【作者】 刘林青；

【导师】 刘金华；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 H5N1亚型禽流感病毒在欧亚大陆广泛流行,对公共卫生安全造成巨大威胁,且该病毒具有人间大流行的潜力,因此,研究H5N1病毒对哺乳动物的致病机制至关重要。妊娠是某些疾病加重的重要风险因素,由于目前H5N1病毒感染孕妇的病例较少,H5N1病毒对孕妇的致病性尚不清楚。本研究以孕鼠为模型动物,探究H5N1病毒感染妊娠期哺乳动物的致病性和病毒的垂直传播能力,对解析H5N1病毒感染妊娠妇女的致病机制具有重要借鉴意义。本研究以clade2.3.4分支H5N1禽流感病毒感染BALB/c孕鼠和非孕鼠,对病毒复制能力、机体先天性免疫反应、脏器病理损伤等方面进行了系统研究。结果表明,攻毒后第3天病毒在孕鼠肺脏中的复制滴度显著高于非孕鼠,并且孕鼠肺脏中病毒阳性的Ⅱ型肺泡上皮细胞数量较多。在病毒感染早期,孕鼠肺脏中与抗病毒相关的细胞因子IFN-γ和TNF-α表达量低于非孕鼠,而感染中后期,孕鼠肺脏中趋化因子MCP-1、MIP-1α和IL-6表达水平显著升高,并有大量炎性细胞浸润,造成严重的肺脏弥散性肺泡损伤。病毒感染后期孕鼠血氧饱和度显著下降,表现出严重的呼吸窘迫综合征症状。整个感染期中,孕鼠雌二醇、孕酮表达水平未见异常。该部分结果表明,H5N1病毒对妊娠期小鼠致病力高于普通雌鼠,病毒在孕鼠肺脏复制能力更强,诱导大量细胞因子表达和炎性细胞浸润,造成组织更严重的病理损伤,因此妊娠是造成H5N1病毒感染哺乳动物病情加重的危险因素。为揭示H5N1病毒在小鼠体内是否具有垂直传播的能力,将H5N1病毒感染BALB/c孕鼠,在攻毒后第3天病毒即出现肺外感染,第4天胎盘可分离到病毒,第5天胎儿可分离到病毒。感染胎盘组织的病毒主要分布在胎盘迷路滋养层内,尤其是母体与胎儿血管网交汇处的滋养细胞及巨噬细胞,在蜕膜层结缔组织间也有少量病毒分布,并且胎鼠肺泡上皮细胞、肝细胞也可检测到病毒,表明H5N1病毒具有经胎盘垂直传播的能力。病毒感染中后期,胎盘组织细胞因子IFN-γ、 TNF-α和IL-6水平显著升高,胎盘出现局灶性坏死,大量炎性细胞浸润,孕鼠出现流产及胎儿坏死等妊娠失败表现。为进一步揭示H5N1病毒发生垂直传播的机制,分别进行外周血单核细胞和多核细胞病毒感染情况、胎盘组织受体分布及胎盘滋养层细胞体外感染研究。结果表明,孕鼠感染H5N1病毒后血液中多核细胞和单核细胞均能被病毒感染,说明血细胞能够作为载体携带病毒到达胎盘组织。小鼠胎盘组织中血管内皮细胞、滋养细胞和绒毛膜板上皮细胞同时表达a-2,3和a-2,6两种唾液酸受体,并且病毒可以有效感染体外培养的原代小鼠胎盘滋养细胞并复制。该部分实验证明H5N1病毒可以以血细胞作为载体到达胎盘,并感染胎盘细胞,病毒对胎盘细胞的直接感染以及胎盘过强的炎症反应造成胎盘组织细胞坏死,结构完整性破坏,病毒得以突破胎盘屏障发生垂直传播,感染胎儿,进而导致妊娠失败。该研究为解析临床上H5N1病毒感染孕妇进而发生垂直传播的机制提供了理论依据。与禽类密切接触的养禽业人员被认为是禽流感感染的高危人群。为了评价该人群感染禽流感的风险,对北京地区的养禽业人员进行了禽流感病毒H5、H7和H9抗体血清学监测和问卷调查。所采集305份血清样本中,H5和H7抗体阳性血清均为0份,H9抗体阳性血清为14份,阳性率为4.59%,说明H9亚型禽流感对公共卫生安全具有一定的威胁性。通过问卷调查发现养禽人员对禽流感的公共卫生安全威胁性普遍认识不足。综上所述,通过对H5N1病毒感染孕鼠致病性和垂直传播机制的研究,以及养禽业人员禽流感病毒血清学调查和认知行为调查,丰富了H5Nl禽流感病毒对哺乳动物致病与传播机制的理论研究,对禽流感病毒跨种间传播的预防和控制具有指导意义。更多还原

【Abstract】 H5N1avian influenza virus spreads widely in Eurasia. The pandemic potential of H5N1virus is a great threat to public health security. It would be helpful for the prevention of H5N1outbreak if we try to explore the pathogenesis of the virus. This study focused on the pathogenesis of H5N1virus infection during pregnancy. Pregnancy is a risk factor for some diseases. The pathogenicity of H5N1virus in pregnant women is not known yet. In this study,We used mouse as a model to research the effect of H5N1on pregnancy.The replication ability of H5N1virus, and body’s innate immune response, organs pathological damage of pregnant and nonpregnant BALB/c mices were studied. The results showed that the virus titers in the lungs of pregnant mice were significantly higher than that of nonpregnant mice at3day post inoculation (dpi). And virus positive type II alveolar epithelial cells in the pregnant mice lungs were more than that of nonpregnant mice. In the early infection, antiviral cytokines IFN-y, TNF-a levels in pregnant mice lungs were significantly higher than that of nonpregnant ones. In the late infection, chemokine MCP-1, MIP-la and IL-6in pregnant mices were significantly increased, and caused lots of inflammatory cells infiltration, with lung diffuse alveolar damage. Oxygen saturation of pregnant mice significantly decreased in the middle and late periods of the infection, with severe symptoms of respiratory distress. H5N1virus infection did not affect the concentration of estradiol and progesterone in serum. The results showed that H5N1virus on pathogenicity in mice during pregnancy was higher than normal female and pregnancy was a risk factor to aggravate infection.To study the vertical transmission ability of H5N1virus, pregnant mice with day13gestation were infected H5N1virus. The viremia appeared at3dpi, viruses were isolated from placenta at4dpi, and fetuses could be infected since5dpi. Immunohistochemical staining showed that the viruses were mainly distributed in the labyrinthine trophoblast. The H5N1virus was found in the placental villus epithelial cells and macrophages in the interface of maternal blood-syncytiotrophoblast interface and a small amount of virus scattered at the decidual layer. Viral antigen was also detected in alveolar epithelial cells and liver cells of fetuses, suggesting that H5N1avian influenza virus transmited vertically through placenta and infected fetuses. Focal necrosis and inflammatory cell infiltration appeared in the placenta of the infected mice. Inflammatory cytokines IFN-y, TNF-a and IL-6in placenta were significantly increased in late virus infection. Pregnancy failure such as miscarriage and necrosis was observed. To explore the mechanism of the vertical transmission of the H5N1virus, we detected the virus in blood cells, receptors distribution in placenta, and virus infection ability in trophoblast cell. Mononuclear cells and multinucleated cells could both be infected by virus, indicating that blood cells could serve as carriers that transport virus to placental tissue. Both the SA-a2,6-Gal and SA-a2,3-Gal receptors were expressed in placental vascular endothelial cells, trophoblast cell, and epithelial cell in chorionic plate. The virus could infect and replicate in trophoblast cell cultured in vitro. The results proved that the H5N1virus could reach the placenta by infecting blood cells as carriers and infect placental cells. The damage induced directly by virus and inflammation caused placental tissue necrosis and structural integrity broken, which facilitated virus breaking through placental barrier and infected fetuses, leading to pregnancy failure.To evaluate the risk of avian influenza infection of poultry workers, we conducted an H5, H7, H9serological study and questionnaire survey in Beijing poultry farms.14persons in305(4.49%) were positive for H9. Questionnaire survey suggested that most of workers had not recognized the threat of bird flu.In summary, this study provides evidence for the transmissibility of the H5N1virus in mammal animal. Studies on the mechanism of the pathogenesis of H5N1virus is significant for avian influenza prevention and control.更多还原