流行性出血热尸检组织中热休克蛋白70mRNA的定位及分布

应用核酸原位分子杂交技术研究了国内不同地区３０例流行性出血热患者尸检组织中病毒ＲＮＡ及ＨＳＰ７０ｍＲＮＡ细胞内定位,同时观察了汉坦病毒感染的ＶｅｒｏＥ６细胞中ＨＳＰ７０的表达情况。结果表明,热休克蛋白７０ｍＲＮＡ在多数组织中均可检测到,分布与病毒ＲＮＡ一致,并且与组织的病理损害有关;体外实验的结果也表明在出血热病毒感染的细胞中有ＨＳＰ７０的高表达。提示热休克蛋白与汉坦病毒的致病以及流行性出血热的发病机理有关。     

人类疱疹病毒7型体外生长特点的研究

研究了人类疱疹病毒７型（ＨＨＶ－７）南京株ＹＹ５在脐血单个核细胞（ＣＢＭＣｓ）及ＳＵＰＴ１细胞株上生长特点。观察病毒感染细胞后不同时间病变效应程度,记数死亡细胞百分率,间接免疫荧光染色记数抗原表达阳性细胞数,并用透射电镜观察病毒感染细胞超微结构变化及病毒复制不同时期的特点。结果发现：ＨＨＶ－７在ＣＢＭＣｓ及ＳＵＰＴ１上出现ＣＰＥ时间迟于ＨＨＶ－６,且ＣＰＥ程度也低于ＨＨＶ－６;ＨＨＶ－７在ＳＵＰＴ     

AHF及NE、人参皂甙对不同年龄大鼠VSMC c-myc基因和HSP_(70)基因表达的影响

用原位杂交法及激光光密度计灰度扫描半定量法,比较了老年组（２４月龄）与青年组（６周龄）Ｗｉｓｔａｒ大鼠离体培养的主动脉ＶＳＭＣ（５－１０代）ｃ－ｍｙｃ与ＨＳＰ７０基因的表达及红细胞抗高血压因子（ＡＨＦ）、去甲肾上腺素（ＮＥ）与人参皂甙对上述基因表达的影响。结果表明：１．增龄导致的ＶＳＭＣ增殖很可能与ｃ－ｍｙｃ基因的过度表达有关,ＮＥ、ＡＨＦ和人参皂甙可以通过调控ｃ－ｍｙｃ基因的表达来影响ＶＳＭＣ增殖。２．增龄伴随的应激能力下降可能与ＨＳＰ７０基因表达的减少有关。３．ＨＳＰ７０基因也可能参与了ＶＳＭＣ增殖与分化的调控,ＮＥ、ＡＨＦ与人参皂甙可能通过调控ＨＳＰ７０基因的表达来影响ＶＳＭＣ的增殖     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及HSP70的表达与病毒复制的关系,在汉滩病毒A9株感染Vero-E6细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞HSP70基因的表达进行了检测.结果表明,汉滩病毒感染细胞4h后即可诱导Vero-E6细胞表达HSP70,表达可持续至感染后5d,且HSP70在细胞内的分布也有改变.提示汉滩病毒可直接诱导HSP70的高表达.     

RT—nested PCR检测肾综合征出血热患者血清病毒核酸

采用异硫氰酸胍－酚－氯仿（ＡＧＰＣ）一步法提取病毒ＲＮＡ,并依据肾综合征出血热病毒（ＨＦＲＳＶ）核蛋白（ＮＰ）编码基因保守区核苷酸序列合成两对巢式引物,建立了逆转录巢式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄＰＣＲ）检测ＨＦＲＳＶＲＮＡ方法,应用此法对ＨＦＲＳＶ感染的ＶｅｒｏＥ６细胞培养液及ＨＦＲＳ患者血清中的病毒ＲＮＡ进行检测。结果显示,感染细胞培养液及３５例ＨＦＲＳ患者血清均为阳性,正常的ＶｅｒｏＥ６     

汉滩病毒感染体外培养神经元损伤及抗体对其保护作用

用体外培养的昆明小鼠胚胎脑皮质神经元,感染汉滩病毒后,　检测原癌基因ＦＯＳ的表达,加入ＨＦＲＳ病人恢复期血清,观察其对神经元的保护作用。将病毒　感染的神经元经免疫组化染色,检测原癌基因ＦＯＳ的快速表达;在病毒感染的神经元内加入Ｈ　ＦＲＳ病人恢复期血清,使用ＭＴＴ比色试验检测神经元存活情况。病毒感染的神经元ＦＯＳ表达明　显　增高,与对照组相比有显著性差异（Ｐ＜０．００１）;　ＨＦＲＳ病人恢复期血清保护病毒感染的神经元　组　与感染对照组神经元活性明显不同,有显著性差异（Ｐ＜０．００１）。　ＨＴＮＶ感染体外培养的神经元　,会使细胞内产生ＦＯＳ的快速表达,ＨＦＲＳ病人恢复期血清对病毒感染的神经元有一定的保护　作用。     

以痘苗病毒天坛株为载体的表达单纯疱疹病毒Ⅰ型糖蛋白D的重组活疫苗的建立

从我国分离到的一株单纯疱疹病毒Ⅰ型（ＨＳＶ－１－１６８株）病毒基因组中,分离出含有糖蛋白Ｄ（ｇＤ）基因的１．２ｋｂ片段,插入带有痘苗病毒天坛株ＴＫ区的质粒ｐＪＳＢ１１７５Ｐ７．５ｋ启动子下游,转染无白血病鸡胚原代细胞,获得带有ＨＳＶ－１－１６８ｇＤ基因的重组痘苗病毒。此株重组病毒在感染细胞膜上表达ＨＳＶ－１－１６８ｇＤ糖蛋白抗原,能与特异性单克隆抗体反应。在感染细胞中表达的膜抗原经ＳＤＳ－ＰＡＧＥ分析,表达分子量为５４ｋＤ糖蛋白。用Ｓｏｕｔｈｅｒｎ杂交分析了重组病毒ＤＮＡ中特异的ｇＤ基因,对作为活疫苗的重组痘苗病毒株进行了一些微生物学活性、免疫原性和毒力等方面的研究。     

汉滩病毒A9株M基因片段在重组痘苗病毒中的表达及鉴定

为发展国产化肾综合征出血热（ＨＦＲＳ）病毒基因工程疫苗,选择了汉滩病毒Ａ９株Ｍ基因片段为目的基因,构建转染质粒ｐＪＳＢＡ９Ｍ。以携带Ｌａｃ基因的重组病毒为亲本,使表达载体ｐＪＳＢ－Ａ９Ｍ上的Ｍ片段与痘苗病毒内的Ｌａｃ基因重组,将Ｌａｃ置换成Ａ９Ｍ片段。用蓝白斑法筛选重组痘苗病毒,经ＰＣＲ扩增证实Ａ９Ｍ片段重组入痘苗病毒基因组内。重组痘苗病毒感染的Ｖｅｒｏ Ｅ６细胞,用抗糖蛋白单克隆抗体（ＨＣＯ２、     

GST融合蛋白在杆状病毒系统中表达的可溶性研究

将构建好的可表达ＧＳＴ融合蛋白的重组病毒ＡｃＭＮＰＶ－ＯＣＣ＾－－ＧＳＴ－６ｘＨｉｓ－Ｅｔｐ２８感染Ｓｆ９细胞,一定时间后取感染了病毒的细胞裂解物上清液进行ＳＤＳ－ＰＡＧＥ分析,结果显示５３ｋＤａ的融合蛋白（ＧＳＴ－６ｘＨｉｓ－Ｅｔｐ２８）呈不溶状态。在原有裂解液的基础上,加固体十二烷基肌氨酸钠致终浓度１．５％,并将Ｔｒｉｔｏｎ Ｘ－１００的比例由１％提高到２％。ＳＤＳ－ＰＡＧＥ结果显示至少有１／     

从病人外周血单个核细胞中检测HHV—6：分离培养和基因扩增

收集婴幼儿急疹及淋巴系统增生性疾病患者外周血单个核细胞进行体外培养,从７例婴幼儿急疹及２例淋巴系统增生性疾病患者中分离出一种病毒,此病毒能在ＰＨＡ激活的人脐血单个核细胞中传代生长,产生典型ＣＰＥ：形成气球样巨细胞。电镜下观察,感染细胞中可见直径１８０ｎｍ左右,有包膜,疱疹样病毒颗粒;血清学试验证明分离株与ＨＳＶ－１,２、ＨＣＭＶ、及ＥＢＶ无抗原交叉,而与ＨＨＶ－６ＧＳ株间存在抗原一致性;多聚酶链反     

汉坦病毒感染诱导乳鼠脑神经细胞表达热休克蛋白70

生后2～3天的昆明乳鼠,每只腹腔接种0.05mL100个半数致死量的陈株汉坦病毒,于接种后不同时间点处死动物,取其脑组织常规固定,石蜡包埋制备5μm连续组织切片,用免疫组化法检测组织中的病毒抗原及热休克蛋白70的表达,用共聚焦显微镜观察二者之间的关系。结果发现,感染了病毒的组织能够稳定地检测到热休克蛋白70的表达,而病毒阴性的组织则检测不到热休克蛋白70的表达,且二者有共定位关系,其分布与组织病变一致。这与在病毒感染的VeroE6细胞及EHF病人尸检组织中得到的结果一致,说明病毒感染可诱导热休克蛋白70的表达,后者可能具有保护组织避免或减轻损伤的作用。     

汉滩病毒核蛋白与热休克蛋白GRP94、HSP27的相互作用

为研究汉滩病毒(Hantaan virus,HTNV)感染诱导乳鼠脑组织热休克蛋白GRP94、HSP27与病毒蛋白的相互关系,选出生2—3d的昆明乳鼠实验性感染汉滩病毒,取8d后发病乳鼠脑组织部分制石蜡切片,用免疫组化结合共聚焦显微镜检测组织中病毒抗原及GRP94、HSP27的表达,部分制匀浆液,用ELISA、免疫共沉淀方法分析病毒抗原和GRP94、HSP27的关系。结果示汉滩病毒感染诱导乳鼠脑组织神经细胞高表达GRP94且与细胞内病毒抗原有共定位关系,但未见HSP27诱导高表达;免疫共沉淀显示汉滩病毒核心抗原(HINV—NP)与GRP94、HSP27呈非共价复合物形式存在。该结果为进一步探讨HSPs在病毒感染复制中的作用以及抗病毒感染方面提供了有意义的实验资料。     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.     

汉滩病毒诱导小鼠骨髓瘤SP2／0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2／0细胞,接种后一定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达。结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞：流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2／0细胞中Fas和FasL表达明显升高。该结果表明汉滩病毒可诱导体外培养SP2／0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关。     

Persistent borna disease virus infection confers instability of HSP70 mRNA in glial cells during heat stress

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.     

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP(+)-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.     

猪流行性腹泻病毒经其附属蛋白抑制细胞凋亡

【背景】orf3位于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV) s基因与e基因之间,是目前发现的PEDV唯一一个附属基因,编码附属蛋白(ORF3蛋白)。我们前期研究初步发现ORF3蛋白对PEDV诱导的细胞凋亡有影响。【目的】研究ORF3蛋白在PEDV侵染复制过程中的毒力作用机制。【方法】实验用3种PEDV:rDR13att-?ORF3 (orf3基因全部敲除)、DR13-ORF3att (携带有C端截短orf3)、rDR13att-ORF3wt(携带全长orf3基因)感染Vero细胞,观察病变情况,再用活细胞成像仪、流式细胞仪、 DNA断裂的原位末端标记法[terminaldeoxynucleotidyltransferase(TDT)-mediated dUTP nick end labeling,TUNEL]等方法检测不同感染时间点的细胞凋亡情况,然后用蛋白质印迹方法分析PEDV感染宿主细胞中主要凋亡相关蛋白(如Caspase-3)的活化或裂解,最后进行转录组测序研究病毒感染细胞中差异基因的表达情况,再用荧光定量PCR验证转录组结果。【结果】rDR13att-?ORF3引起较多的细胞病变,活细胞成像仪的动态观察结果显示,3种病毒侵染的细胞凋亡水平随着时间的延长均高于正常阴性细胞,但敲除orf3的病毒感染细胞后细胞凋亡率比其他两种病毒更高;敲除orf3病毒感染细胞凋亡率显著高于其他两种病毒;病毒rDR13att-?ORF3感染细胞后TUNEL阳性细胞数比DR13-ORF3att和rDR13att-ORF3wt更多;表达ORF3蛋白的重组PEDV可以抑制Caspase-3的活化;ORF3蛋白对受感染细胞Heat shock 70 kD protein 1B (HSP70)基因转录有促进作用,荧光定量PCR结果表明rDR13att-ORF3wt感染细胞的HSP70表达量高于rDR13att-?ORF3感染细胞。【结论】PEDV通过ORF3蛋白抑制细胞凋亡,而且这种作用可能是通过抑制Caspase-3的活化或增加HSP70的产生来完成的。     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。