登革病毒对人血管内皮细胞产生细胞因子的影响

目的了解登革病毒感染对人血管内皮细胞产生几种细胞因子的影响。方法用登革病毒Ⅱ型（ＤＶ－２）及ＬＰＳ单独作用或联合作用于人脐静脉内皮细胞（ＨＵＶＥＣ），于作用后４，２４，４８，７２和９６小时分别收集培养上清液，用ＥＬＩＳＡ法检测ＧＭ－ＣＳＦ，ＩＬ－６和ＴＮＦα的含量。实验数据采用方差分析处理。结果ＤＶ－２感染能使ＨＵＶＥＣ分泌ＧＭ－ＣＳＦ和ＩＬ－６的能力增强。病毒感染后４小时，ＧＭ－ＣＳＦ和ＩＬ－６的水平开始升高，并分别于感染后７２和２４小时达高峰，然后开始下降。然而，ＤＶ－２感染并不能提高ＨＵＶＥＣ分泌ＴＮＦα的能力。ＬＰＳ单独作用或ＤＶ－２与ＬＰＳ联合作用均可提高ＨＵＶＥＣ分泌ＧＭ－ＣＳＦ和ＩＬ－６的能力，但对分泌ＴＮＦα的能力没有明显的促进作用。结论登革病毒感染能提高ＨＵＶＥＣ分泌ＧＭ－ＣＳＦ和ＩＬ－６的能力。细胞因子在登革病毒的致病与免疫过程中可能起重要作用。     

黄芪多糖对嗜中性粒细胞与内皮细胞粘附及粘附分子的影响

目的：通过研究黄芪多糖对嗜中性粒细胞与血管内皮细胞粘附及粘附分子的影响,探讨黄芪多糖对炎症反应的作用及其机制。方法：以黄芪多糖或黄芪多糖加ＩＬ－１ＴＮＦ处理人嗜中性粒细胞或人脐静脉内皮细胞（ＨＵＶＥＣ）后,用蛋白染料染色法研究药物对嗜中性粒细胞与内皮细胞粘附的作用,用细胞ＥＬＩＳＡ法、ＡＰＡＡＰ免疫细胞化学染色法研究药物对ＨＵＶＥＣ表面ＩＣＡＭ－１及嗜中性粒细胞表面ＣＤ１８表达的影响。结果：黄芪多糖（４０μｇｍＬ－１ｍｇｍＬ）作用于ＨＵＶＥＣ,能促进ＨＵＶＥＣ与嗜中性粒细胞粘附,并与ＩＬ－…     

健胎液、川芎嗪对缺氧人脐静脉内皮细胞作用的实验研究

目的研究健胎液和川芎嗪对缺氧人脐静脉内皮细胞（ ＨＵＶＥＣ）分泌血管活性物质－内皮素（ ＥＴ）、前列环素（ＰＧＩ２）、一氧化氮（ＮＯ）的影响,以及该方药对缺氧ＨＵＶＥＣ形态结构上的保护作用。方法将ＨＵＶＥＣ分成五组;正常组、缺氧组、血清组、含药血清组、川芎嗪组。观察各组细胞的形态改变,检测细胞上清液ＥＴ、ＰＧＩ２、ＮＯ。结果健胎液可显著提高缺氧ＨＵＶＥＣ合成分泌ＰＧＩ２、ＮＯ（ＰＭ０．０５～０．０１）,抑制ＥＴ的释放（Ｐ４ｈ＜０．０５）;川芎嗪显著促进ＰＧＩ２的合成分泌（Ｐ＜０．０５）。从超微结构上观察．健胎液和川芎嗪能明显减轻缺氧对ＨＵＶＥＣ的细胞器和细胞核的损伤,尤以健胎液保护线粒体免受缺氧损伤的作用更显著。结论健胎液对缺氧ＨＵＶＥＣ的保护作用优于川芎嗪,这与健胎液对内皮源性的血管活性物质的整体调节效应有关。     

小檗碱对中性粒细胞与血管内皮细胞粘附的影响及机制探讨

目的：探讨小檗碱对中性粒细胞及血管内皮细胞粘附作用及粘附分子的影响。方法：以人脐静脉内皮细胞（ＨＵＶＥＣ）为模型,用蛋白染料染色法观察小檗碱对中性粒细胞（ＰＭＮ）与ＨＵＶＥＣ粘附作用的影响;用细胞ＥＬＩＳＡ法、ＡＰＡＡＰ细胞免疫化学染色法观察小檗碱对ＰＭＮ－ＨＵＶＥＣ粘附过程中粘附分子ＩＣＡＭ－１及ＣＤ１８表达的影响。结果：小檗碱（０－３２～８μｇｍＬ）作用于ＨＵＶＥＣ,可抑制ＰＭＮ与ＨＵＶＥＣ间的粘附,对ＩＬ－１、ＴＮＦ（１０００μｇｍＬ）诱导的ＰＭＮ－ＨＵＶＥＣ粘附增强亦有抑制作用;而小…     

六种细胞因子对登革病毒感染人血管内皮细胞的影响

用不同浓度的ＴＮＦα，ＩＦＮα，ＧＭ－ＣＳＦ，ＩＬ－１β，ＩＬ－２和ＩＬ－６等六种细胞因子分别作用于登革病毒Ⅱ型（ＤＶ２）感染人脐静脉内皮细胞（ＨＵＶＥＣ）的不同环节（病毒感染前，感染同时，感染后）。于感染后４８小时收集感染上清液，用微量蚀斑法滴定病毒产量。结果发现：ＩＬ－２、ＩＬ－６和ＧＭ－ＣＳＦ可以促进ＤＶ２在ＨＵＶＥＣ中复制，而ＩＦＮα，ＴＮＦα及ＩＬ－１β对ＤＶ２的复制则有抑制作用。并发现细胞因子对病毒复制的影响与细胞因子的浓度和作用环节有关，ＧＭ－ＣＳＦ对病毒复制的影响与浓度成反比，而其它五种细胞因子对病毒的作用则呈剂量依赖性。六种细胞因子在实验浓度范围内均不造成血管内皮细胞形态和数量的改变。     

健胎液对人脐静脉内皮细胞分泌血管活性物质的影响

目的：探讨健胎液防治胎儿宫内生长迟缓的机理，应用血清药理学方法研究健胎液对缺氧人脐静脉内皮细胞（ＨＵＶＥＣ）分泌血管活性物质内皮素（ＥＴ）、前列环素（ＰＧＩ_２）、一氧化氮（ＮＯ）的影响，以及该方药对缺氧ＨＵＶＥＣ形态结构上的保护作用。方法：将ＨＵＶＥＣ随机分成四组：正常组、缺氧组、血清组、健胎液组，观察细胞形态的改变；检测内皮素（ＥＴ）、前列环素（ＰＧＩ_２）和一氧化氮（ＮＯ），进行统计学处理。结果：健胎液可显著提高缺氧ＨＵＪＶＥＣ合成分泌ＰＧＩ_２、ＮＯ（Ｐ ＜ ０．０５～０．０１），同时，ＥＴ的释放显著降低（Ｐ_４ｈ＜ ０．０５）。从超微结构上观察，健胎液能明显减轻缺氧对ＨＵＶＥＣ的细胞器和细胞核的损伤，尤以保护线粒体免受缺氧损伤的作用更显著。结论；通过增强血管内皮细胞对缺氧的耐受性，调节血管性物质的分泌是健胎液防治胎儿宫内生长迟缓的机制之一。     

IVC RECONSTRUCTION OF THE DOGS WITH DACRON SEEDED WITH AUTOLOGOUS VENOUS FRAGMENTS

ＩＶＣＲＥＣＯＮＳＴＲＵＣＴＩＯＮＯＦＴＨＥＤＯＧＳＷＩＴＨＤＡＣＲＯＮＳＥＥＤＥＤＷＩＴＨＡＵＴＯＬＯＧＯＵＳＶＥＮＯＵＳＦＲＡＧＭＥＮＴＳＩＶＣＲＥＣＯＮＳＴＲＵＣＴＩＯＮＯＦＴＨＥＤＯＧＳＷＩＴＨＤＡＣＲＯＮＳＥＥＤＥＤＷＩＴＨＡＵＴＯＬＯＧＯ...     

山莨菪碱抗血栓形成的机制研究

观察中药山莨菪碱对内毒素脂多糖所致血管内皮细胞表达纤溶酶原激活物抑制剂１的作用和作用机制。方法用胰蛋白酶消化培养人脐静脉内皮细胞（ＨＵＶＥＸ）;采用酶联免疫吸附试验法和Ｎｏｒｔｈｅｒｎ印迹方法观察ＨＵＶＥＧＣ条件培养液ＰＡＩ－１蛋白量及ｍＲＮＡ表达用免疫细胞学检测ＨＵＶＥＣ核因子ＮＦ－ｋＢ的核内转移情况,结晶显著啬培养ＨＵＶＥＣ的ＰＡＩ－１蛋白和ｍＲＮＡ表达;然而当山莨菪碱和ＬＰＳ同时作用于ＨＵＶ     

前列环素对内皮素生物学效应的拮抗作用

为探讨内皮素与前列环素间相互作用关系及生理意义，本工作在整体动物，离体血管条及培养的血管平滑肌细胞上发发现：ＰＧＩ２有效拮抗ＥＴ１引起的大鼠升压作用；剂量依赖地逆转ＥＴ１对小鼠的致死作用；抑制ＥＴ１促培养的大鼠ＶＳＭＣ增殖效应；减少大鼠离体系膜上动脉ＥＴ１的基础释放及凝血酶刺激的ＥＴ１释放。结果表明：ＰＧＩ２能有效拮抗ＥＴ的生物学效应，并提示血管内皮细胞所释放的ＰＧＩ２和ＥＴ间存在反馈调节。     

CHANGES OF 6-K-PGF_(1a) RELEASE FROM THE LUMINAL SURFACE OF DACRON SEEDED WITH AUTOLOOUS VENOUS TISSUE FRAGMENTS

ＣＨＡＮＧＥＳＯＦ６－Ｋ－ＰＧＦ１ａＲＥＬＥＡＳＥＦＲＯＭＴＨＥＬＵＭＩＮＡＬＳＵＲＦＡＣＥＯＦＤＡＣＲＯＮＳＥＥＤＥＤＷＩＴＨＡＵＴＯＬＯＯＵＳＶＥＮＯＵＳＴＩＳＳＵＥＦＲＡＧＭＥＮＴＳＣＨＡＮＧＥＳＯＦ６－Ｋ－ＰＧＦ１ａＲＥＬＥＡＳＥＦＲＯＭＴＨ...     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.     

In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection

Electrical resistance across human umbilical vein endothelial cells (HUVECs) was measured using an electrical cell sensor system. The transendothelial electrical resistance (TEER) value was used to estimate the permeability through endothelial cells in vitro. Decrease in the TEER value was associated with increase in the passage of albumin through endothelial cells in the albumin permeability assay. The effects of cytokines and dengue virus infection on the permeability of HUVECs were examined by measuring the TEER value. Tumor necrosis factor alpha (TNF-alpha) at 1 and 0.1 microg/ml decreased the TEER value, but TNF-alpha at lower dose did not. Interferon-gamma (IFN-gamma) at 1 microg/ml also decreased the TEER value. In contrast, interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) or interferon-beta (IFN-beta) did not decrease the TEER value. The decrease in the TEER value was associated with the morphological changes of HUVECs. Dengue virus infection at a multiplicities of infection (m.o.i.) of 5 pfu/cell decreased the TEER value. Infection at an m.o.i. of 0.5 pfu/cell did not decrease the TEER value; however, addition of 0.01 microg/ml of TNF-alpha to these infected endothelial cells decreased the TEER value. The results suggest that TNF-alpha and dengue virus infection decrease synergistically the TEER value of endothelial cells. The TEER method is easy, reliable and can be applicable to further analysis of the increase in the permeability of endothelial cells in vitro induced by inflammatory cytokines and dengue virus infection.     

Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

FasL/Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype‐2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype‐2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1–2 were constantly very low, whereas Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors 2–4 decreased after dengue virus serotype‐2 infection. This result suggested that dengue virus serotype‐2 may inhibit Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors‐induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype‐2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus‐induced apoptosis of vascular endothelial cells in vitro. J. Med. Virol. 82:1392–1399, 2010. © 2010 Wiley‐Liss, Inc.     

Human influenza virus infection and apoptosis induction in human vascular endothelial cells

Acute encephalopathy accompanying influenza virus infection results in brain and systemic organ failure mainly through vasogenic edema with high levels of inflammatory cytokines, such as blood tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as the cytochrome c apoptosis marker. A highly virulent strain of avian influenza virus causes fatal infection in chickens by infecting vascular endothelial cells in systemic organs, inducing apoptosis therein. To verify the possibility of apoptosis induction by human influenza virus in infected human vascular endothelial cells, purified influenza virus-infected human umbilical vein endothelial cells (HUVECs) were examined using a tissue culture method. When pre-treated with TNF-alpha, influenza virus (Philippine strain, H3N2) promoted TNF-alpha induced apoptosis of HUVECs. Viral replication was confirmed in HUVECs infected with the Philippine strain in the absence of TNF-alpha by measurement of the amount of infective virus in the culture supernatant using the tissue culture infectious dose (TCID) method, immunohistochemistry and real-time PCR. The number of influenza virus genomes in the infected HUVECs at 24 hr post-infection increased about fivefold compared to that just after virus adsorption. Many TUNEL-positive influenza virus-infected HUVECs were observed using the TUNEL method. Furthermore, cleaved caspase 3 was also detected in influenza virus-infected cells by immunofluorescence staining. These results demonstrated that human influenza virus can infect and replicate in human vascular endothelial cells and induce apoptosis therein.     

DENV-2 诱导HUVECs 自噬和影响细胞活力的研究

目的:通过利用登革域型病毒(DENV-2)感染原代人脐静脉内皮细胞(HUVECs)后,研究病毒诱导HUVECs 产生自噬和对细胞活力的影响。方法:利用DENV-2 NGC 株感染HUVECs,Real-time PCR 检测HUVECs 中DENV-2 NS1 基因部分系列的表达,给予自噬诱导剂雷帕霉素(RAPA)和自噬抑制剂氯喹(CQ)预处理观察细胞自噬通量变化,激光共聚焦显微镜观察自噬体形成;CCK-8 检测细胞活力;Western blot 检测自噬通量标志性蛋白LC3、P62 的蛋白表达量。结果:DENV-2 感染HUVECs 组病毒NS1 基因在各时间点均有表达;被感染的HUVECs 在24 h 时细胞活力下降不明显,36、48 h 细胞活力分别下降至未处理组82.46%和78.47%,差异具有显著统计学意义(P<0.05);HUVECs 被DENV-2 感染后,LC3-Ⅱ蛋白量表达明显增高,自噬底物P62 表达明显降低,36、48 h 时间点相较未处理组具有统计学意义(P<0.05),与RAPA 处理组结果相似(P>0.05)。CQ 处理组LC3-Ⅱ及P62 蛋白量表达均明显升高,36、48 h 时间点相较未处理组具有显著性差异(P<0.05)。HUVECs被DENV-2 感染36 h,激光共聚焦显微镜观察到LC3-Ⅱ表达明显高于未处理组,自噬诱导剂RAPA 处理HUVECs 后,LC3-Ⅱ表达明显增加,自噬强度更加明显。DENV-2 与CQ 联合处理组相较DENV 单独处理组,36 h 细胞活力下降了13.61%,差异具有显著统计学意义(P<0.05)。结论:DENV-2 感染可抑制HUVECs 的生长;而DENV-2 诱导HUVECs 产生的自噬却有利于HUVECs 的存活。     

Time course of virus-specific macromolecular synthesis during rubella virus infection in Vero cells

Virus specific macromolecular synthesis was studied in Vero cells infected with plaque-purified rubella virus under one-step multiplication conditions. Under these conditions, the rate of virus production was found to increase rapidly until 24 hr postinfection after which time the rate of virus production rose more slowly, reaching a peak level at 48 hr postinfection. This peak rate of virus production was maintained through 72 hr postinfection. A majority of the cells remained alive through 96 hr postinfection, although a 20 to 30% decrease in the number of living cells occurred between 24 and 48 hr postinfection, the time period at which cytopathic effect was first observed. The virus structural proteins were first detected intracellularly at 16 hr postinfection. The rate of synthesis of these proteins was already maximal at 16 hr postinfection and remained constant through 48 hr postinfection. By immunofluorescence, cells expressing virus proteins were first observed at 12 hr postinfection. At 24 hr postinfection, 35 to 50% of the cells in the infected culture were exhibiting immunofluorescence, at 36 hr postinfection, 65 to 90% of the cells were exhibiting immunofluorescence, and at 48 hr postinfection, all of the cells were exhibiting immunofluorescence. The virus genomic and subgenomic RNA species were first detectable by 12 hr postinfection. The rate of synthesis of both of these species peaked at 26 hr postinfection. Rubella virus infection was found to have no effect on total cell RNA synthesis. However, a modest inhibition of total cell protein synthesis which reached 40% by 48 hr postinfection was observed. When Northern analysis of RNA extracted from infected cells was performed, a negative-polarity, virus-specific RNA probe hybridized only to the virus genomic and subgenomic RNA species. A positive-polarity, virus-specific RNA probe hybridized predominantly to a negative-polarity RNA of genome length indicating that both the genomic and subgenomic RNAs are synthesized from a genome-length negative-polarity template. Defective interfering (DI) RNAs were not detected in infected cells through 96 hr postinfection or in cells onto which virus released through 96 hr postinfection was passaged. Thus, the generation of DI particles by rubella virus appears to play no role in the slow, noncytopathic replication of this virus or in the ability of rubella virus-infected cells to survive for extended periods of time.     

登革病毒促进人树突状细胞分泌TNF-α、IL-6、IFN-γ的动态观察

目的：研究登革病毒（DV）对人树突状细胞（DC）产生细胞因子的影响。 方法： 人外周新鲜血常规分离单核细胞，经细胞因子GM-CSF、IL-4诱导培养DC，形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型感染DC，于作用后6、12、24、48、72 h分别收集上清液和细胞，间接免疫荧光法检测细胞上病毒抗原表达，ELISA法检测登革病毒感染后细胞因子TNF-α、IL-6、IFN-γ水平的动态变化。 结果： 人外周血经GM-CSF、IL-4诱导培养1周即可得到典型树突状细胞。间接免疫荧光法证明感染的DC胞浆和胞膜上携带登革病毒抗原，DV感染使DC分泌TNF-α、IL-6能力显著大于对照组（P<0.01），但其分泌IFN-γ的能力无明显改变。 结论： 树突状细胞是登革病毒的靶细胞，登革病毒感染可促进树突状细胞分泌TNF-α、IL-6。树突状细胞可能参与机体抗登革病毒感染的免疫防御机制。     

2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响

 目的：探讨2型登革病毒（DENV2）感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法：用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果：DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论：登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。     

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae)

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.