上,我们筛选了两株病毒作为模型探索H5N1亚型禽流感病毒对哺乳动物致病力的分子机制。这两株病毒分别是分离自人的A/Anhui/2/2005(AH/2/05)和分离自鸡的A/chicken/Sichuan/81/2005 (CK/SC/81/05),它们基因组高度同源,仅有27个氨基酸不同,但对小鼠致病力截然不同,前者对小鼠高度致死,后者仅在小鼠体内局限性复制,致病力差异约10000倍。我们利用流感病毒反向遗传操作技术构建了一系列重组病毒,并在小鼠体内分析这些病毒的致病力。结果发现单个基因替换不能明显改变AH/2/05和CK/SC/81/05对小鼠的致病力,而PB1和PA基因同时替换则可改变这两株病毒对小鼠的致病型。我们进一步对一系列PA和PB1基因的突变株进行了研究,发现PB1基因622、709、PA基因315位的3个氨基酸共同改变是上述两株病毒的致病力差异的决定性因素。我们进一步的研究发现这些位点氨基酸的改变可影响病毒在哺乳动物细胞的聚合酶活性,部分揭示了其影响致病力的分子机制。本研究首次揭示聚合酶PB1和PA中与H5N1禽流感病毒对哺乳动物致病力的关键氨基酸位点,这些结果对评估H5N1亚型禽流感病毒分离株对公共卫生的风险性具有指导意义。更多还原

【Abstract】 H5N1 influenza viruses are important pathogens for both veterinary and public health. In 1997, highly pathogenic H5N1 avian influenza virus caused outbreaks in chickens in Hong Kong and was transmitted to humans, causing the deaths of 6 of 18 people infected. Up-to-data, about 500 human cases of H5N1 infections with greater than 50% mortality have been reported in many countries, posing huge challenge to the public health. To understand the genetic determinants of viral pathogenicity and their ability to cross species barriers to mammalian hosts is, therefore, a research priority on influenza viruses.The H5N1 viruses are divided into distinct phylogenetic clades based on their HA genes. The clade 2.3.4 is one of the widely distributed clade and the viruses in this clade continue to infect poultry and humans in China and several Southeast Asia countries. Our previous studies revealed clade 2.3.4 viruses isolated in China formed multiple genotypes due to the big diversity detected in their NA and internal genes. We tested 16 clade 2.3.4 viruses isolated from China in a mammalian mouse model. Our results indicated that these viruses are different in their replication and virulence in mice, and they could be classified into high, middle and low pathotypes.Based on the results described above, we selected two viruses as models to explore the virulence determinants of the clade 2.3.4 viruses in mammalian hosts. The two viruses, A/Anhui/2/2005(AH/2/05) and A/chickens/Sichuan/81/2005 (CK/SC/81/05), are genetically very similar and only differ by 27 amino acids in their entire genome. However, the two viruses are significantly different in their virulence to mice. The AH/2/05 virus is highly lethal in mice, with a 50% mouse lethal dose (MLD50) of 1.5 log10EID50 (50% egg infectious dose), while the CK/SC/81/05 is quite mild, and it mainly replicates in the lung of mice, with a MLD50 of 5.5 log10EID50. We used reverse genetics to generate a series of reassortants of these two viruses and tested them in mice. The results indicated that none of the single-gene-switch contributed to the observed difference of the two viruses in mice, but the switch of both the PA and PB1 genes could completely change the pathotypes of two viruses in mice. We found that the changes of two amino acids at position 622 and 709 of PB1 and one amino acid at position 315 of PA contributed together to the pathogenicity difference of the two viruses in mice. We further demonstrated that the changes in these sites affect the polymerase activities of the two viruses in mammalian cells, which partially elucidate the mechanism of their virulence in mammalian hosts. Our results here are the first to indicate that the mutations in PA and PB1 proteins are critical for the pathogenicity of H5N1 influenza in mammalian hosts, and the amino acids we identified here could be used for assessing the virulence potential of H5N1 field isolates.更多还原