Rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system

A genetically modified cell line (Vero–ICP10-EGFP) was constructed for detection of herpes simplex virus (HSV) by a simple, rapid and direct method. The cell line was developed by stable transfection of Vero cell with a plasmid encoding the green fluorescent protein (GFP) driven by the promoter of the HSV-2 ICP10 gene. As early as 6 h after infection with HSV, fluorescence-emitting cells can be observed under a fluorescence microscope. A single infected cell emitting fluorescence can be observed with soft agar overlay by inverted fluorescence microscopy. No induction of detectable fluorescence was seen following infections with human cytomegalovirus (HCMV), varicella zoster virus (VZV), coxsackievirus A16 and enterovirus 71. Analysis by flow cytometry also demonstrated that intensity of the triggered fluorescence is proportional to the titer of HSV inoculated. Taken together, this novel GFP reporter system could become a useful means for rapid detection and quantification of HSV in clinical specimens.     

人髓核细胞中不同滴度慢病毒介导绿色荧光蛋白基因的表达

背景：慢病毒作为基因载体感染椎间盘髓核细胞的感染效率研究具有实际应用价值。 目的：测定不同滴度慢病毒介导的绿色荧光蛋白基因感染人髓核细胞的感染条件及感染参数。 方法：采用酶消化法分离培养人髓核细胞,利用基因重组技术构建高滴度慢病毒介导的绿色荧光蛋白基因,按照不同感染复数感染第2代人髓核细胞。 结果与结论：第2代髓核细胞在慢病毒介导的绿色荧光蛋白基因的感染复数为1,10,50,100时,感染后第4天荧光显微镜观察,流式细胞术测定其感染效率分别为32.1%,41.1%,54.2%和86.8%,继续传代培养3代,第5代椎间盘髓核细胞绿色荧光蛋白的表达仍能保持在60%以上。表明慢病毒介导的绿色荧光蛋白基因在人髓核细胞中可以持续高效表达。     

Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry: increase of class I MHC expression on uninfected cells and decrease on infected cells

Summary Cultured human foreskin fibroblasts (HFF) were infected with different multiplicities of infection (moi 0.001-0.1) of human cytomegalovirus (HCMV) strain AD 169 or a clinical isolate. Percentage of infected cells was determined by analysis of immediate early (IEA), early (EA), and late (LA) virus antigen expression with flow cytometry or by immunoperoxidase staining. Changes in the expression of class I MHC surface molecules were demonstrated by comparing the mean fluorescence intensities of infected HFF cultures with those of mock infected cell cultures by flow cytometry. At day three post infection single fluorescence analysis showed that infected HFF cultures split into low and high density class I MHC bearing cells. The addition of anti-interferon reduced the expression of class I MHC, distinctly. The assumption that infected cells down-regulate and uninfected cells up-regulate their expression of class I MHC molecules was demonstrated by double fluorescence analysis both with flow cytometry and fluorescence microscopy. Analysis of class I MHC-antigen expression versus immediate (IEA, mab E13), early (EA, mab 9221), or late (LA, mab BM219) virus antigen expression yielded three cell populations of HCMV infected HFF cultures three days post infection: 1. uninfected cells with an increase of class I MHC, 2. high density class I MHC, IEA and/or EA expressing cells, and 3. low class I MHC, IEA, EA and LA expressing cells.     

Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system

Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins.     

Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.     

利用同源重组构建大鼠甘油二酯激酶γ(DGKγ)慢病毒过表达载体

文题释义： 甘油二酯激酶γ(DGKγ)：属于第Ⅰ类DGK亚型,主要分布于神经系统,参与浦肯野细胞树突的发育、突触可塑性调节、神经上皮细胞的增殖和迁移、神经元分化等功能活动。 同源重组技术：一种利用同源重组的原理,进行无缝克隆的技术。该技术无需考虑插入片段的酶切位点,可将插入片段PCR产物定向克隆至任意载体的任意位点。同源重组技术还能实现多个DNA片段的一步组装,操作简单,重组效率高。 背景：慢病毒载体作为外源性转基因载体已被广泛应用,但是大鼠甘油二酯激酶γ(diacylglycerol kinase γ,DGKγ)基因慢病毒载体未见报道。 目的：用同源重组的方法构建大鼠DGKγ慢病毒过表达载体。 方法：提取成年SD大鼠脑组织总RNA,以反转录得到的cDNA作为模板,通过PCR反应分段扩增大鼠DGKγ基因CDS区5'端1 029 bp和3'端1 362 bp,用同源重组技术将这2个片段与线性化载体进行定向连接,构建CMV-rat DGKγ-GFP慢病毒载体并进行PCR扩增及测序鉴定。经293T细胞包装后产生慢病毒,收集慢病毒感染293T细胞,荧光显微镜下观察细胞中GFP的表达并应用实时荧光定量PCR和Western blotting法检测细胞中DGKγ mRNA和蛋白的表达。 结果与结论：CMV-rat DGKγ-GFP慢病毒载体经PCR扩增和测序鉴定构建成功;经CMV-rat DGKγ-GFP慢病毒感染后的293T细胞,荧光显微镜下呈GFP阳性,实时荧光定量PCR显示DGKγ mRNA的表达较空载体组显著升高(P < 0.01),Western blotting显示DGKγ蛋白表达较空载体组极显著升高(P < 0.001)。提示：成功构建了大鼠DGKγ慢病毒过表达载体,DGKγ在293T细胞有效高表达。 ORCID: 0000-0001-9352-3043(李蕾) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.     

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.     

携带绿色荧光蛋白基因的单轮感染活性HIV假病毒的建立及其活性检测

目的构建携带绿色荧光蛋白基因的HIV假病毒载体,并研究该载体包装的HIV假病毒的感染活性,为进一步进行HIV生物学研究与中和抗体实验室评价搭建安全的技术平台.方法将增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因插入到骨架质粒pNL43 Luc R-E-的nef基因读码框,获得携带EGFP基因的质粒pNL43 EGFP R-E-;通过将pNL43 EGFP R-E-与编码HIV膜蛋白的质粒共转染293T细胞,收集上清,获得携带EGFP基因的HIV假病毒.该假病毒感染CD4^＋CCR5^＋的HOS细胞后可表达绿色荧光,这样通过流式细胞仪可测定表达绿色荧光的细胞数与细胞感染率.结果携带绿色荧光蛋白基因的HIV假病毒感染CD4^＋CCR5^＋的HOS细胞后,被感染的细胞可以表达绿色荧光蛋白,细胞的感染率与病毒加入量呈线性关系.结论获得了携带绿色荧光蛋白基因的HIV假病毒载体,并建立了具有单轮感染活性的HIV假病毒感染的检测方法.     

Hepatitis B Virus Vector Carries a Foreign Gene into Liver Cells In Vitro

Hepatitis B viruses (HBV) specifically target the liver, where they efficiently infect quiescent hepatocytes. Thus, HBV virus has potential to be used as vectors for liver-directed gene transfer. We constructed a new HBV-based vector system. It is composed of transfer vector for transferring a foreign gene, green fluorescence protein (GFP) gene, and a helper vector. When the transfer vector and the helper vector were cotransfected into HepG2 cells, the recombinant HBV (rHBV) particles were generated by trans-complementation between two vectors. The rHBV particles carrying the foreign gene were identified by the Southern blot assay. To test gene delivery and the transduction of the rHBV, we infected primary human hepatocytes and immortalized, HepG2 cells with rHBV in vitro. The results using fluorescence microscopy confirmed that the inserted GFP gene was successfully transferred and expressed both in primary human hepatocytes and HepG2 cells.     

Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.     

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.     

重组慢病毒表达载体介导HIV-1 Vpr蛋白调控KSHV潜伏感染的初探

目的 利用慢病毒载体系统包装含人类免疫缺陷病毒1型(HIV-1)病毒蛋白R(Vpr)的重组慢病毒,使之感染原发性渗出性淋巴瘤(PEL)细胞BCBL-1,并检测Vpr蛋白对细胞中卡波济肉瘤相关疱疹病毒(KSHV)潜伏感染与裂解性复制的影响.方法 从实验室先前构建的重组真核表达质粒pCI-neo-Vpr中扩增出Vpr基因,插入到pHAGE-CMV-MCS-IzsGreen载体中构建成重组慢病毒质粒pHAGE-Vpr,利用脂质体将其与包装质粒psPAX2及包膜质粒pMD2.G共转染293T细胞,荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)的表达,293T细胞培养上清经0.45 μm滤器过滤后即获得重组病毒悬液.慢病毒系列稀释后感染293T细胞,荧光计数法测定病毒滴度,逆转录PCR(RT-PCR)检测Vpr基因在293T细胞中的转录.以感染复数(MOI)为1的病毒量感染靶细胞BCBL-1,荧光显微镜观察靶细胞中GFP的表达,并利用RT-PCR和和免疫印迹(Western blot)技术分别检测Vpr基因在靶细胞中的转录和表达情况,同时检测KSHV裂解期基因复制与转录激活子(Rta)mRNA转录及蛋白表达.结果 经酶切鉴定、核酸序列测定和293T细胞中GFP表达证实成功包装了携带HIV-1Vpr基因的重组慢病毒,滴度为4×107TU/ml.重组慢病毒感染BCBL-1细胞后,细胞中有GFP表达,RT-PCR和Western blot均能够在相应位置检测到目的 基因Vpr的条带,并且Vpr降低了KSHV RtamRNA转录及蛋白表达水平.结论 重组慢病毒介导的HIV-1 Vpr蛋白过表达能够抑制KSHV裂解性复制、增强病毒的潜伏感染.     

Evaluation of a flow cytometric model for monitoring HIV antigen expression in vitro.

Using flow cytometry, monoclonal antibodies to the HIV proteins p24, gp41 and p17 were evaluated for their ability to detect HIV antigens associated with HIV-infected T cells. Mixtures containing varying ratios of HIV-infected and uninfected cells were subjected to analysis with these monoclonal antibodies. In most cases, the monoclonal antibodies identified the correct ratio of HIV-infected cells to uninfected cells in the mixtures tested. An HIV anti-p24 monoclonal antibody was selected for further studies. Flow cytometric analysis was performed on various populations of cells including uninfected, acutely infected and chronically infected cells. Based on cell population fluorescence intensity three distinct regions were identified. In the first region were cells having low level fluorescence that were considered negative for HIV antigens, a profile detected in uninfected cells, and in the majority of cells in the first days following acute HIV infection. In the second region were those cells exhibiting strong fluorescence such as chronically infected cells or acutely infected cells several days after infection. A third region was identified containing cells that were intermediate in fluorescence intensity. Cells exhibiting intermediate intensity fluorescence appeared to have low concentrations of HIV p24 antigen associated with them either through viral adsorption and uptake or through low level virus expression. These intermediate region cells appeared in the early stages following acute infection, and also when chronically infected cells and uninfected cells were permeabilized together, suggesting a 'leaching' of HIV proteins from highly infected cells to uninfected cells. This leaching type of phenomenon could present problems in determining gating parameters for positive cells since uninfected cells that have associated HIV antigens exhibit higher fluorescence intensity than uninfected cells.     

Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression

A hallmark of all herpesvirus is the ability to exist in either a latent, or lytic, state of replication, enabling the lifelong infection of its host. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) can efficiently establish a latent infection in a variety of cell types in vitro, making it a valuable model for the study of latency and reactivation. To facilitate the identification of KSHV lytic replication, and allow subsequent experiments with live cells, a recombinant virus, rKSHV.219, was constructed using JSC-1 cells that expresses the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-1α promoter, and with the gene for puromycin resistance as a selectable marker. rKSHV.219 from JSC-1 cells was used to infect Vero cells for purification of the recombinant virus. Vero cells were also used for the production of rKSHV.219 at levels of 10⁵-10⁶ infectious units (IU) of virus per milliliter using a combination of KSHV/RTA expressed from a baculovirus vector, BacK50, and butyrate. Virus produced from Vero cells was used to infect human fibroblasts (HF), 293, DU145, T24, HaCaT, and HEp-2 cells, and in all cells except 293 cells, only a latent infection was established with GFP expression, but no RFP expression. In 293 cells, 10-15% of cells showed lytic gene expression. Both primary and immortalized microvascular endothelial cells (MVEC) were also infected with rKSHV.219, and reduced spontaneous lytic replication was found in immortalized cells. In all cells used in this study, rKSHV.219 efficiently established latent infections from which the virus could be reactivated to productive lytic replication. This work also demonstrated strong synergy between KSHV/RTA and butyrate for the activation of KSHV lytic replication and the production of infectious virus.     

Identification of simian varicella virus gene 21 promoter region using green fluorescent protein

Clinical, pathological, immunological and virological features of simian varicella virus (SVV) infection in primates closely resemble those of varicella zoster virus (VZV) infection in humans. In ganglia infected latently of humans and monkeys, gene 21 of VZV and SVV is transcribed, respectively. We determined the nucleotide sequence of the intragenic region between SVV genes 20 and 21 to identify the putative promoter region for SVV gene 21. A recombinant clone was prepared in which the gene encoding green fluorescent protein (GFP) was inserted ten base pairs upstream of the predicted translational start site for SVV gene 21. SVV-infected monkey kidney cells transfected with the recombinant clone showed the presence of green fluorescence, whereas transfection of these cells with a construct containing the GFP gene in the opposite orientation, produced no fluorescence. The recombinant clone containing GFP flanked by SVV sequences can be used to prepare a SVV mutant in which the virus gene 21 promoter drives GFP. Such a mutant will be useful in analyzing varicella pathogenesis and latency in experimentally infected animals, studies not possible in humans.     

Flow cytometry assay for intracellular rabies virus detection

Following previous studies reporting microbiological diagnosis by flow cytometry, the possibility of using this method was examined to monitor infection of susceptible cell lines by a fixed rabies virus strain (Pasteur Virus strain-PV) or a wild rabies virus strain (WRS). Suspensions of BHK-21 and C6 cells were infected with viruses and a time course of virus infection was established. Sequentially, at several time points, infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry. A Becton Dickinson FACSCalibur flow cytometer was used to analyze the percentage of cells infected and the kinetics of the infection process was determined. As early as 12 h after inoculation with both rabies virus strains, significant levels (P<0.01) of infection (from 4.7 to 7.1%) were detected by flow cytometry. The maximum level of infection was obtained at 48 h in C6 cells (88%) with both viruses. The advantages of this method for examination of intracellular virus infection are discussed.     

趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达**

背景：趋化因子受体7(chemokine receptor-7, CCR7)是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受。 目的：构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达。 方法：采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体。将CCR7 DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7。采用脂质体转染法将慢病毒系统3质粒(重组慢病毒质粒LV-CCR7、包装质粒     ΔNRF及包膜质粒pVSVG)共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达。 结果与结论：实验成功扩增出小鼠CCR7 DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293 FT细胞后,24 h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108 U/L以上,获得携带CCR7基因的重组慢病毒。慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态。结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达。 关键词：趋化因子受体7;未成熟树突状细胞;慢病毒载体;真核表达;组织工程 doi:10.3969/j.issn.1673-8225.2012.01.029     

Ebola virus infection of human PBMCs causes massive death of macrophages, CD4 and CD8 T cell sub-populations in vitro

Ebola virus causes an often fatal disease characterized by poor immune response and high inflammatory reaction in the patients. One of the causes for poor immunity is virus-mediated apoptosis of lymphocytes in the host. In this study, we infected human PBMCs with Ebola Zaire virus and study apoptosis of different cell types using flow cytometry. We have shown that Ebola virus causes bystander death of CD4 and CD8 T cells. Cells infected with virus had 30-40% active caspase 3(+), annexin-V(+) and Bcl2(low) phenotype by day 8 PI as compared to inactivated virus-treated cells. 60-70% of the macrophages were also dead by day 8 PI and had similar phenotype. Our data also showed that virus may induce death signals in Fas(+)/FasL(+) T lymphocytes and macrophages but did not upregulate Fas/FasL expression in these cells. Lastly, CD4, CD8 and CD14 cells were purified after infection and were studied for death signals by RNAse protection assay. We found an upregulation of TRAIL mRNA in CD4 and CD8 T cells on day 7 PI. A two-fold increase in CD4 T cells and three-fold increase in CD8 T cells were observed in TRAIL mRNA levels as compared to uninfected controls and inactive virus-treated cells. Surprisingly, we did not find any difference in TRAIL mRNA levels between infected macrophages and uninfected controls. These data suggest that Ebola virus evades the immune response by causing massive lymphocyte death. In addition, they may give an explanation on why the host is unable to produce a good antibody response in the absence of CD4 T cells.     

The hypersensitive response to cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell

Cucumber mosaic virus (CMV) expressing the green fluorescent protein (GFP), and lacking either the 3a movement protein or the coat protein (CP), failed to induce a hypersensitive response producing local lesions in inoculated leaves of Chenopodium amaranticolor. Cytological analysis showed that both viral-encoded proteins are required for cell-to-cell movement of the virus and the simultaneous appearance of cellular necrosis. In the absence of either or both proteins, infection was confined to single, non-necrotized, epidermal cells. CMV with a mutation in the 3a protein (M8 CMV) could infect tobacco systemically but did not induce necrotic lesions in C. amaranticolor. In this host, the mutated 3a protein was unable to promote viral movement out of the initially infected epidermal cell. Movement-deficient CMV expressing wild-type (WT) 3a protein as a fusion to the GFP, as well as WT CP, also failed to induce necrosis. Finally, single epidermal cells infected with a movement-deficient CMV expressing WT 3a protein, WT CP, and free GFP did not show necrosis. These data indicate that viral movement out of the initially infected epidermal cell, and not the simultaneous expression in this cell of the 3a protein and the CP, is required for the induction of cell death.