登革热Ⅱ型病毒受体的初步研究

本实验用甲基纤维素复盖层的c_6/36单层细胞微量蚀斑法的间接免疫荧光法,初步观察了登革热Ⅱ型病毒与靶细胞的联接反应情况。同时还制备了四种细胞的可溶性膜制剂,并研究了它们对登革热Ⅱ型病毒的抑制活性。结果提示,对登革热Ⅱ型病毒敏感的细胞表面存在登革热Ⅱ型病毒受体,在触发病毒感染阶段起重要作用。     

登革热2型病毒感染人内皮细胞的机制

登革热病毒(DEN)通过两种机制感染宿主细胞,即与病毒IgG形成复合物结合于Fc受体,或是病毒糖蛋白直接与宿主细胞特异性受体结合。第一种机制已做研究,发现登革热出血/休克综合征的病毒滴度的增高与这种IgG-Fc介导的抗体增高有关,而对第二种机制的研究较少,且一直未发现DEN的宿主细胞受体。     

热带瘟疫--登革热

结合登革热疫情流行状况,介绍了登革病毒及其传播途径,登革热的特征病理变化和类型,提出了预防的措施。     

登革病毒的分子进化和流行趋势

登革热是一种最流行的蚊媒传播传染病 ,近二十年来其流行呈上升趋势 ,本文从现代分子生物学和分子进化角度 ,对登革热的流行趋势进行综述。     

登革病毒感染对妊娠结局的影响

登革热(dengue fever)是一种由登革病毒感染引起的蚊媒传染病,主要流行于热带、亚热带地区,如东南亚、美洲和西太平洋地区等。近年来,在中国登革热的流行范围明显扩大,多省市曾多次暴发不同规模的疫情。有临床证据表明,妊娠期登革病毒感染与不良妊娠结局(adverse pregnancy outcome)及胎儿先天缺陷的关系十分密切。妊娠期感染登革病毒可能增加孕妇发生产后出血、产妇死亡等妊娠并发症发生的风险,也会增加胎儿流产、早产、宫内死亡、低出生体重、先天缺陷和新生儿围产期死亡的可能性。因此,研究登革病毒感染与不良妊娠结局的关联,探索登革热的致病机制,对于控制登革热和改善妊娠结局有十分重要的意义。现就登革热对妊娠结局的影响作一概述。     

产生抗登革热Ⅰ型病毒单克隆抗体杂交瘤细胞株的建立

用登革热Ⅰ型病毒(夏威夷株)兔疫的小白鼠脾细胞与小鼠骨髓瘤细胞融合,获得7个产生抗登革热Ⅰ型病毒单克隆抗体的杂交瘤细胞系,这些杂交瘤细胞上清液及小鼠腹水抗体均用间接兔疫荧光法检测,其中3个杂交瘤细胞系产生的抗体对登革热Ⅰ型病毒具有型特异性。     

基于ceRNA网络识别登革热诊断标志物

通过比较登革热患者和健康人群转录组数据,识别差异基因,构建失调ceRNA网络,筛选关键基因富集分析,解析潜在生物学功能,助力登革热诊断标志物的研究。从GEO数据库下载登革热外周血芯片数据,识别差异基因并进行富集分析。结合miRNA-mRNA互作数据,利用超几何算法和皮尔森相关性计算方法识别登革热失调ceRNA互作对,使用Cytoscape软件可视化ceRNA网络与模块挖掘,对网络模块进行功能富集及外部数据验证表达模式。筛选出251个差异基因,发现其富集在细胞周期等生物学通路中。经外部数据验证,网络模块基因的表达趋势与训练集数据大致相同,表明模块基因在登革热疾病中的潜在诊断效能。本研究可为确定有效的疾病诊断分子标志物提供思路。     

Science:科学家或开发出新型的登革热疫苗疗法

<正>发表在国际杂志Science上的一篇研究论文中,来自范德堡大学等处的研究人员通过研究解析了一种人类单克隆抗体的结构,在动物模型中这种人类单克隆抗体可以中和潜在致死性的登革热病毒;该研究或为开发新型有效的疗法或疫苗来抵御登革热提供新的思路。登革热病毒是一种蚊媒病毒,其每年会感染3.9亿人,登革热同时也是热带地区人们死亡的主要原因。研究者James Crowe博士表示,目前科     

穿琥宁注射液治疗登革热疗效观察

目的观察穿琥宁治疗登革热的临床疗效。方法28例登革热患者随机分为穿琥宁治疗组和对照组,观察疗效及热程,白细胞(WBC)和血小板(PLT)复常时间。结果穿琥宁治疗组在总病程上显著短于对照组,P<0.05,在WBC、PLT复常方面,2组差异无显著性。结论穿琥宁可有效治疗登革热。     

海南岛125株登革病毒的分离与鉴定

1985年9月儋县发生一次由Ⅱ型登革病毒引起的登革热和登革出血热的爆发流行,至1986年10月疫情已由儋县波及本岛十县二市。根据记载,国外登革热流行期间,常伴多个型别登革病毒同时传播。为掌握本岛此次登革热流行时除Ⅱ型病毒外,是否尚有其他型别流行,于1986年1月至10月登革热流行期间,取十县二市278例急性期病人血清,接种C6/36白纹伊蚊细胞系,进行病毒分离,共获得125株病毒。均用抗登革     

RNA干扰抑制登革病毒复制的研究

为了研究RNA干扰(RNAi)对Ⅰ型登革病毒(DENV-1)在白纹伊蚊C6/36细胞内复制的影响,本研究设计并合成针对I型登革病毒Pr M基因的小干扰RNA,以脂质体法转染入C6/36细胞后,用DENV-1感染已转染的细胞,观察细胞病变效应,MTT法检测细胞存活率,荧光定量RT-PCR检测登革病毒RNA含量。结果表明:转染siRNA的C6/36细胞在受登革病毒攻击7天后仍无明显细胞病变效应,细胞存活率比对照组提高2.26倍,细胞内登革病毒RNA拷贝数比对照组降低约97.54%。说明利用RNA干扰技术能有效抑制登革病毒核酸在C6/36细胞内复制,并对细胞具有一定保护作用,为登革热的防治提供了新的思路。     

Detection of Dengue Virus Antigens in Cultured Cells by Using Protein A-Gold-Silver Staining (pAgs) Method

A protein A-gold-silver (pAgs) staining was developed to detect dengue virus antigens in cultured cells. The method can be carried out in either newly-subcultured or monolayered cells. Dengue virus-inoculated C6/36 clone of Aedes albopictus cells and human endothelial cells appeared brown-yellowish color on the peripheral membrane of the infected cells. In many cases, the infected C6/36 cells appeared darker than that of the infected endothelial cells. The positive results from the inoculated C6/36 cells usually appeared as early as 2 days post-inoculation for types 1, 2, and 4 of dengue viruses and 3 days for the dengue 3 virus. The same batch of specimens detected by direct immunofluorescence antibody test (DFA) showed positive 4 days post-inoculation for the types 2, 3, and 4 of dengue viruses and 6 days for the dengue 1 virus. The result also showed that all pAgs-positive specimens were also DFA-positive, but not vice versa. It suggested that pAgs is not only sensitive but also specific for dengue virus detection from inoculated cultured cells.     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.     

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.     

国内首次自患者标本中同时分离的登革2型和3型病毒的鉴定

采用间接免疫荧光方法 ,检测患者血清标本中的抗登革病毒IgM和IgG抗体 ;同时将病人急性期血清接种C6 36细胞进行病毒分离。从分离的病毒悬液中提取RNA ,进行RT PCR扩增和序列测定。结果显示 ,该患者血清中存在抗登革病毒的IgM和IgG抗体。从病人血清中分离的病毒 ,经RT PCR和序列测定证实为登革 2型和 3型病毒的特异序列。表明该患者为登革 2型和 3型病毒混合感染     

应用DNA-RNA斑点杂交法检测登革病毒核酸

新近制备了大量纯化的pEH920 DNA,该质粒DNA插入了登革病毒2型核酸片段的互补DNA。以[a-~(32)P]dCTP按缺口转译法标记pEH 920 DNA作为探针,以感染病毒的蚊细胞c_6/36培养上清作标本,应用DNA-RNA斑点杂交法检测了登革病毒核酸。结果显示同位素标记探针(pEH 920)与登革病毒2型标本反应最强,具有一定的型特异性。但与其它血清型登革病毒也呈一定交叉反应。初步探讨了探针的敏感性,至少可检出TCID_(50)625的登革病毒2型核酸。     

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.