登革病毒2型及其E蛋白与宿主细胞氧化还原状态的关系
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摘要
登革病毒(dengue virus,DV)是黄病毒属的单股正链RNA病毒,分为四个血清型(DV1~4)。DV主要通过埃及伊蚊和白纹伊蚊传播,每种血清型都可引起人类登革热(classical dengue fever, DF)和登革出血热/登革休克综合征(dengue hemorrhagic fever/dengue shock syndrome,DHF/DSS)。近年来,随着旅游业的发展和地球温暖化,DF和DHF/DSS流行、暴发越来越频繁。DHF/DSS患者病死率高,发病机制不清。
较多的临床研究显示肝细胞是DV的重要靶细胞之一,肝损害在DHF/DSS发病过程中占有重要地位:1)肝组织中可以检测到DV抗原,并能分离到具有感染性的病毒;2)DV还可以感染体外培养的HepG2,HuH-7,HA22T,Hep3B,PLC等人类肝癌细胞株,培养上清中也可检测到成熟的病毒颗粒;3)DHF/DSS病人常常出现肝肿大,尸检可见肝组织脂肪变性,枯否氏细胞肥大等。血清转氨酶水平升高,凝血酶含量下降,其幅度变化与肝损害程度及出血、休克的发生密切相关。据文献报道,DV可以直接感染肝细胞,引起肝脏损伤,换言之,肝脏可能是DV重要的靶器官之一。然而,在过去相当长的时间里,学者们对DV感染后凝血系统、血管内皮细胞及炎症因子变化做了较多的研究,与之相比,肝细胞损伤机制所受关注较少,如能发现病毒复制对肝细胞哪些方面产生了影响,找到宿主细胞参与病毒复制的关键分子,不仅对DV感染机制的阐明有重要意义,同时也为进一步研究抗病毒药物奠定重要的基础。
生理情况下,活性氧(reactive oxygen species,ROS)在体内维持在有利无害的极低水平,参与多种生命过程。当机体受到不利条件时,ROS在体内增多并引起组织细胞氧化损伤的病理过程,则称为氧化应激(Oxidative Stress,OS)。新近研究表明细胞氧化还原状态(redox state)的平衡参与病毒复制和致病过程。病毒由于缺乏能量代谢系统和必要的酶类而不能独立生存,严格依赖于宿主细胞。进入细胞后利用细胞的合成代谢系统进行病毒大分子合成,因而扰乱了宿主细胞的自身代谢和生理功能,导致细胞的氧化还原平衡被打破。细胞通过产生还原型谷胱苷肽(reduced glutathione,GSH),超氧化物歧化酶(SOD),硫氧还蛋白,触酶等抗氧化分子,保持它的还原状态。其中含三个半胱氨酸的GSH在真核细胞抗氧化方面最为重要。多种病毒如人免疫缺陷病毒(human immunodeficiency virus,HIV)、丙型肝炎病毒(hepatitis C virus,HCV)等体内外感染均可改变细胞内氧化还原状态,使其处于促氧化状态(pro-oxidant),即OS。病毒可通过增加细胞内氧化剂含量,或抑制抗氧化剂合成来影响宿主细胞氧化平衡,表现为氧化水平升高,抗氧化能力下降。Peterhans等最先于1979年发现用RNA病毒感染宿主细胞后,吞噬细胞可以产生ROS。他们用仙台病毒(Sendai Virus,SV)感染小鼠的脾细胞,最先证实病毒感染能使细胞产生ROS。1987年他们又发现流感病毒和副粘病毒体外可激活单核细胞和多核白细胞产生ROS。HIV感染时,细胞质、外周血淋巴细胞和肺表皮细胞浸出液中的抗氧化剂水平降低,含高水平GSH的T淋巴细胞被选择性丢失。Hennet等人发现感染A型流感病毒的小鼠肺脏内抗氧化物质,如GSH和维生素C和E,都有很大程度的下降。这说明病毒感染与氧化应激密切相关。此外,陆续报道的还有1型单纯疱疹病毒(herpessimplex virus-1,HSV-1)、HCV等,这些病毒感染均可使细胞内的GSH下降。研究认为病毒感染过程中的氧化还原状态的改变是GSH消耗的结果,不同病毒感染不同细胞对GSH消耗程度、持续时间和诱导机制不尽相同。副流感病毒在引起上皮细胞病变时,GSH的水平急剧降低。而HIV感染人巨噬细胞时只有慢性感染建立后才可观察到明显的抗氧化水平的降低。病毒感染所致的细胞促氧化状态,可能是在病毒与宿主细胞相互作用过程中,触发了某些因子,最终引起细胞内氧化还原状态改变,进而诱导细胞凋亡或细胞过度增殖等病理过程。上述研究提示病毒感染可能参与宿主细胞的氧化应激过程。进一步对病理生理条件下GSH代谢的研究,将有助于人们对疾病发生机制的认识,同时为疾病的治疗提供新的思路。目前对于DV感染引起宿主细胞氧化还原状态改变的报道较少。
最近,有学者报道感染DV的病人体内氧化还原状态发生改变。因此推测,与其他急性病毒感染相似,DV感染对肝脏的直接损害作用可能是通过影响肝细胞的氧化还原状态引起的。在DV的10个蛋白中,E蛋白是位于DV表面的结构蛋白,是病毒颗粒的主要包膜蛋白。E蛋白在毒粒侵染细胞过程中起重要作用,E蛋白上可能具有某些宿主细胞表面受体结合的配体。E蛋白作为病毒的吸附蛋白,在病毒进入细胞过程中发挥关键作用。那么,E蛋白对宿主细胞的氧化还原状态又有何影响?鉴于以上的研究背景,本研究拟通过检测登革病毒2型(DV2)感染和稳定表达E蛋白的人肝癌细胞株HepG2细胞内外GSH的变化,以及药物处理改变细胞内GSH水平后病毒滴度的变化,反映DV2感染引起的宿主细胞氧化还原状态的改变及其对病毒感染的影响,初步探讨DV2感染与细胞内氧化还原状态的关系。期望本研究结果为深入阐明DHF/DSS的发病机制、DV的致病机理和进一步设计新型靶向抗病毒药物提供理论依据。
本研究主要结果与结论如下:
1. DV2感染对HepG2细胞内外GSH水平的影响
感染组加入DV2以MOI=10感染HepG2细胞,模拟感染组则加入56℃灭活30min的病毒液,同等条件置于37℃,以感染起始记为0时,在感染10min、20min、30min、40min、60min/1h、2h、6h、12h、24h、48h(后5个时相点吸附后1h,更换病毒维持液)时,分别用PBS充分洗涤细胞,胰蛋白酶消化,取出细胞。经过四次快速冻融,离心取上清用于GSH的测定。取相同数量的细胞经超声裂解用于测定细胞蛋白浓度。结果发现,病毒感染后10min、20min、30min、40min、60min/1h,HepG2细胞内GSH水平与模拟感染组比较,呈下降趋势,其中以30min下降最为明显,为16.82±0.86nmol/mg(n=5),与模拟感染组23.14±1.41nmol/mg(n=5)和感染组的其他时相点比较均有显著差异(P<0.01)。病毒感染后2h、6h、12h、24h、48h,HepG2细胞内GSH水平与模拟感染组比较,仍呈下降趋势,其中以2h,24h时下降较为明显,分别为29.51±3.16nmol/mg(n=5)和17.75±3.32nmol/mg(n=5),与相应时相点的模拟感染组35.45±3.55nmol/mg(n=5)和22.91±4.15nmol/mg(n=5)以及感染组的其它时相点比较,差异显著(P<0.05),其后逐渐恢复,到48h时已与模拟感染组无显著差异(P>0.05)。感染后30min上清中的GSH含量为47.86±3.00nmol/ml(n=4),较模拟感染组升高33.09%,且有显著差异(P<0.05),而模拟感染组与病毒原液则无显著差异(P>0.05)。以上结果说明DV2感染能够使HepG2细胞内GSH水平下降,改变宿主细胞的氧化还原状态,且呈现阶段性变化,推测可能与病毒的复制过程有关。结合感染后同一时相点细胞外GSH水平的升高和文献报道,推测DV2感染后30min细胞内GSH水平的快速降低可能是由于DV2感染所致细胞膜通透性增加、GSH的外漏引起的。
2. DV2 E蛋白对宿主细胞内外GSH水平的影响
首先构建了稳定表达E蛋白的HepG2细胞株pRe-E/HepG2,并且通过PCR、酶切、核苷酸测序及间接免疫荧光法进行了鉴定和检测,确认了该细胞中E蛋白的表达。同时构建了稳定转染空质粒的细胞株pRe-E/HepG2作为对照。
然后测定了pRe-E/HepG2细胞内外GSH的水平。结果:pRe-E/HepG2细胞内GSH水平与pRe/HepG2细胞对照组比较,呈下降趋势。pRe-E/HepG2细胞内GSH平均浓度为25.61±2.23nmol/mg(n=4),pRe/HepG2细胞内的GSH平均浓度为33.38±1.07nmol/mg(n=4),与pRe/HepG2细胞比较,pRe-E/HepG2细胞内GSH下降了24.3%(P<0.01)。取对数生长期pRe-E/HepG2和pRe/HepG2细胞的培养上清0.5ml测定GSH浓度,发现pRe-E/HepG2细胞上清中GSH含量平均值为36.72±1.40nmol/ml(n=4),pRe/HepG2细胞上清中GSH含量平均为57.41±2.00nmol/ml(n=4),前者相较后者明显降低,两者差异显著(P<0.05),前者较后者平均下降了36.35%。以上结果表明,稳定表达E蛋白的pRe-E/HepG2细胞较稳定转染空质粒的pRe/HepG2细胞,细胞内外GSH浓度均下降显著,提示E蛋白在宿主细胞中的表达不仅可以改变细胞内的氧化还原状态,还可以使宿主细胞外的GSH水平降低,在DV2感染诱使的细胞氧化还原状态变化的过程中,E蛋白可能起重要作用。
3. BSO及外源性GSH处理对DV2感染的影响
依据MTT实验结果并参照文献,选择BSO的处理浓度为0.2mM和1mM,外源性GSH的处理浓度为10mM和20mM。
在不感染病毒的情况下,培养上清中加入0.2mM、1mM BSO处理18h,可使细胞内GSH含量由空白对照组的24.53±2.59nmol/mg(n = 5)分别下降至14.29±1.48nmol/mg(n=5)、14.05±1.93nmol/mg(n=5)(P<0.05),且两种浓度下降幅度无显著差异(P>0.05),幅度平均为42.24%(n=5)。在DV2感染+BSO处理组,感染前18h分别用0.2mM、1mM BSO预处理HepG2细胞,然后用DV2以MOI=1感染HepG2细胞,并维持BSO浓度至感染24h,空白对照组不加药物处理。感染后24h收取病毒上清,噬斑试验测定病毒滴度(PFU/ml)。结果发现0.2mM与1mM BSO处理组病毒滴度较空白对照组分别上升了119.79%(n=5)和127.51%(n=5),与空白对照组比较差别显著(P<0.05),但两种浓度BSO处理组的病毒滴度上升幅度无显著差异(P>0.05)。
在不感染病毒的情况下,培养上清中加入10mM、20mM GSH溶液与空白对照组相比,细胞内GSH含量均无显著差异(P>0.05);在DV2感染+GSH处理组,从感染起始到感染24h维持上清内GSH浓度分别为10mM、20mM,空白对照组不加药物处理。感染后24h收取病毒上清,测定病毒滴度(PFU/ml)。结果发现较空白对照组而言,10mM与20mM GSH处理组病毒滴度分别下降40.24%(n=5)和56.62%(n=5),两种浓度GSH处理组均与空白对照组差异显著(P<0.05),且20mM GSH处理组病毒滴度下降更为明显(P<0.05);以上结果说明不仅DV2可以引起细胞内的氧化还原状态改变,细胞内的氧化还原状态反过来也可以影响DV2的复制和增殖。
以上结果为阐明DV的致病机理和进一步设计靶向抗病毒药物提供了初步的实验依据。
Dengue virus(DV) is a member of mosquito-borne flaviviruses and possesses a positive-sense RNA genome consisting of one single open-reading frame(ORF). DV had four serotypes which are closely related antigenically(DV1~4). It’s transmitted by the mosquito Aedes aegypti and Aedes albopictus. Infection with DV can result in classical dengue fever(DF) and/or dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS). Epidemics of DHF/DSS have been more frequent since the 1980s in the tropics and subtropics worldwide such as Southeast Asia and South PRC. Therefore, DV infection has become a severe problem of the public health. DHF/DSS have high case fatality, and the pathogenesis of DHF/DSS is not well understood.
The liver has been increasingly recognized as a significant target organ in the pathogenesis of DV infection. Hepatic lesion plays an important role in DHF/DSS. DV antigen can be found in hepatic tissue, and infectious virus also can be isolated from liver. DV can infect cultural cells, such as HepG2, HuH-7, HA22T, Hep3B, PLC. Viral particles can be detected in supernatant.
Mammalian cell mainly produce energe by aerobic respiration. About 1～3% of the gaining oxygen turns into superoxide anion or its active derivate, called reactive oxygen species(ROS). ROS are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes.
Viral multiplication occurs exclusively within the host cell and thus influences on numerous factors that control cell machinery and metabolism. Several findings have demonstrated the involvement of the intracellular redox balance in the establishment of viral infection and the progression of viral-induced diseases. Reducing conditions are normally maintained within the cell by molecules, such as glutathione(GSH), superoxide dismutase, thioredoxin, and catalase, which constitute the system developed by cells to counteract oxidation.
GSH is a cysteine-containing tripeptide(y-glutamyl-cysteinyl-glycine), found in eukaryotic cells, which has a number of important functions in cell physiology. GSH provides cells with a large supply of reducing equivalents, thus acting as a major cellular antioxidant, via metabolic interconversion with its oxidized disulphide form(GSSG).
It has been reported that many viruses, such as sendai virus(SV), human immunodeficiency virus(HIV), influenza A virus, herpessimplex virus-1(HSV-1) and hepatitis C virus(HCV), were associated with oxidative stress resulting in a decrease in the total concentration of GSH. Whether and how antioxidants decrease in cells infected DV is rarely reported. DV E protein located at the superficial of DV particle is one of DV’s structural proteins, which plays an important role in initiation of infection. In the present study, we have investigated the intracellular and extracellular GSH content of HepG2 cells infected with dengue virus 2(DV2) or stably transfected by E protein, and assayed extracellular DV2 titers of cells treated by BSO or exogenous GSH, in order to understand the relationship between redox state changes in host cells and dengue virus infection.
RESULTS:
1. DV2 infection can induce changes of intracellular and extracellular GSH level of HepG2 cells
In order to study the effect of viral infection on the intracellular level of GSH, HepG2 cells were infected with DV2 and then level of intracellular GSH was assayed. One set of cells was mock-infected and used as control. Intracellular GSH content in DV2 infected HepG2 cells was detected at different time points(10min, 20min, 30min, 40min, 60min/1h, 2h, 6h, 12h, 24h, 48h after infection) by spectrophotometry quantitation. We found that GSH content shows a decreasing tendency after DV2 infection and the lowest values of intracellular GSH levels were seen at 30min, 2h, 24 h after infection respectively, and they showed significant difference from others(P<0.01).
In order to investigate whether the loss of intracellular GSH at 30min after DV2 infection was induced by membrane perturbation upon virus fusion, we assayed the GSH level in supernantant of infected and mock-infected HepG2 cells 30min after DV2 infection. At 30min after infection, GSH levels in each supernatant or stock virus suspension were assayed. We found a significantly increased GSH levels in supernatants of infected cell by 33.09% compared with that in supernatants mock-infected cell(P<0.05). But there is no significant difference between the GSH level of mock-infected cell supernatants and stock virus suspension. It suggested that DV2 infection might lead to the GSH depletion in HepG2 cells and GSH might be lost from cells undergoing viral infection, and the transient GSH depletion seen at 30min after DV2 asorption may due to GSH leakage.
2. DV2 E protein has effects on the intracellular and extracellular GSH level of HepG2 cells
It was proved that DV2 infection have effect on the intracellular level of GSH. We presumed that DV2 proteins maybe also affect intracellular level of GSH. We studied the levels of GSH in HepG2 cells expressing DV2 E protein(pRe-E/HepG2) and compared them with control cells(pRe/HepG2). Firstly, HepG2 cell stably expressing DV2 E protein or control plasmid were identified by PCR, restriction enzyme, nucleotide sequencing and indirect immunofluorescence(IFL) and named as pRe-E/HepG2 and pRe/HepG2 respectively.
In order to study the role of DV2 E protein in redox state of host cell, we assayed the intracellular and extracellular GSH level of pRe-E/HepG2 and pRe/HepG2 cells respectively. We found that GSH levels were significantly decreased in pRe-E/HepG2 cells by 24.3% compared with control cells pRe/HepG2. And GSH levels were significantly decreased in supernatant of pRe-E/HepG2 cells compared with that of control cells pRe/HepG2. This indicated that DV2 E protein stably expressing in host cells can decrease both the level of intracellular and extracellular GSH.
3. The effect of BSO or exogenous GSH on DV2 infection
In order to investigate the effect of intracellular GSH level on DV2 infection, HepG2 cells were treated with BSO or exogenous GSH. One set of cells were pre-treated with BSO for 18h and infected with DV2, then maintained in medium in the presence of BSO for 24h. Another set of cells were infected with DV2 and treated with exogenous GSH simultaneously, then maintained in medium in the presence of GSH for 24h. Treatment with BSO can reduce the synthesis of endogenous GSH by 42.24%(n=5). Treatment with GSH can maintain the level of intracellular GSH during infection. The supernatant of both groups was collected at 24h after infection and DV2 titer was detected by plaque assay (PFU/ml).We found that the virus titer significantly increased by treatment with BSO, and the virus titer significantly decreased by treatment with GSH. It indicated that not only DV2 infection can induce change of cellular redox state, but also cellular redox state affects DV2 infection.
In conclusion, our data presented may give a clue to interpreting some critical points of the complex interrelationship between GSH status and viral replication. Thus, based on this favourable profile, further research concerning the potential clinical efficacy of GSH against DV infection is warranted.
较多的临床研究显示肝细胞是DV的重要靶细胞之一,肝损害在DHF/DSS发病过程中占有重要地位:1)肝组织中可以检测到DV抗原,并能分离到具有感染性的病毒;2)DV还可以感染体外培养的HepG2,HuH-7,HA22T,Hep3B,PLC等人类肝癌细胞株,培养上清中也可检测到成熟的病毒颗粒;3)DHF/DSS病人常常出现肝肿大,尸检可见肝组织脂肪变性,枯否氏细胞肥大等。血清转氨酶水平升高,凝血酶含量下降,其幅度变化与肝损害程度及出血、休克的发生密切相关。据文献报道,DV可以直接感染肝细胞,引起肝脏损伤,换言之,肝脏可能是DV重要的靶器官之一。然而,在过去相当长的时间里,学者们对DV感染后凝血系统、血管内皮细胞及炎症因子变化做了较多的研究,与之相比,肝细胞损伤机制所受关注较少,如能发现病毒复制对肝细胞哪些方面产生了影响,找到宿主细胞参与病毒复制的关键分子,不仅对DV感染机制的阐明有重要意义,同时也为进一步研究抗病毒药物奠定重要的基础。
生理情况下,活性氧(reactive oxygen species,ROS)在体内维持在有利无害的极低水平,参与多种生命过程。当机体受到不利条件时,ROS在体内增多并引起组织细胞氧化损伤的病理过程,则称为氧化应激(Oxidative Stress,OS)。新近研究表明细胞氧化还原状态(redox state)的平衡参与病毒复制和致病过程。病毒由于缺乏能量代谢系统和必要的酶类而不能独立生存,严格依赖于宿主细胞。进入细胞后利用细胞的合成代谢系统进行病毒大分子合成,因而扰乱了宿主细胞的自身代谢和生理功能,导致细胞的氧化还原平衡被打破。细胞通过产生还原型谷胱苷肽(reduced glutathione,GSH),超氧化物歧化酶(SOD),硫氧还蛋白,触酶等抗氧化分子,保持它的还原状态。其中含三个半胱氨酸的GSH在真核细胞抗氧化方面最为重要。多种病毒如人免疫缺陷病毒(human immunodeficiency virus,HIV)、丙型肝炎病毒(hepatitis C virus,HCV)等体内外感染均可改变细胞内氧化还原状态,使其处于促氧化状态(pro-oxidant),即OS。病毒可通过增加细胞内氧化剂含量,或抑制抗氧化剂合成来影响宿主细胞氧化平衡,表现为氧化水平升高,抗氧化能力下降。Peterhans等最先于1979年发现用RNA病毒感染宿主细胞后,吞噬细胞可以产生ROS。他们用仙台病毒(Sendai Virus,SV)感染小鼠的脾细胞,最先证实病毒感染能使细胞产生ROS。1987年他们又发现流感病毒和副粘病毒体外可激活单核细胞和多核白细胞产生ROS。HIV感染时,细胞质、外周血淋巴细胞和肺表皮细胞浸出液中的抗氧化剂水平降低,含高水平GSH的T淋巴细胞被选择性丢失。Hennet等人发现感染A型流感病毒的小鼠肺脏内抗氧化物质,如GSH和维生素C和E,都有很大程度的下降。这说明病毒感染与氧化应激密切相关。此外,陆续报道的还有1型单纯疱疹病毒(herpessimplex virus-1,HSV-1)、HCV等,这些病毒感染均可使细胞内的GSH下降。研究认为病毒感染过程中的氧化还原状态的改变是GSH消耗的结果,不同病毒感染不同细胞对GSH消耗程度、持续时间和诱导机制不尽相同。副流感病毒在引起上皮细胞病变时,GSH的水平急剧降低。而HIV感染人巨噬细胞时只有慢性感染建立后才可观察到明显的抗氧化水平的降低。病毒感染所致的细胞促氧化状态,可能是在病毒与宿主细胞相互作用过程中,触发了某些因子,最终引起细胞内氧化还原状态改变,进而诱导细胞凋亡或细胞过度增殖等病理过程。上述研究提示病毒感染可能参与宿主细胞的氧化应激过程。进一步对病理生理条件下GSH代谢的研究,将有助于人们对疾病发生机制的认识,同时为疾病的治疗提供新的思路。目前对于DV感染引起宿主细胞氧化还原状态改变的报道较少。
最近,有学者报道感染DV的病人体内氧化还原状态发生改变。因此推测,与其他急性病毒感染相似,DV感染对肝脏的直接损害作用可能是通过影响肝细胞的氧化还原状态引起的。在DV的10个蛋白中,E蛋白是位于DV表面的结构蛋白,是病毒颗粒的主要包膜蛋白。E蛋白在毒粒侵染细胞过程中起重要作用,E蛋白上可能具有某些宿主细胞表面受体结合的配体。E蛋白作为病毒的吸附蛋白,在病毒进入细胞过程中发挥关键作用。那么,E蛋白对宿主细胞的氧化还原状态又有何影响?鉴于以上的研究背景,本研究拟通过检测登革病毒2型(DV2)感染和稳定表达E蛋白的人肝癌细胞株HepG2细胞内外GSH的变化,以及药物处理改变细胞内GSH水平后病毒滴度的变化,反映DV2感染引起的宿主细胞氧化还原状态的改变及其对病毒感染的影响,初步探讨DV2感染与细胞内氧化还原状态的关系。期望本研究结果为深入阐明DHF/DSS的发病机制、DV的致病机理和进一步设计新型靶向抗病毒药物提供理论依据。
本研究主要结果与结论如下:
1. DV2感染对HepG2细胞内外GSH水平的影响
感染组加入DV2以MOI=10感染HepG2细胞,模拟感染组则加入56℃灭活30min的病毒液,同等条件置于37℃,以感染起始记为0时,在感染10min、20min、30min、40min、60min/1h、2h、6h、12h、24h、48h(后5个时相点吸附后1h,更换病毒维持液)时,分别用PBS充分洗涤细胞,胰蛋白酶消化,取出细胞。经过四次快速冻融,离心取上清用于GSH的测定。取相同数量的细胞经超声裂解用于测定细胞蛋白浓度。结果发现,病毒感染后10min、20min、30min、40min、60min/1h,HepG2细胞内GSH水平与模拟感染组比较,呈下降趋势,其中以30min下降最为明显,为16.82±0.86nmol/mg(n=5),与模拟感染组23.14±1.41nmol/mg(n=5)和感染组的其他时相点比较均有显著差异(P<0.01)。病毒感染后2h、6h、12h、24h、48h,HepG2细胞内GSH水平与模拟感染组比较,仍呈下降趋势,其中以2h,24h时下降较为明显,分别为29.51±3.16nmol/mg(n=5)和17.75±3.32nmol/mg(n=5),与相应时相点的模拟感染组35.45±3.55nmol/mg(n=5)和22.91±4.15nmol/mg(n=5)以及感染组的其它时相点比较,差异显著(P<0.05),其后逐渐恢复,到48h时已与模拟感染组无显著差异(P>0.05)。感染后30min上清中的GSH含量为47.86±3.00nmol/ml(n=4),较模拟感染组升高33.09%,且有显著差异(P<0.05),而模拟感染组与病毒原液则无显著差异(P>0.05)。以上结果说明DV2感染能够使HepG2细胞内GSH水平下降,改变宿主细胞的氧化还原状态,且呈现阶段性变化,推测可能与病毒的复制过程有关。结合感染后同一时相点细胞外GSH水平的升高和文献报道,推测DV2感染后30min细胞内GSH水平的快速降低可能是由于DV2感染所致细胞膜通透性增加、GSH的外漏引起的。
2. DV2 E蛋白对宿主细胞内外GSH水平的影响
首先构建了稳定表达E蛋白的HepG2细胞株pRe-E/HepG2,并且通过PCR、酶切、核苷酸测序及间接免疫荧光法进行了鉴定和检测,确认了该细胞中E蛋白的表达。同时构建了稳定转染空质粒的细胞株pRe-E/HepG2作为对照。
然后测定了pRe-E/HepG2细胞内外GSH的水平。结果:pRe-E/HepG2细胞内GSH水平与pRe/HepG2细胞对照组比较,呈下降趋势。pRe-E/HepG2细胞内GSH平均浓度为25.61±2.23nmol/mg(n=4),pRe/HepG2细胞内的GSH平均浓度为33.38±1.07nmol/mg(n=4),与pRe/HepG2细胞比较,pRe-E/HepG2细胞内GSH下降了24.3%(P<0.01)。取对数生长期pRe-E/HepG2和pRe/HepG2细胞的培养上清0.5ml测定GSH浓度,发现pRe-E/HepG2细胞上清中GSH含量平均值为36.72±1.40nmol/ml(n=4),pRe/HepG2细胞上清中GSH含量平均为57.41±2.00nmol/ml(n=4),前者相较后者明显降低,两者差异显著(P<0.05),前者较后者平均下降了36.35%。以上结果表明,稳定表达E蛋白的pRe-E/HepG2细胞较稳定转染空质粒的pRe/HepG2细胞,细胞内外GSH浓度均下降显著,提示E蛋白在宿主细胞中的表达不仅可以改变细胞内的氧化还原状态,还可以使宿主细胞外的GSH水平降低,在DV2感染诱使的细胞氧化还原状态变化的过程中,E蛋白可能起重要作用。
3. BSO及外源性GSH处理对DV2感染的影响
依据MTT实验结果并参照文献,选择BSO的处理浓度为0.2mM和1mM,外源性GSH的处理浓度为10mM和20mM。
在不感染病毒的情况下,培养上清中加入0.2mM、1mM BSO处理18h,可使细胞内GSH含量由空白对照组的24.53±2.59nmol/mg(n = 5)分别下降至14.29±1.48nmol/mg(n=5)、14.05±1.93nmol/mg(n=5)(P<0.05),且两种浓度下降幅度无显著差异(P>0.05),幅度平均为42.24%(n=5)。在DV2感染+BSO处理组,感染前18h分别用0.2mM、1mM BSO预处理HepG2细胞,然后用DV2以MOI=1感染HepG2细胞,并维持BSO浓度至感染24h,空白对照组不加药物处理。感染后24h收取病毒上清,噬斑试验测定病毒滴度(PFU/ml)。结果发现0.2mM与1mM BSO处理组病毒滴度较空白对照组分别上升了119.79%(n=5)和127.51%(n=5),与空白对照组比较差别显著(P<0.05),但两种浓度BSO处理组的病毒滴度上升幅度无显著差异(P>0.05)。
在不感染病毒的情况下,培养上清中加入10mM、20mM GSH溶液与空白对照组相比,细胞内GSH含量均无显著差异(P>0.05);在DV2感染+GSH处理组,从感染起始到感染24h维持上清内GSH浓度分别为10mM、20mM,空白对照组不加药物处理。感染后24h收取病毒上清,测定病毒滴度(PFU/ml)。结果发现较空白对照组而言,10mM与20mM GSH处理组病毒滴度分别下降40.24%(n=5)和56.62%(n=5),两种浓度GSH处理组均与空白对照组差异显著(P<0.05),且20mM GSH处理组病毒滴度下降更为明显(P<0.05);以上结果说明不仅DV2可以引起细胞内的氧化还原状态改变,细胞内的氧化还原状态反过来也可以影响DV2的复制和增殖。
以上结果为阐明DV的致病机理和进一步设计靶向抗病毒药物提供了初步的实验依据。
Dengue virus(DV) is a member of mosquito-borne flaviviruses and possesses a positive-sense RNA genome consisting of one single open-reading frame(ORF). DV had four serotypes which are closely related antigenically(DV1~4). It’s transmitted by the mosquito Aedes aegypti and Aedes albopictus. Infection with DV can result in classical dengue fever(DF) and/or dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS). Epidemics of DHF/DSS have been more frequent since the 1980s in the tropics and subtropics worldwide such as Southeast Asia and South PRC. Therefore, DV infection has become a severe problem of the public health. DHF/DSS have high case fatality, and the pathogenesis of DHF/DSS is not well understood.
The liver has been increasingly recognized as a significant target organ in the pathogenesis of DV infection. Hepatic lesion plays an important role in DHF/DSS. DV antigen can be found in hepatic tissue, and infectious virus also can be isolated from liver. DV can infect cultural cells, such as HepG2, HuH-7, HA22T, Hep3B, PLC. Viral particles can be detected in supernatant.
Mammalian cell mainly produce energe by aerobic respiration. About 1～3% of the gaining oxygen turns into superoxide anion or its active derivate, called reactive oxygen species(ROS). ROS are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes.
Viral multiplication occurs exclusively within the host cell and thus influences on numerous factors that control cell machinery and metabolism. Several findings have demonstrated the involvement of the intracellular redox balance in the establishment of viral infection and the progression of viral-induced diseases. Reducing conditions are normally maintained within the cell by molecules, such as glutathione(GSH), superoxide dismutase, thioredoxin, and catalase, which constitute the system developed by cells to counteract oxidation.
GSH is a cysteine-containing tripeptide(y-glutamyl-cysteinyl-glycine), found in eukaryotic cells, which has a number of important functions in cell physiology. GSH provides cells with a large supply of reducing equivalents, thus acting as a major cellular antioxidant, via metabolic interconversion with its oxidized disulphide form(GSSG).
It has been reported that many viruses, such as sendai virus(SV), human immunodeficiency virus(HIV), influenza A virus, herpessimplex virus-1(HSV-1) and hepatitis C virus(HCV), were associated with oxidative stress resulting in a decrease in the total concentration of GSH. Whether and how antioxidants decrease in cells infected DV is rarely reported. DV E protein located at the superficial of DV particle is one of DV’s structural proteins, which plays an important role in initiation of infection. In the present study, we have investigated the intracellular and extracellular GSH content of HepG2 cells infected with dengue virus 2(DV2) or stably transfected by E protein, and assayed extracellular DV2 titers of cells treated by BSO or exogenous GSH, in order to understand the relationship between redox state changes in host cells and dengue virus infection.
RESULTS:
1. DV2 infection can induce changes of intracellular and extracellular GSH level of HepG2 cells
In order to study the effect of viral infection on the intracellular level of GSH, HepG2 cells were infected with DV2 and then level of intracellular GSH was assayed. One set of cells was mock-infected and used as control. Intracellular GSH content in DV2 infected HepG2 cells was detected at different time points(10min, 20min, 30min, 40min, 60min/1h, 2h, 6h, 12h, 24h, 48h after infection) by spectrophotometry quantitation. We found that GSH content shows a decreasing tendency after DV2 infection and the lowest values of intracellular GSH levels were seen at 30min, 2h, 24 h after infection respectively, and they showed significant difference from others(P<0.01).
In order to investigate whether the loss of intracellular GSH at 30min after DV2 infection was induced by membrane perturbation upon virus fusion, we assayed the GSH level in supernantant of infected and mock-infected HepG2 cells 30min after DV2 infection. At 30min after infection, GSH levels in each supernatant or stock virus suspension were assayed. We found a significantly increased GSH levels in supernatants of infected cell by 33.09% compared with that in supernatants mock-infected cell(P<0.05). But there is no significant difference between the GSH level of mock-infected cell supernatants and stock virus suspension. It suggested that DV2 infection might lead to the GSH depletion in HepG2 cells and GSH might be lost from cells undergoing viral infection, and the transient GSH depletion seen at 30min after DV2 asorption may due to GSH leakage.
2. DV2 E protein has effects on the intracellular and extracellular GSH level of HepG2 cells
It was proved that DV2 infection have effect on the intracellular level of GSH. We presumed that DV2 proteins maybe also affect intracellular level of GSH. We studied the levels of GSH in HepG2 cells expressing DV2 E protein(pRe-E/HepG2) and compared them with control cells(pRe/HepG2). Firstly, HepG2 cell stably expressing DV2 E protein or control plasmid were identified by PCR, restriction enzyme, nucleotide sequencing and indirect immunofluorescence(IFL) and named as pRe-E/HepG2 and pRe/HepG2 respectively.
In order to study the role of DV2 E protein in redox state of host cell, we assayed the intracellular and extracellular GSH level of pRe-E/HepG2 and pRe/HepG2 cells respectively. We found that GSH levels were significantly decreased in pRe-E/HepG2 cells by 24.3% compared with control cells pRe/HepG2. And GSH levels were significantly decreased in supernatant of pRe-E/HepG2 cells compared with that of control cells pRe/HepG2. This indicated that DV2 E protein stably expressing in host cells can decrease both the level of intracellular and extracellular GSH.
3. The effect of BSO or exogenous GSH on DV2 infection
In order to investigate the effect of intracellular GSH level on DV2 infection, HepG2 cells were treated with BSO or exogenous GSH. One set of cells were pre-treated with BSO for 18h and infected with DV2, then maintained in medium in the presence of BSO for 24h. Another set of cells were infected with DV2 and treated with exogenous GSH simultaneously, then maintained in medium in the presence of GSH for 24h. Treatment with BSO can reduce the synthesis of endogenous GSH by 42.24%(n=5). Treatment with GSH can maintain the level of intracellular GSH during infection. The supernatant of both groups was collected at 24h after infection and DV2 titer was detected by plaque assay (PFU/ml).We found that the virus titer significantly increased by treatment with BSO, and the virus titer significantly decreased by treatment with GSH. It indicated that not only DV2 infection can induce change of cellular redox state, but also cellular redox state affects DV2 infection.
In conclusion, our data presented may give a clue to interpreting some critical points of the complex interrelationship between GSH status and viral replication. Thus, based on this favourable profile, further research concerning the potential clinical efficacy of GSH against DV infection is warranted.
引文
1. Diamond MS, Edgil D, Roberts TG, et al.Infection of human cells by dengue virus is modulated by different cell types and viral strains. J Virol, 2000, 74(17):7814-23.
2. Felix A, Rey. Dengue virus envelope glycoprotein structure: New insight into its interaction during viral entry. Proc Nati Acad Sci USA, 2003, 100(12):6899–901.
3. Suksanpaisan L, Smith DR. Analysis of saturation binding and saturation infection for dengue serotypes 1 and 2 in liver cells. Intervirology, 2003, 46(1):50-5.
4. An J, Kimura-kuroda J, Hirabayashi, et al. Development of a novel mouse model for dengue virus infection. Virology, 1999, 263:70-7.
5. Lin YL, Lei HY, Lin YS, et al. Heparin inhibits dengue-2 virus infection of five human liver cell lines. Antiviral Res, 2002, 56(1):93-6.
6. Fridovich I. Superoxide dismutases advances in Enzymology & Related Areas of Molecular. Biology, 1986, 58:61-97.
7. Wu G, Fang YZ, Yang S,et al. Glutatione metabolism and its implications for health. J Nutrition, 2004, 134(3):489-92.
8. Bode AM, Liang HQ, Green EH, et al. Ascorbic acid recycling in Nb2 lymphoma cell: implications for tumor progression. Free Radic Biol Med, 1999, 26(1-2):136-47.
9. Jenner P. Oxidative stress in Parkinson'sdisease. Ann Neurol, 2003, 53:26-38.
10. Elliott SJ, Koliwad SK. Redox control of ion channel activity in vascular endothelial cells by glutathione. Microcirculation, 1997, 36:341-7.
11.石京山,江文德。外源性谷胱苷肽在大鼠的药代动力学和器官组织分布。上海医科大学学报,1996,23(2):122-5.
12. Akaike T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA, 1996, 93(6):2448-53.
13. Waris G, Turkson J, Hassanein T, et al. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol, 2005, 79(3):1569-80.
14. Okuda M, Li K, Beard MR, et al. Mitochondrial injury, oxidative stress, and antioxidantgene expression are induced by hepatitis C virus core protein. Gastroenterology, 2002, 122(2):366-75.
15. Ray G, Kumar V, Kapoor AV, et al. Status of antioxidants and other biochemical abnormalities in children with dengue fever. J Trop Pediatr, 1999, 45:4-7.
16. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB, 2003, 17(6):758-60.
17. Lin YL, Liu CC, Chuang JI, et al. Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells. J Virol, 2000, 276(1):114-26.
18. Gil L, Martinez G, Tapanes R, et al. Oxidative stress in adult dengue patients. Am J Trop Med Hyg, 2004, 71(5):652-7.
19. Peterhans E. Sendal virus stimulates chemiluminescence in mouse spleen cells. Biochem Biophys Res Commun, 1979, 91:383-92.
20. Peterhans E, Grob M, Bürge T, et al. Virus-induced formation of reactive oxygen intermediates in phagocytic cells. Free Radical Res Commun, 1987, 3:39-46.
21. Edeas MA, Emerit I, Khalfoun Y, et al. Clastogenic factors in plasma of HIV-1 infected patients act ivate HIV-1 replication in vitro: inhibition by superoxide dismutase. Free Radic Biol Med, 1997, 23:571-8.
22. Hennet T, Peterhans E, Stocker, R. Alterations in antioxidant defences in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
23. Palamara AT, Perno CF, Ciriolo MR, et al. Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication. Antiviral Research, 1995, 27:237-53.
24. Abdalla MY, Ahmad IM, Spitz DR, et al. Hepatitis C virus core and non-structural proteins lead to different effects on cellular antioxidant defenses. J Med Virol, 2005, 76:489-97.
25. Ciriolo MR, Palamara AT, Incerpi S, et al. Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection. J Biol Chem, 1997, 272(5):2700-8.
26. Ceme K M, Dede S, Bayiroglu F, et al. Relationship between antioxidant capacity and oxidative stress in children with acute hepatitis A. World J Gastroenterol, 2006,12(38):6212-5.
27. Droge W, Schulze-Osthoff K, Mihm S, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology. J FASEB, 1994, 8(14):1131-8.
28. Suliman HB, Ryan LK, Bishop L, et al. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase. Am J Physiol(Lung Cell Mol Physiol), 2001, 280(1):L69–78.
29. Jahoor F, Jackson A, Gazzard B, et al. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol, 1999, 276:E205-11.
30. Beck MA. The influence of antioxidant nutrients on viral infection. Nutr Rev, 1998, 1:S140–6.
31. Rayman MP. The importance of selenium to human health. Lancet, 2000, 356:233–41.
32. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on effect of sodium selenite in prevention of Keshan disease. J Chin Med, 1979, 92:471-6.
33. Beck MA, Kolbeck PC, Rohr L, et al. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol, 1994. 43:166-70.
34. Staal JT, Roederer M, Herzenberg LA, et al. Intracellular thiols regulate activation of nuclear factor-B and transcription of human immunodeficiency virus. Proc Natl Acad Sci USA, 1990, 87:9943-7.
35. Baruchel S, Wainberg MA, The role of oxidative stress in disease progression individuals infected by the human immunodeficiency virus. J Leukoc Biol, 1992, 52:111-4.
36. Nelson HK, Shi Q, Van Dael P, et al. Host nutritional selenium status as a driving force for influenza virus mutations. J FASEB, 2001, 15:1846-8.
37. Beck MA, Nelson HK, Shi Q, et al. Selenium deficiency increases the pathology of an influenza virus infection. J FASEB, 2001, 15:1481-3.
38. Staal FJ, Roederer M, Israelski DM, et al. Intracellular glutathione levels in T cell subsets decrease in HIV-infected indivi- duals. AIDS Res Hum Retroviruses, 1992, 8(2):305-11.
39. Tatu UC, Hammond, Helenius A. Folding and oligomerization. of influenzahemagglutinin in the ER and the intermediate compartment. J EMBO, 1995, 14:1340-8.
40. Ozawa M, Asano A, Okada Y. Importance of interpeptide disulfide bond in a viral glycoprotein with hemagglutination and neuraminidase activities. FEBS Lett, 1976, 70(1):145-9.
41. Yu SC, Ching LL, Chang HT, et al. Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus. J Virology, 1999, 6257-64.
42. Allen DS, Harry D. Glutathione is required for efficient production of infectious picornavirus virions. Virology, 2006, 353:258-67.
43. Mohankumar K, Ramasamy P. White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus. Virus Research, 2006, 115:69-75.
44. Hidaka L, Hino K, Korenaga M, et al. Stronger Neo-Minophagen C, a glycyrrhizin-containing preparation, protects liver against carbon tetrachloride-induced oxidative stress in transgenic mice expressing the hepatitis C virus polyprotein. Liver lnt, 2007. 27(6): 845-53.
45. Garaci E, Palamara AT, di Francesco P, et al. Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus. Biochem Biophys Res Commun, 1992, 188:1090-6.
1. Wulf D. Free radicals in the physiological control of cell function. Physiol Rev, 2002, 82(1):47-95.
2. Storz P. Reactive oxygen species in tumor progression. Front Biosci, 2005, 10:1881-96.
3. Ialoi C, Apel K, Danon A. Reactive oxygen signaling: the latest news. Curt Opin Plant Biol, 2004, 7(3):323-8.
4. Block G, Dietrich M, Norkus EP, et al. Factors associated with oxidative stress in human populations. Am J Epidemiol, 2002, 156:274-85.
5. Muller F. Reactive oxygen intermediates and human immunodeficiency virus (HIV) infection. Free Radic Biol Med, 1992, 13:651-57.
6. Pace GW, Leaf CC. The role of oxidative stress in HIV disease. Free Radic Biol Med, 1995, 19:523-8.
7. Talior I, Yarkoni M, Bashan N, et al. Increased glucose uptake promotes oxidative stress and PKC-delta activation in adipocytes of obese-insulin-resistant mice. Am J Physiol Endocrinol Metab, 2003, 285:E295-302.
8. Novalija E, Kevin LG, Camara AK, et al. Reactive oxygen species precede the epsilon isoform of protein kinase C in the anesthetic preconditioning signaling cascade. Anesthesiology, 2003, 99:421-28.
9. Torre D, Pugliese A, Speranza F. Role of nitric oxide in HIV-1 infection: friend or foe? Lancet Infect Dis, 2002, 2:273-80.
10. Schulze-Osthoff K, Baker AC, Vanhaesebroeck B, et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. J Biol Chem, 1992, 167:5317-23.
11. Flohe L, Brigelius-Flohe R, Saliou C, et al. Redox regulation of NF-KAPPA B activation. Free Radic Biol Med, 1997, 22:1115-26.
12. Goldstone SD, Hunt NH. Redox regulation of the mitogen-activated protein kinase pathway during lymphocyte activation. Biochim Biophys Acta. 1997, 1355:353-60.
13. Dobmeyer TS, Findhammer S, Dobmeyer JM, et al. Ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: Role for lymphocyte loss in HIV infection.Free Radic Biol Med, 1997, 22:775-85.
14. Elliott SJ, Koliwad SK. Redox control of ion channel activity in vascular endothelial cells by glutathione. Microcirculation, 1997, 36:341-7.
15. France M, Rene G, Pierre L, et al. Blood and plasma glutathione measured in subjects by HPLC: Relation to sex ,aging, biological Variables, and life habits. Clin Chemistry, 1995, 41:1509-17.
16. Rita P, Elena DV, Giuliana C, et al. HPLC with o-phthalaldehyde precolumn Derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue. Clin Chem, 1995, 41(3):448-54.
17.石京山,江文德。外源性谷胱苷肽在大鼠的药代动力学和器官组织分布。上海医科大学学报,1996,23(2):122-5。
18. De Quay B, Malinverni R, Lauterburg BH. Glutathione depletion in HIV infected patients:role of cysteine deficiency and effect of oral N-acetylcysteine.AIDS, 1992, 6:815-9.
19. Halliwell B. Free radicals, antioxidants and human disease: curiosity, cause, or consequence? The Lancet, 1994, 344:721-4.
20. Halliwell B. Oxidants and human disease: Some new concept. J FASEB, 1987, 1:358-64.
21. Akaike T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA. 1996, 93(6):2448-53.
22. Waris G, Turkson J, Hassanein T, et al. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol. 2005, 79(3):1569-80.
23. Okuda M, Li K, Beard, MR, et al. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology, 2002, 122(2):366-75.
24. Ray G, Kumar V, Kapoor AV et al. Status of antioxidants and other biochemical abnormalities in children with dengue fever. J Trop Pediatr, 1999, 45:4-7.
25. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB. 2003, 17(6):758-60.
26. Lin YL, Liu CC, Chuang, JI, et al. Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue- 2-virus-infected human liver cells. J Virol, 2000, 276(1):114-26.
27. Gil L, Martinez G, Tapanes R, et al. Oxidative stress in adult dengue patients. Am J Trop Med Hyg, 2004, 71(5):652-7.
28. Palamara AT, Perno CF, Ciriolo MR, et al. Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication. J Antiviral Res, 1995, 27(3):237-53.
29. Ciriolo MR, Palamara AT, Incerpi S, et al. Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection. J Biol Chem, 1997, 272(5):2700-8.
30. Ceme KM, Dede S, Bayiroglu F, et al. Relationship between antioxidant capacity and oxidative stress in children with acute hepatitis A. World J Gastroenterol, 2006, 12(38):6212-5.
31. Droge W, Schulze-Osthoff K, Mihm S, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology. J FASEB, 1994, 8(14):1131-8.
32. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB, 2003, 17(6):758-60.
33. Peterhans E. Sendal virus stimulates chemiluminescence in mouse spleen cells. Biochem Biophys Res Commun, 1979, 91:383-92.
34. Peterhans E, Grob M, Bürge T, et al. Virus-induced formation of reactive oxygen intermediates in phagocytic cells. Free Radical Res Commun, 1987, 3:39-46.
35. Hennet T, Peterhans E, Stocker R. Alterations in antioxidant defenses in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
36. Suliman HB, Ryan LK, Bishop L, et al. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase. Am J Physiol(Lung Cell Mol. Physiol.), 2001, 280:L69-78.
37. Hennet T, Peterhans E, Stocker R. Alterations in antioxidant defense in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
38. Jacoby DB, Choi AMK, Influenza virus induces expression of antioxidant genes in human epithelial cells. Free Radic Biol Med, 1994, 16:821-4.
39. Xie B, Zhou JF, Lu Q, et al. Oxidative stress in patients with acute coxsackie virus myocarditis. Biomed Environ Sci, 2002, 15:48-57.
40. Li Q, Bolli R, Qiu Y, et al. Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation. 2001, 103:1893-8.
41. Jareno EJ, Roma J, Romero B, et al. Serum malondialdehyde correlates with therapeutic efficiency of high activity antiretroviral therapies (HAART) in HIV-1 infected children. Free Radic Res, 2002, 36:341-4.
42. Beck MA, Williams-Toone D, Levander OA. Coxsackie virus B3-resistant mice become susceptible in Se/ vitamin E deficiency. Free Radic Biol Med, 2003, 34:1263-70.
43. Beck MA. The influence of antioxidant nutrients on viral infection. Nutr Rev, 1998, 1:S140-6.
44. Rayman MP. The importance of selenium to human health. Lancet, 2000, 356:233-41.
45. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on effect of sodium selenite in prevention of Keshan disease. J Chin Med, 1979, 92:471-6.
46. Beck MA, Kolbeck PC, Rohr L, et al. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol, 1994. 43:166-70.
47. Nelson HK, Shi Q, Van Dael P, et al. Host nutritional selenium status as a driving force for influenza virus mutations. J FASEB, 2001, 15:1846-8.
48. Beck MA, Nelson HK, Shi Q, et al. Selenium deficiency increases the pathology of an influenza virus infection. J FASEB, 2001, 15:1481-3.
49. Smith AD, Dawson H. Glutathione is required for efficient production of infectious picornavirus virions. Virology, 2006, 353:258-67.
50. Uehara M, Sato N. Impaired ability of neutrophils to produce oxygen-derived free radicals in patients with chronic liver disease and hepatocellular carcinoma. Hepatology, 1994, 20:326-30.
51. Liu RH, Baldwin B, Tennant BC, et al. Elevated formation of nitrate and N-nitrosodi-methylamine in woodchuck s associated with chronic woodchuck hepatitis virus infection. Cancer Res, 1991, 51:3925-9.
52. Valyi-Nagy T, Olson SJ, Valyi-Nagy K, et al. Herpes simplex virus type 1 latency in the murine nervous system is associated with oxidative damage to neurons. Virology, 2000,278:309-21.
53. Milatovic D, Zhang Y, Olson SJ, et al. Herpes simplex virus type1 encephalitis is associated with elevated levels of F2-isoprostanes and F4-neuroprostanes. J Neurovirol, 2002, 8:295-305.
54. Schar P. Spontaneous DNA damage, genome instability and cancer when DNA replication escapes control. Cell, 2001, 104:329-32.
55. Benencia F, Courreges MC, Gamba G, et al. Effect of aminoguanidine, a nitric oxide synthase inhibitor,on ocular infection with herpes simplex virus in Balb/c mice. Invest Ophthalmol Vis Sci, 2001, 42:1277-84.
56. Lim YJ, Chang SE, Choi JH, et al. Expression of inducible nitric oxide synthase in skin lesions of acute herpes zoster. J Dermatol Sci, 2002, 29:201-5.
57. Brown DR, Wong BS, Hafiz F, et al. Normal prion protein has an activity like that of superoxide dismutase. Biochem J, 1999, 344:1-5.
58. Thackray AM, Bujdoso R. PrP (c) expression influences the establishment of herpes simplex virus type 1 latency. J Virol, 2002, 76:2498-509.
59. Pata C, Yazar A, Altintas E, et al. Serum levels of intercellular adhesion molecule-1 and nitric oxide in patients with chronic hepatitis related to hepatitis C virus: connection fibrosis. Hepatogastroenterology, 2003, 50:794-7.
60.方平,吴同生。古拉定治疗急性甲型病毒性肝炎疗效观察。安徽医学,2001,22(6):68。
61. Dalpke AH, Thomssen R, Ritter K. Oxidative injury to endothelial cells due to Epstein-Barr virus-induced autoantibodies against manganese superoxide dismutase. J Med Virol, 2003, 71:408-16.
62. Cao JX, Teoh ML, Moon M, et al. Leporipoxvirus Cu-Zn superoxide dismutase homologs inhibit cellular superoxide dismutase, but are not essential for virus replication or virulence. Virology, 2002, 296:125-35.
63. Edeas MA, Emerit I, Khalfoun Y, et al. Clastogenic factors in plasma of HIV 21 infected patients activate HIV-1 replication in vitro: inhibit ion by superoxide dismutase. F ree Radic Biol Med, 1997, 23:571-8.
64. Trotti R, Rondanelli M, Anesi A, et al. Increased erythrocyte glutathione peroxidase activity and serum tumor necrosis factor-alpha in HIV-infected patients: relationship to on-going prothrombotic state. J Hematother Stem Cell Res, 2002, 11:369-75.
65. Gil L, Martinez G, Gonzalez I, et al. Contribution to characterization of oxidative stress in HIV/AIDS patients. Pharmacol Res, 2003, 47:217-24.
66. Treitinger A, Spada C, Verdi JC, et al. Decreased antioxidant defence in individuals infected by the human immunodeficiency virus. Eur J Clin Invest, 2000, 30:454-9.
67. Wang M, Howell JM, Libbey JE, et al. Manganese superoxide dismutase induction during measles virus infection. J Med Virol, 2003, 70:470-4.
68. Staal FJ, Roederer M, Israelski DM, et al. Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals. J AIDS Res Hum Retroviruses, 1992, 8(2):305-11.
69. Staal JT, Roederer M, Herzenberg LA, et al. Intracellular thiols regulate activation of nuclear factor-B and transcription of human immunodeficiency virus. Proc Natl Acad Sci USA, 1990, 87:9943-7.
70. Baruchel S, Wainberg MA. The role of oxidative stress in disease progression individuals infected by the human immunodeficiency virus. J Leukoc Biol, 1992, 52:111-4.
71. Pocidalo MA, Aillet F, Virelizier JL, et al. Influence of redox status of lymphocyets and monocytes on HIV t ranscription and replication. Immunobiol, 1995, 193:204-9.
72. Roederer M, Ela SW, Staal FJT, et al. N-acetylcysteine: A new app roach to anti-HIV therapy. AIDS Res. and Human Retrovirus, 1992, 8:209-16.
1. Malavige GN, Fernando S, Fernando DJ. Dengue viral infections. J Postgrad Med, 2004, 80(948):588–601.2.
2.肖维威,马文丽,郑文岭等.登革病毒的基因组结构及其基因检测.中华检验医学杂志, 2003, 26:188-9.
3. Gibbons RV, Vaughn DW. Dengue: an escalating problem. BMJ, 2002, 324:1563–6.
4. Chambers TJ, Hahn CS, Galler R, et al. Flavivirus genome orgnization, expression, and replication. Annu Rev Microbiol, 1990, 44:649.
5. Falgout B, Pethel M, Zhang YM, et al. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructrual proteins. J Virol, 1991, 65:2467.
6. Sven M, Sandra S, Ralf B. Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-Structural Protein 4B. J Biol Chem, 2006, 281(13):8854-63.
7. Ju-Tao G, Junpei H, Christoph S. West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon. J Virol, 2005, 79(3):1343–50.
8. Jorge L, Mu?oz J, Maudry LR, et al. Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses. J Virol, 2005, 79(13):8004–13.
9. Lin RJ, Chang BL, Yu HP, et al. Blocking of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a protein tyrosine phosphatase-mediated mechanism. J Virol, 2006, 80(12):5908-18。
10. Best SM, Morris KL, Shannon JG, et al. Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J Virol, 2005, 79(20):12828-39.
11.陈建忠,朱海红。IFNα/β信号传导研究进展.国外医学·流行病学传染病学分册,2003,30(5):266-8。
12. Carlos TS, Fearns R, Randall RE. Interferon-induced alterations in the pattern of parainfluenza virus 5 transcription and protein synthesis and the induction of virusinclusion bodies. J Virol, 2005, 79(22):14112-21.
13. Wansley EK, Dillon PJ, Gainey MD, et al. Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells. J Virol, 2005, 335(1):131-44.
14. Blindenbacher A, Duong FH, Hunziker L, et al. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology, 2003, 124(5):1465-75.
15. Jorge L, Mu?oz J, Gilma G, et al. Inhibition of interferon signaling by dengue virus. PNAS, 2003, 100(24):14333–8.
16. Wen JL, Xiang JW, Vladislav V, et al. Inhibition of Interferon Signaling by the New York 99 Strain and Kunjin Subtype of West Nile Virus Involves Blockage of STAT1 and STAT2 Activation by Nonstructural Proteins. J Virol, 2005, 79(3):1934–42.
17. Meleri J, Andrew D, Linda H, et al. Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression. J Virol, 2005, 79(9):5414–20.
18. Ramaswamy ML, Shi MM, Monick GW, et al. Specific inhibition of type I interferon signal transduction by respiratory syncytial virus. Cell Mol Biol, 2004,30:893–900.
19. Andrejeva J, Young DF, Goodbourn S, et al. Degradation of STAT1 and STAT2 by the V proteins of simian virus 5 and human parainfluenza virus type 2, respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons. J Virol, 2002, 76:2159–67.
20. Parisien J.P, Lau JF, Rodriguez JJ, et al. The V protein of human parainfluenza virus 2 antagonizes type I interferon responses by destabilizing signal transducer and activator of transcription 2. J Virol, 2001, 283:230–9.
21. Indira U, Alex C, Aruna S, et al. Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gene Viro, 2006, 87:2605–14.
22. Kathryn AH, Luella RM, Lara EG, et al. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4. J Virol, 2003, 312:222–32.
23. Wei FT, Yuki E, Masayuki T, et al. Molecular Basis for Adaptation of a ChimericDengue Type-4/Japanese Encephalitis Virus to Vero Cells. Microbiol.Immunol, 2005, 49(3):285-94.
24. Joseph EB, Gracielle GM, Cai-Yen F, et al. Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells. Vaccine, 2003, 21:4317–27.
25. Leitmeyer KC, Vaughn DW, Watts DM, et al. Dengue virus structural differences that correlate with pathogenesis. J Virol, 1999, 73(6):4738-47.
26. Dunster LM, Wang H, Ryman KD, et al. Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus. J Virol, 1999, 261(2):309–18.
27. Jennings AD, Gibson CA, Miller BR, et al. Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. J Infect Dis, 1994, 169(3):512–8.
28. Ni H, Chang GJ, Xie H, et al. Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14. J Gen Virol, 1995, 76(2):409–13.
2. Felix A, Rey. Dengue virus envelope glycoprotein structure: New insight into its interaction during viral entry. Proc Nati Acad Sci USA, 2003, 100(12):6899–901.
3. Suksanpaisan L, Smith DR. Analysis of saturation binding and saturation infection for dengue serotypes 1 and 2 in liver cells. Intervirology, 2003, 46(1):50-5.
4. An J, Kimura-kuroda J, Hirabayashi, et al. Development of a novel mouse model for dengue virus infection. Virology, 1999, 263:70-7.
5. Lin YL, Lei HY, Lin YS, et al. Heparin inhibits dengue-2 virus infection of five human liver cell lines. Antiviral Res, 2002, 56(1):93-6.
6. Fridovich I. Superoxide dismutases advances in Enzymology & Related Areas of Molecular. Biology, 1986, 58:61-97.
7. Wu G, Fang YZ, Yang S,et al. Glutatione metabolism and its implications for health. J Nutrition, 2004, 134(3):489-92.
8. Bode AM, Liang HQ, Green EH, et al. Ascorbic acid recycling in Nb2 lymphoma cell: implications for tumor progression. Free Radic Biol Med, 1999, 26(1-2):136-47.
9. Jenner P. Oxidative stress in Parkinson'sdisease. Ann Neurol, 2003, 53:26-38.
10. Elliott SJ, Koliwad SK. Redox control of ion channel activity in vascular endothelial cells by glutathione. Microcirculation, 1997, 36:341-7.
11.石京山,江文德。外源性谷胱苷肽在大鼠的药代动力学和器官组织分布。上海医科大学学报,1996,23(2):122-5.
12. Akaike T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA, 1996, 93(6):2448-53.
13. Waris G, Turkson J, Hassanein T, et al. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol, 2005, 79(3):1569-80.
14. Okuda M, Li K, Beard MR, et al. Mitochondrial injury, oxidative stress, and antioxidantgene expression are induced by hepatitis C virus core protein. Gastroenterology, 2002, 122(2):366-75.
15. Ray G, Kumar V, Kapoor AV, et al. Status of antioxidants and other biochemical abnormalities in children with dengue fever. J Trop Pediatr, 1999, 45:4-7.
16. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB, 2003, 17(6):758-60.
17. Lin YL, Liu CC, Chuang JI, et al. Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells. J Virol, 2000, 276(1):114-26.
18. Gil L, Martinez G, Tapanes R, et al. Oxidative stress in adult dengue patients. Am J Trop Med Hyg, 2004, 71(5):652-7.
19. Peterhans E. Sendal virus stimulates chemiluminescence in mouse spleen cells. Biochem Biophys Res Commun, 1979, 91:383-92.
20. Peterhans E, Grob M, Bürge T, et al. Virus-induced formation of reactive oxygen intermediates in phagocytic cells. Free Radical Res Commun, 1987, 3:39-46.
21. Edeas MA, Emerit I, Khalfoun Y, et al. Clastogenic factors in plasma of HIV-1 infected patients act ivate HIV-1 replication in vitro: inhibition by superoxide dismutase. Free Radic Biol Med, 1997, 23:571-8.
22. Hennet T, Peterhans E, Stocker, R. Alterations in antioxidant defences in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
23. Palamara AT, Perno CF, Ciriolo MR, et al. Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication. Antiviral Research, 1995, 27:237-53.
24. Abdalla MY, Ahmad IM, Spitz DR, et al. Hepatitis C virus core and non-structural proteins lead to different effects on cellular antioxidant defenses. J Med Virol, 2005, 76:489-97.
25. Ciriolo MR, Palamara AT, Incerpi S, et al. Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection. J Biol Chem, 1997, 272(5):2700-8.
26. Ceme K M, Dede S, Bayiroglu F, et al. Relationship between antioxidant capacity and oxidative stress in children with acute hepatitis A. World J Gastroenterol, 2006,12(38):6212-5.
27. Droge W, Schulze-Osthoff K, Mihm S, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology. J FASEB, 1994, 8(14):1131-8.
28. Suliman HB, Ryan LK, Bishop L, et al. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase. Am J Physiol(Lung Cell Mol Physiol), 2001, 280(1):L69–78.
29. Jahoor F, Jackson A, Gazzard B, et al. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol, 1999, 276:E205-11.
30. Beck MA. The influence of antioxidant nutrients on viral infection. Nutr Rev, 1998, 1:S140–6.
31. Rayman MP. The importance of selenium to human health. Lancet, 2000, 356:233–41.
32. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on effect of sodium selenite in prevention of Keshan disease. J Chin Med, 1979, 92:471-6.
33. Beck MA, Kolbeck PC, Rohr L, et al. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol, 1994. 43:166-70.
34. Staal JT, Roederer M, Herzenberg LA, et al. Intracellular thiols regulate activation of nuclear factor-B and transcription of human immunodeficiency virus. Proc Natl Acad Sci USA, 1990, 87:9943-7.
35. Baruchel S, Wainberg MA, The role of oxidative stress in disease progression individuals infected by the human immunodeficiency virus. J Leukoc Biol, 1992, 52:111-4.
36. Nelson HK, Shi Q, Van Dael P, et al. Host nutritional selenium status as a driving force for influenza virus mutations. J FASEB, 2001, 15:1846-8.
37. Beck MA, Nelson HK, Shi Q, et al. Selenium deficiency increases the pathology of an influenza virus infection. J FASEB, 2001, 15:1481-3.
38. Staal FJ, Roederer M, Israelski DM, et al. Intracellular glutathione levels in T cell subsets decrease in HIV-infected indivi- duals. AIDS Res Hum Retroviruses, 1992, 8(2):305-11.
39. Tatu UC, Hammond, Helenius A. Folding and oligomerization. of influenzahemagglutinin in the ER and the intermediate compartment. J EMBO, 1995, 14:1340-8.
40. Ozawa M, Asano A, Okada Y. Importance of interpeptide disulfide bond in a viral glycoprotein with hemagglutination and neuraminidase activities. FEBS Lett, 1976, 70(1):145-9.
41. Yu SC, Ching LL, Chang HT, et al. Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus. J Virology, 1999, 6257-64.
42. Allen DS, Harry D. Glutathione is required for efficient production of infectious picornavirus virions. Virology, 2006, 353:258-67.
43. Mohankumar K, Ramasamy P. White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus. Virus Research, 2006, 115:69-75.
44. Hidaka L, Hino K, Korenaga M, et al. Stronger Neo-Minophagen C, a glycyrrhizin-containing preparation, protects liver against carbon tetrachloride-induced oxidative stress in transgenic mice expressing the hepatitis C virus polyprotein. Liver lnt, 2007. 27(6): 845-53.
45. Garaci E, Palamara AT, di Francesco P, et al. Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus. Biochem Biophys Res Commun, 1992, 188:1090-6.
1. Wulf D. Free radicals in the physiological control of cell function. Physiol Rev, 2002, 82(1):47-95.
2. Storz P. Reactive oxygen species in tumor progression. Front Biosci, 2005, 10:1881-96.
3. Ialoi C, Apel K, Danon A. Reactive oxygen signaling: the latest news. Curt Opin Plant Biol, 2004, 7(3):323-8.
4. Block G, Dietrich M, Norkus EP, et al. Factors associated with oxidative stress in human populations. Am J Epidemiol, 2002, 156:274-85.
5. Muller F. Reactive oxygen intermediates and human immunodeficiency virus (HIV) infection. Free Radic Biol Med, 1992, 13:651-57.
6. Pace GW, Leaf CC. The role of oxidative stress in HIV disease. Free Radic Biol Med, 1995, 19:523-8.
7. Talior I, Yarkoni M, Bashan N, et al. Increased glucose uptake promotes oxidative stress and PKC-delta activation in adipocytes of obese-insulin-resistant mice. Am J Physiol Endocrinol Metab, 2003, 285:E295-302.
8. Novalija E, Kevin LG, Camara AK, et al. Reactive oxygen species precede the epsilon isoform of protein kinase C in the anesthetic preconditioning signaling cascade. Anesthesiology, 2003, 99:421-28.
9. Torre D, Pugliese A, Speranza F. Role of nitric oxide in HIV-1 infection: friend or foe? Lancet Infect Dis, 2002, 2:273-80.
10. Schulze-Osthoff K, Baker AC, Vanhaesebroeck B, et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. J Biol Chem, 1992, 167:5317-23.
11. Flohe L, Brigelius-Flohe R, Saliou C, et al. Redox regulation of NF-KAPPA B activation. Free Radic Biol Med, 1997, 22:1115-26.
12. Goldstone SD, Hunt NH. Redox regulation of the mitogen-activated protein kinase pathway during lymphocyte activation. Biochim Biophys Acta. 1997, 1355:353-60.
13. Dobmeyer TS, Findhammer S, Dobmeyer JM, et al. Ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: Role for lymphocyte loss in HIV infection.Free Radic Biol Med, 1997, 22:775-85.
14. Elliott SJ, Koliwad SK. Redox control of ion channel activity in vascular endothelial cells by glutathione. Microcirculation, 1997, 36:341-7.
15. France M, Rene G, Pierre L, et al. Blood and plasma glutathione measured in subjects by HPLC: Relation to sex ,aging, biological Variables, and life habits. Clin Chemistry, 1995, 41:1509-17.
16. Rita P, Elena DV, Giuliana C, et al. HPLC with o-phthalaldehyde precolumn Derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue. Clin Chem, 1995, 41(3):448-54.
17.石京山,江文德。外源性谷胱苷肽在大鼠的药代动力学和器官组织分布。上海医科大学学报,1996,23(2):122-5。
18. De Quay B, Malinverni R, Lauterburg BH. Glutathione depletion in HIV infected patients:role of cysteine deficiency and effect of oral N-acetylcysteine.AIDS, 1992, 6:815-9.
19. Halliwell B. Free radicals, antioxidants and human disease: curiosity, cause, or consequence? The Lancet, 1994, 344:721-4.
20. Halliwell B. Oxidants and human disease: Some new concept. J FASEB, 1987, 1:358-64.
21. Akaike T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA. 1996, 93(6):2448-53.
22. Waris G, Turkson J, Hassanein T, et al. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol. 2005, 79(3):1569-80.
23. Okuda M, Li K, Beard, MR, et al. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology, 2002, 122(2):366-75.
24. Ray G, Kumar V, Kapoor AV et al. Status of antioxidants and other biochemical abnormalities in children with dengue fever. J Trop Pediatr, 1999, 45:4-7.
25. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB. 2003, 17(6):758-60.
26. Lin YL, Liu CC, Chuang, JI, et al. Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue- 2-virus-infected human liver cells. J Virol, 2000, 276(1):114-26.
27. Gil L, Martinez G, Tapanes R, et al. Oxidative stress in adult dengue patients. Am J Trop Med Hyg, 2004, 71(5):652-7.
28. Palamara AT, Perno CF, Ciriolo MR, et al. Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication. J Antiviral Res, 1995, 27(3):237-53.
29. Ciriolo MR, Palamara AT, Incerpi S, et al. Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection. J Biol Chem, 1997, 272(5):2700-8.
30. Ceme KM, Dede S, Bayiroglu F, et al. Relationship between antioxidant capacity and oxidative stress in children with acute hepatitis A. World J Gastroenterol, 2006, 12(38):6212-5.
31. Droge W, Schulze-Osthoff K, Mihm S, et al. Functions of glutathione and glutathione disulfide in immunology and immunopathology. J FASEB, 1994, 8(14):1131-8.
32. Nencioni L, Iuvara A, Aquilano K, et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. J FASEB, 2003, 17(6):758-60.
33. Peterhans E. Sendal virus stimulates chemiluminescence in mouse spleen cells. Biochem Biophys Res Commun, 1979, 91:383-92.
34. Peterhans E, Grob M, Bürge T, et al. Virus-induced formation of reactive oxygen intermediates in phagocytic cells. Free Radical Res Commun, 1987, 3:39-46.
35. Hennet T, Peterhans E, Stocker R. Alterations in antioxidant defenses in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
36. Suliman HB, Ryan LK, Bishop L, et al. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase. Am J Physiol(Lung Cell Mol. Physiol.), 2001, 280:L69-78.
37. Hennet T, Peterhans E, Stocker R. Alterations in antioxidant defense in lung and liver of mice infected with influenza A virus. J Gen Virol, 1992, 73:39-46.
38. Jacoby DB, Choi AMK, Influenza virus induces expression of antioxidant genes in human epithelial cells. Free Radic Biol Med, 1994, 16:821-4.
39. Xie B, Zhou JF, Lu Q, et al. Oxidative stress in patients with acute coxsackie virus myocarditis. Biomed Environ Sci, 2002, 15:48-57.
40. Li Q, Bolli R, Qiu Y, et al. Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation. 2001, 103:1893-8.
41. Jareno EJ, Roma J, Romero B, et al. Serum malondialdehyde correlates with therapeutic efficiency of high activity antiretroviral therapies (HAART) in HIV-1 infected children. Free Radic Res, 2002, 36:341-4.
42. Beck MA, Williams-Toone D, Levander OA. Coxsackie virus B3-resistant mice become susceptible in Se/ vitamin E deficiency. Free Radic Biol Med, 2003, 34:1263-70.
43. Beck MA. The influence of antioxidant nutrients on viral infection. Nutr Rev, 1998, 1:S140-6.
44. Rayman MP. The importance of selenium to human health. Lancet, 2000, 356:233-41.
45. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on effect of sodium selenite in prevention of Keshan disease. J Chin Med, 1979, 92:471-6.
46. Beck MA, Kolbeck PC, Rohr L, et al. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol, 1994. 43:166-70.
47. Nelson HK, Shi Q, Van Dael P, et al. Host nutritional selenium status as a driving force for influenza virus mutations. J FASEB, 2001, 15:1846-8.
48. Beck MA, Nelson HK, Shi Q, et al. Selenium deficiency increases the pathology of an influenza virus infection. J FASEB, 2001, 15:1481-3.
49. Smith AD, Dawson H. Glutathione is required for efficient production of infectious picornavirus virions. Virology, 2006, 353:258-67.
50. Uehara M, Sato N. Impaired ability of neutrophils to produce oxygen-derived free radicals in patients with chronic liver disease and hepatocellular carcinoma. Hepatology, 1994, 20:326-30.
51. Liu RH, Baldwin B, Tennant BC, et al. Elevated formation of nitrate and N-nitrosodi-methylamine in woodchuck s associated with chronic woodchuck hepatitis virus infection. Cancer Res, 1991, 51:3925-9.
52. Valyi-Nagy T, Olson SJ, Valyi-Nagy K, et al. Herpes simplex virus type 1 latency in the murine nervous system is associated with oxidative damage to neurons. Virology, 2000,278:309-21.
53. Milatovic D, Zhang Y, Olson SJ, et al. Herpes simplex virus type1 encephalitis is associated with elevated levels of F2-isoprostanes and F4-neuroprostanes. J Neurovirol, 2002, 8:295-305.
54. Schar P. Spontaneous DNA damage, genome instability and cancer when DNA replication escapes control. Cell, 2001, 104:329-32.
55. Benencia F, Courreges MC, Gamba G, et al. Effect of aminoguanidine, a nitric oxide synthase inhibitor,on ocular infection with herpes simplex virus in Balb/c mice. Invest Ophthalmol Vis Sci, 2001, 42:1277-84.
56. Lim YJ, Chang SE, Choi JH, et al. Expression of inducible nitric oxide synthase in skin lesions of acute herpes zoster. J Dermatol Sci, 2002, 29:201-5.
57. Brown DR, Wong BS, Hafiz F, et al. Normal prion protein has an activity like that of superoxide dismutase. Biochem J, 1999, 344:1-5.
58. Thackray AM, Bujdoso R. PrP (c) expression influences the establishment of herpes simplex virus type 1 latency. J Virol, 2002, 76:2498-509.
59. Pata C, Yazar A, Altintas E, et al. Serum levels of intercellular adhesion molecule-1 and nitric oxide in patients with chronic hepatitis related to hepatitis C virus: connection fibrosis. Hepatogastroenterology, 2003, 50:794-7.
60.方平,吴同生。古拉定治疗急性甲型病毒性肝炎疗效观察。安徽医学,2001,22(6):68。
61. Dalpke AH, Thomssen R, Ritter K. Oxidative injury to endothelial cells due to Epstein-Barr virus-induced autoantibodies against manganese superoxide dismutase. J Med Virol, 2003, 71:408-16.
62. Cao JX, Teoh ML, Moon M, et al. Leporipoxvirus Cu-Zn superoxide dismutase homologs inhibit cellular superoxide dismutase, but are not essential for virus replication or virulence. Virology, 2002, 296:125-35.
63. Edeas MA, Emerit I, Khalfoun Y, et al. Clastogenic factors in plasma of HIV 21 infected patients activate HIV-1 replication in vitro: inhibit ion by superoxide dismutase. F ree Radic Biol Med, 1997, 23:571-8.
64. Trotti R, Rondanelli M, Anesi A, et al. Increased erythrocyte glutathione peroxidase activity and serum tumor necrosis factor-alpha in HIV-infected patients: relationship to on-going prothrombotic state. J Hematother Stem Cell Res, 2002, 11:369-75.
65. Gil L, Martinez G, Gonzalez I, et al. Contribution to characterization of oxidative stress in HIV/AIDS patients. Pharmacol Res, 2003, 47:217-24.
66. Treitinger A, Spada C, Verdi JC, et al. Decreased antioxidant defence in individuals infected by the human immunodeficiency virus. Eur J Clin Invest, 2000, 30:454-9.
67. Wang M, Howell JM, Libbey JE, et al. Manganese superoxide dismutase induction during measles virus infection. J Med Virol, 2003, 70:470-4.
68. Staal FJ, Roederer M, Israelski DM, et al. Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals. J AIDS Res Hum Retroviruses, 1992, 8(2):305-11.
69. Staal JT, Roederer M, Herzenberg LA, et al. Intracellular thiols regulate activation of nuclear factor-B and transcription of human immunodeficiency virus. Proc Natl Acad Sci USA, 1990, 87:9943-7.
70. Baruchel S, Wainberg MA. The role of oxidative stress in disease progression individuals infected by the human immunodeficiency virus. J Leukoc Biol, 1992, 52:111-4.
71. Pocidalo MA, Aillet F, Virelizier JL, et al. Influence of redox status of lymphocyets and monocytes on HIV t ranscription and replication. Immunobiol, 1995, 193:204-9.
72. Roederer M, Ela SW, Staal FJT, et al. N-acetylcysteine: A new app roach to anti-HIV therapy. AIDS Res. and Human Retrovirus, 1992, 8:209-16.
1. Malavige GN, Fernando S, Fernando DJ. Dengue viral infections. J Postgrad Med, 2004, 80(948):588–601.2.
2.肖维威,马文丽,郑文岭等.登革病毒的基因组结构及其基因检测.中华检验医学杂志, 2003, 26:188-9.
3. Gibbons RV, Vaughn DW. Dengue: an escalating problem. BMJ, 2002, 324:1563–6.
4. Chambers TJ, Hahn CS, Galler R, et al. Flavivirus genome orgnization, expression, and replication. Annu Rev Microbiol, 1990, 44:649.
5. Falgout B, Pethel M, Zhang YM, et al. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructrual proteins. J Virol, 1991, 65:2467.
6. Sven M, Sandra S, Ralf B. Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-Structural Protein 4B. J Biol Chem, 2006, 281(13):8854-63.
7. Ju-Tao G, Junpei H, Christoph S. West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon. J Virol, 2005, 79(3):1343–50.
8. Jorge L, Mu?oz J, Maudry LR, et al. Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses. J Virol, 2005, 79(13):8004–13.
9. Lin RJ, Chang BL, Yu HP, et al. Blocking of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a protein tyrosine phosphatase-mediated mechanism. J Virol, 2006, 80(12):5908-18。
10. Best SM, Morris KL, Shannon JG, et al. Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J Virol, 2005, 79(20):12828-39.
11.陈建忠,朱海红。IFNα/β信号传导研究进展.国外医学·流行病学传染病学分册,2003,30(5):266-8。
12. Carlos TS, Fearns R, Randall RE. Interferon-induced alterations in the pattern of parainfluenza virus 5 transcription and protein synthesis and the induction of virusinclusion bodies. J Virol, 2005, 79(22):14112-21.
13. Wansley EK, Dillon PJ, Gainey MD, et al. Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells. J Virol, 2005, 335(1):131-44.
14. Blindenbacher A, Duong FH, Hunziker L, et al. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology, 2003, 124(5):1465-75.
15. Jorge L, Mu?oz J, Gilma G, et al. Inhibition of interferon signaling by dengue virus. PNAS, 2003, 100(24):14333–8.
16. Wen JL, Xiang JW, Vladislav V, et al. Inhibition of Interferon Signaling by the New York 99 Strain and Kunjin Subtype of West Nile Virus Involves Blockage of STAT1 and STAT2 Activation by Nonstructural Proteins. J Virol, 2005, 79(3):1934–42.
17. Meleri J, Andrew D, Linda H, et al. Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression. J Virol, 2005, 79(9):5414–20.
18. Ramaswamy ML, Shi MM, Monick GW, et al. Specific inhibition of type I interferon signal transduction by respiratory syncytial virus. Cell Mol Biol, 2004,30:893–900.
19. Andrejeva J, Young DF, Goodbourn S, et al. Degradation of STAT1 and STAT2 by the V proteins of simian virus 5 and human parainfluenza virus type 2, respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons. J Virol, 2002, 76:2159–67.
20. Parisien J.P, Lau JF, Rodriguez JJ, et al. The V protein of human parainfluenza virus 2 antagonizes type I interferon responses by destabilizing signal transducer and activator of transcription 2. J Virol, 2001, 283:230–9.
21. Indira U, Alex C, Aruna S, et al. Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gene Viro, 2006, 87:2605–14.
22. Kathryn AH, Luella RM, Lara EG, et al. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4. J Virol, 2003, 312:222–32.
23. Wei FT, Yuki E, Masayuki T, et al. Molecular Basis for Adaptation of a ChimericDengue Type-4/Japanese Encephalitis Virus to Vero Cells. Microbiol.Immunol, 2005, 49(3):285-94.
24. Joseph EB, Gracielle GM, Cai-Yen F, et al. Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells. Vaccine, 2003, 21:4317–27.
25. Leitmeyer KC, Vaughn DW, Watts DM, et al. Dengue virus structural differences that correlate with pathogenesis. J Virol, 1999, 73(6):4738-47.
26. Dunster LM, Wang H, Ryman KD, et al. Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus. J Virol, 1999, 261(2):309–18.
27. Jennings AD, Gibson CA, Miller BR, et al. Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. J Infect Dis, 1994, 169(3):512–8.
28. Ni H, Chang GJ, Xie H, et al. Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14. J Gen Virol, 1995, 76(2):409–13.