RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells

Chikungunya has emerged as one of the most important arboviral infection of public health significance. Recently several parts of Indian Ocean islands and India witnessed explosive, unprecedented epidemic. So far, there is no effective antiviral or licensed vaccine available against Chikungunya infection. RNA interference mediated inhibition of viral replication has emerged as a promising antiviral strategy. In this study, we examined the effectiveness of small interfering RNAs (siRNAs) against the inhibition of Chikungunya virus replication in Vero cells. Two siRNAs against the conserved regions of nsP3 and E1 genes of Chikungunya virus were designed. The siRNA activity was assessed by detecting both the infectious virus and its genome. The results indicated a reduction of virus titer up to 99.6% in siRNA transfected cells compared to control. The viral inhibition was most significant at 24 h (99%), followed by 48 h (65%) post infection. These results were also supported by the quantitative RT-PCR assay revealing similar reduction in Chikungunya viral genomic RNA. The siRNAs used had no effect on the expression of house keeping gene indicating non-interference in cellular mechanism. The specific and marked reduction in viral replication against rapidly replicating Chikungunya virus achieved in this study offers a potential new therapeutic approach. This is the first report demonstrating the effectiveness of siRNA against in vitro replication of Chikungunya virus.     

Selective inhibition of hepatitis B virus replication by RNA interference

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.     

RNA干扰引起的非特异性免疫反应及其机制研究探讨

基因的表达失控是疾病发生的主要原因之一,干扰靶基因的表达可能成为有效的治疗手段。RNA干扰技术是近年兴起的基因调控干预方法,其基础,特别是应用研究极受关注,人们期待RNA干扰能成为肿瘤、病毒感染等难治疾病的临床治疗手段。然而,这一新兴技术在应用研究过程中显现出诸多问题,如细胞毒性、引起机体非特异性反应等等。就RNA干扰引起的非特异性免疫反应展开综述,探讨其机制,期望为RNA干扰的应用研究提供一些思考。     

RNA干扰抗病毒感染

RNA干扰是由双链RNA诱导的、关闭同源序列基因表达的机制。它是一种自然存在于植物、线虫、果蝇等真核细胞生物中的抵抗病毒感染方式。随着在哺乳动物细胞培养中成功地诱导RNA干扰,利用RNA干扰预防、治疗病毒感染已成为新的研究热点,并取得了有希望的成果。在未来,有望成为抗病毒感染的有效方法。     

RNA干扰在疾病治疗上的应用

RNA干扰(RNA interference,RNAi)是一种双链RNA分子在mRNA水平上引发的特异性基因沉默现象。RNAi在基因治疗方面表现出了光明的前景,已成功地应用于多种疾病的临床治疗。本文主要介绍了RNAi在疾病治疗上的应用及研究进展。     

siRNA抑制乙肝病毒的研究进展

乙型肝炎作为一种发病率高、死亡率高的传染性疾病,已严重威胁人类健康,乙肝病毒（hepatitis B virus,HBV）是诱发乙型肝炎的重要病因。目前,最主要的治疗方法是运用抗病毒药物控制病情,但这些药物都不能完全治愈乙型肝炎且复发率高。近年来,RNA干扰技术（RNA interference, RNAi）逐渐成为有效、快速治疗乙型肝炎的新疗法。利用RNA干扰技术体外合成针对HBV基因的siRNA,选择适当的载体将其运送至靶细胞,使HBV基因沉默,从而抑制病毒复制,可有效达到治疗乙肝的效果。本文围绕siRNA沉默HBV基因的设计原理、递送载体、靶向策略、以及治疗效果与应用前景等方面进行了系统综述,为今后siRNA治疗乙肝的临床应用提供参考。     

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。     

RNA interference in immune cells by use of osmotic delivery of siRNA

Delivery of siRNA to immune cells has been one of the major obstacles to widespread application of RNAi in the immunology field. Here, we report that osmotic delivery of siRNA can be used to silence genes in macrophage RAW264.7 without incurring either cytotoxic or immunomodulatory activity. We also showed usefulness of the osmotic delivery in other types of cells including T cell DO11.10. By repeated osmotic delivery of siRNA, long-term gene silencing was readily achieved. When TLR4 was disrupted in RAW264.7 cells for 48 h and the cells were stimulated with the TLR4 ligand LPS, a significant decrease in TNFalpha production was observed. DNA microarray-based gene expression profile analysis showed that gene silencing by osmotic delivery of siRNA was target-specific and the delivery method itself had little influence on overall gene expression.     

RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

Epidemiologic interference of virus populations

Summary There are a few simulation studies for interference models in the literature but the present paper discusses an analytical model for the competition of two interfering virus populations in a community. The mathematical model consist of eight coupled differential equations which have up to four equilibrium points. Criteria for local stability are given.This paper has been read at the workshop on Nonlinear Models in Biology and Medicine, 23rd Biometric Colloquium, German Region of the Biometric Society, Nuremberg, March 1977     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。     

RNA干涉的研究进展

生物体内导入双链RNA后会引起体内同源基因特异性的沉默,这种现象称为RNA干涉,本主要介绍RNA干涉的研究历史,作用机制和应用等方面的情况。     

Inhibition of gammaherpesvirus replication by RNA interference

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.     

Interaction of the innate immune system with positive-strand RNA virus replication organelles

The potential health risks associated with (re-)emerging positive-strand RNA (+RNA) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. The innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (PAMPs). Viral RNA is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (IFNs) and pro-inflammatory cytokines. However, +RNA viruses developed various methods to avoid detection and downstream signaling, including isolation of viral RNA replication in membranous viral replication organelles (ROs). These structures therefore play a central role in infection, and consequently, loss of RO integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. This review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ROs and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein 1A/1B-light chain 3 (LC3) and ubiquitin, resulting in the recruitment of IFN-inducible GTPases. Further studies should evaluate whether this process forms a general effector mechanism in +RNA virus infection, thereby creating the opportunity for development of novel antiviral therapies.     

Replication of hepatitis C virus RNA on autophagosomal membranes

Previous studies indicated that hepatitis C virus (HCV) perturbs the autophagic pathway to induce the accumulation of autophagosomes in cells. To understand the role of autophagosomes in the HCV life cycle, we established a stable Huh7 hepatoma cell line that contained an HCV subgenomic RNA replicon and also expressed a GFP-LC3 fusion protein. The GFP-LC3 protein is localized to autophagosomes during autophagy and served as a convenient marker for autophagosomes. Our results indicate that the silencing of the expression of LC3 or Atg7, two protein factors critical for the formation of autophagosomes, suppresses the replication of HCV RNA. Confocal microscopy studies revealed the localization of HCV NS5A and NS5B proteins, which are two important components of the HCV RNA replication complex, and nascent HCV RNA to autophagosomes. The association of the HCV RNA replication complex with the autophagosomal membranes was further confirmed by co-immunoprecipitation and immunoelectron microscopy studies. Interestingly, inhibition of Class III PI3K activity had no effect on the autophagosomes induced by HCV. These results indicate that HCV induces autophagosomes via a Class III PI3K-independent pathway and uses autophagosomal membranes as sites for its RNA replication.     

RNAi及其抑制口蹄疫病毒复制的研究进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double strand RNA,dsRNA)介导的序列特异的RNA降解过程。已经证明,在植物和昆虫细胞中RNAi是其主要的抗病毒免疫机制,至今为止几乎没有发现在病毒感染哺乳动物细胞过程中诱发有效的抗病毒RNAi反应。因此,人们希望能够利用人工方法在哺乳动物细胞中建立有效的抗病毒RNAi防御策略。迄今为止,对多种哺乳动物病毒的研究结果令人振奋。主要围绕RNAi的分子基础、基本策略及其在抑制口蹄疫病毒复制中的研究现状作了综述。