【作者】 汪海波；

【导师】 陈志伟；

【作者基本信息】 武汉大学 ， 微生物学， 2009， 博士

【摘要】 1.云南省I型HIV分子流行病学研究自1989年在静脉吸毒人群中发现第一例HIV-1感染者以来,云南省始终拥有最多的HIV-1感染者,然而,其分子流行病学未被全面的研究过。首先我们运用免疫学方法确认了从1989年至2004年以来累计103015例HIV-1感染者,从中我们挑选了863例感染者作为我们这次的研究对象。这些HIV-1感染者代表了云南省16个州县、四种感染途径、11个民族和10种职业。对其核心蛋白gag区p17基因的分析,我们发现了3种主要流行的亚型C/CRF07_BC/CRF08_BC(占53%),CRF01_AE(占40.5%)和B(占6.5%)。通过对那些已知危险因素分析,发现有90.9%的静脉吸毒者携带C/CRF07_BC/CRF08_BC亚型病毒,而有85.4%的CRF01_AE亚型病毒感染者是通过性接触被感染的。在傣族感染者中,CRF01_AE亚型没有发现明显的种族隔离现象。在地理分布上面,C/CRF07_BC/CRF08_BC亚型病毒分布于整个云南省,而CRF01_AE亚型病毒主要分布于与缅甸接壤的州县。另外,对其逆转录酶基因的分析,我们发现C/CRF07_BC/CRF08_BC亚型病毒实际上是一组病毒的重组体,包含C, CRF08_BC, CRF07_BC和新BC重组亚型病毒。本研究首次对云南省全省范围进行了HIV-1分子流行病学研究,我们发现了C/CRF07_BC/CRF08_BC和CRF01_AE是云南省的主要流行株,其中在通过性传播途径感染HIV-1的感染者中主要流行CRF01_AE亚型属于一个新的发现,表明在整个中国普通人群当中可能存在一个新的HIV-1病毒亚型的流行趋势。我们的研究结果将大力推进对HIV-1进化的研究及艾滋病疫苗的开发。2.针对B’亚型的SHIV/中国恒河猴模型的建立在中国和东南亚流行的一个主要的HIV-1亚型——B’亚型的持续蔓延,要求我们建立一个合适的动物模型来研究该种病毒的传播和致病机理。鉴于B’HIV-1和现有的其他SHIV株在env序列上存在巨大的差异,我们构建了一个新的SHIV病毒株——SHIVB’WHU。该病毒的体外特性已经在庄柯博士的毕业论文中探讨过,但是其体内特性还没有被研究过。因此,我们利用传统的体内传代方法将SHIVB’WHU在中国恒河猴体内连续传代(四代)以增强它的感染能力。我们还研究了不同组织内的病毒变异株以期弄清楚病毒急性感染期内复制和适应的位点,这一点在以前的所有SHIV株上都没有被很好的研究过。我们通过检测猴子体内的病毒载量发现,SHIVB’WHU的感染能力在传代后确实增强了,然而有趣的是,感染能力的增强并没有改变它的CCR5嗜性,而且跟从外周血、小肠和其他一些组织的淋巴细胞中扩增得到的env序列变异没有相关性(我们只发现最小限度的env序列变异)。我们认为这种最小限度的env序列变异可能跟急性感染期内缺少很强的选择压力有关。另外,在第三代和第四代猴子的小肠中我们没有发现CD4+T细胞显著减少的现象。我们的研究结果表明SHIVB’WHU是可以复制和适应中国恒河猴的,由此建立的SHIVB’WHU/中国恒河猴模型将是一个更为合适的研究疫苗和杀微生物制剂的工具。更多还原

【Abstract】 1. HIV-1 epidemic in Yunnan province of ChinaDating back to the first epidemic among injection drug users in 1989, the Yunnan province has had the highest number of human immunodeficiency virus type 1 (HIV-1) infections in China. However, the molecular epidemiology of HIV-1 in Yunnan has not been fully characterized. Using immunoassays, we identified 103,015 accumulated cases of HIV-1 infections in Yunnan between 1989 and 2004. We studied 863 patients representing Yunnan’s 16 prefectures from four risk groups,11 ethnic populations, and ten occupations. We identified three major circulating subtypes:C/CRF07_BC/CRF08_BC (53%), CRF01_AE (40.5%), and B (6.5%) by analyzing the sequence of p17, which is part of the gag gene. For patients with known risk factors,90.9% of injection drug users had C/CRF07_BC/CRF08_BC viruses, whereas 85.4% of CRF01_AE infections were acquired through sexual transmission. No distinct segregation of CRF01_AE viruses was found among the Dai ethnic group. Geographically, C/CRF07_BC/CRF08 BC was found throughout the province, while CRF01_AE was largely confined to the prefectures bordering Myanmar. Furthermore, C/CRF07_BC/CRF08_BC viruses were found to consist of a group of viruses, including C, CRF08_BC, CRF07_BC, and new BC recombinants, based on the characterization of their reverse transcriptase genes. This is the first report of a province-wide HIV-1 molecular epidemiological study in Yunnan. While C/CRF07_BC/CRF08_BC and CRF01_AE are codominant, the discovery of many sexually transmitted CRF01_AE cases is new and suggests that this subtype may lead to a new epidemic in the general Chinese population. We discuss implications of our results for understanding the evolution of the HIV-1 pandemic and for vaccine development.2. The establishment of B’SHIV/Chinese rhesus macaques modelThe increasing prevalence of HIV-1 subtype B’, one of the major genotypes in China and Southeast Asia, calls for efforts to develop a relevant animal model to study viral transmission and pathogenesis. Since there are significant sequence differences between B’HIV-1 and other SHIVs in env gene, a new SHIV, designated SHIVB’WHU, was generated. The in vitro characteristics have been discussed by Dr. Ke Zhuang in her Ph.D dissertation. However, the in vivo characteristics have not been studied. Serial passages of SHIVB’WHU were conducted in Chinese macaques to enhance viral infectivity. Moreover, viral variants in different tissues were studied to understand the site of viral replication and adaptation during the acute phase of infection, which has not been previously studied. The infectivity of SHIVB’WHU was enhanced in macaques during the serial passage as determined by viral load measurement. Interestingly, the enhanced infectivity did not result in altered CCR5-tropism and was unlikely associated with sequence variation in env genes recovered from lymphocytes of peripheral blood, small intestines and various tissues. The minimal variation in env is probably due to the lack of strong selection pressure during the acute phase of SHIVB’WHU infection in Chinese macaques. Significant CD4+ T cell loss was not found in small intestines of P3-P4 infected animals. Our data indicate that SHIVB’WHU is replication-competent and has adapted in Chinese rhesus macaques. The model of SHIVB’WHU/Chinese rhesus macaque can be used as a more appropriate model for vaccine research and microbicide evaluation.更多还原