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Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression  [PDF]
Jana Nejepinska, Radek Malik, Susan Wagner, Petr Svoboda
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0087517
Abstract: In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells—expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.
Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells  [PDF]
Jenny Calvén, Yuliana Yudina, Lena Uller
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062718
Abstract: Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ1 production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 μg/ml dsRNA, 0.1–1 μg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ1 mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ1 protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ1 mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ1.
Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells  [PDF]
Yuye Li, Jiong Xie, Siyu Wu, Jun Xia, Peifen Zhang, Chao Liu, Ping Zhang, Xi Huang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055108
Abstract: Background Dengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses. Methodology/Principal Findings By RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-β in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-κB, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production. Conclusions/Significance PKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.
A Review Of Chikungunya  [cached]
Bincy Thomas,A.R.Tekade,Pande V.V
Pharmaceutical Reviews , 2007,
Abstract: Vaccines and new drugs against chikungunya are urgently needed. Chikungunya is generally not fatal.However, in 2005-2007, many deaths have been associated with chikungunya worldwide and still it is continuing. Currently there is no vaccine and fully satisfied drugs to treat all symptoms of chikungunya.Treatment are just symptomatic and in many cases improper use of NSAIDS leads to more complications. Every year 1000s are getting infected and many are dying. So it is high time to turn health organisation’s concentration to find a solution for this health issue.IntroductionThe word chikungunya meaning "that which bends up", which is derived from its arthritic symptoms. It is the hottest topic of discussion among public today, so is necessary to know the causes, symptoms, treatment and preventive measures of this viral fever. Usually chikungunya virus or CHIKV is spread by mosquito bites from Aedes aegypti mosquitoes, Aedes albopictus (Tiger mosquito), Aedes luteocephalus, or A. taylori.. There is no reported case of human-human transmission of CHIKV. The outbreak of infection and studies about it reveals that people living near to forest and water stores are more prone to this viral desease, since these areas provide better environment for mosquito breeding.High fever and arthritic pain, especially severe pain on extremes is characteristic of chikungunya fever. In allopathic, treatment is based on the symptoms and no preventive drugs are available. Siddha system claims some medicines for both treatment and prevention, but is not scientifically proven. By taking proper precaution against mosquito bites by using insecticides, repellents and other measures, transmission of CHIKV can be prevented to greater extent.
Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism  [PDF]
Shridhar Bale,Jean-Philippe Julien equal contributor,Zachary A. Bornholdt equal contributor,Christopher R. Kimberlin equal contributor,Peter Halfmann,Michelle A. Zandonatti,John Kunert,Gerard J. A. Kroon,Yoshihiro Kawaoka,Ian J. MacRae,Ian A. Wilson,Erica Ollmann Saphire
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002916
Abstract: Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.
Vimal Kumar Birendra,Singh Vishwabhan,Joon Uttam,Suvagiya Vishal
International Research Journal of Pharmacy , 2012,
Abstract: Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). Chikungunya is a re-emerging arthropod-borne viral disease caused by Chikungunya virus (CHIKV) belonging to the Togaviridae family of genus Alphavirus. It is a virus with a single stranded, positive sense RNA, as its genome. It is maintained in a sylvatic and urban cycle involving humans and the mosquito species Aedes aegypti and Aedes albopictus. It has a major health impact on humans as it causes fever, rashes, arthralgia and myalgia. Polyarthralgia is the most important feature of CHIKV infection which primarily affects the small joints of the wrists and fingers along with the large joints like shoulders and knees. Currently, there are no vaccines or treatment regimens available for CHIKV infection. The molecular mechanism underlying the chronic polyarthralgia observed in patients is not well understood. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. In this review we have summarized the CHIKV organization, replication, epidemiology, clinical manifestations and pathogenesis with emphasis on the arthralgia.
Gor czka chikungunya
Ilona Cebula-Byrska,Eugeniusz J. Kucharz
Reumatologia , 2011,
Abstract: Gor czka chikungunya jest tropikaln chorob arbowirusow wyst puj c g ównie w po udniowej Azji i wschodniej Afryce.Wirus chikungunya spowodowa w 2005 r. masowe zachorowaniana wyspach Oceanu Indyjskiego oraz w zachodnich nadbrze nychrejonach Indii i w Malezji. Mi dzy 1995 a 2009 r. odnotowanook. 100 przypadków tej choroby w Stanach Zjednoczonych (u osóbpowracaj cych z podró y). Pierwszy przypadek gor czki chikungunyaw Europie stwierdzono w okolicach Rimini w 2007 r., co sta osi powodem zwrócenia przez Europejczyków wi kszej uwagi nat chorob . atwo rozprzestrzeniania si gor czki chikungunyai mo liwo wywo ania epidemii wynika m.in. z faktu, i jest onaprzenoszona przez komary z rodzaju Aedes. Choroba objawia si wysok gor czk , trwaj c kilka dni, plamist lub wybro czynow wysypk na skórze tu owia i kończyn, przewlek ym bólem lubzapaleniem stawów kolanowych, skokowych oraz drobnych stawówkończyn, bólem mi ni i zapaleniem pochewek ci gnistych.U 12% chorych zapalenie stawów mo e trwa 3–5 lat (tab. I, II).Infekcja wirusem chikungunya jest chorob samo ograniczaj c si , aczkolwiek rzadko mo e si objawia klinicznie w postacipowa nych, zagra aj cych yciu powik ań, m.in. neurologicznych,gastrologicznych (piorunuj ce zapalenie w troby) czy powik ańkrwotocznych (zw aszcza u dzieci). W gor czce chikungunya niema swoistego leczenia przeciwwirusowego ani szczepionki. Leczeniejest objawowe. Jedyn znan metod zapobiegawcz jest unikanienara enia na uk ucia przez komary (moskitiery, repelenty).
RIG-I and dsRNA-Induced IFNβ Activation  [PDF]
Stéphane Hausmann, Jean-Baptiste Marq, Caroline Tapparel, Daniel Kolakofsky, Dominique Garcin
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003965
Abstract: Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral pppRNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5′ tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.
Characterization of Reemerging Chikungunya Virus  [PDF]
Marion Sourisseau,Clémentine Schilte,Nicoletta Casartelli,Céline Trouillet,Florence Guivel-Benhassine,Dominika Rudnicka,Nathalie Sol-Foulon,Karin Le Roux,Marie-Christine Prevost,Hafida Fsihi,Marie-Pascale Frenkiel,Fabien Blanchet,Philippe V Afonso,Pierre-Emmanuel Ceccaldi,Simona Ozden,Antoine Gessain,Isabelle Schuffenecker,Bruno Verhasselt,Alessia Zamborlini,Ali Sa?b,Felix A Rey,Fernando Arenzana-Seisdedos,Philippe Desprès,Alain Michault,Matthew L Albert,Olivier Schwartz
PLOS Pathogens , 2007, DOI: 10.1371/journal.ppat.0030089
Abstract: An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.
Ribavirin therapy for Chikungunya arthritis
Rajan Ravichandran, Manju Manian.
Journal of Infection in Developing Countries , 2008,
Abstract: Background: Chikungunya is an acute viral infection presenting with a febrile episode and severe arthralgia, swelling of soft tissues, especially around the ankles. Many patients recover with nonspecific treatment of analgesics. Some patients continue to have subacute crippling arthritis in the legs affecting their mobility. This study was undertaken to see the effect of the antiviral drug Ribavirin in the clinical outcome of these patients.Methodology: Ten patients who continued to have crippling lower limb pains and arthritis for at least two weeks after a febrile episode were taken up for the drug study. Ten similar patients during the same period were included as controls. In the study group Ribavirin was given at 200 mg twice a day for seven days. Both groups were followed up for four weeks Results: All patients in the drug group reported improvement in the joint pains with six of them capable of walking freely. The soft tissue swelling also reduced in eight. In three patients the pain returned after mobilization. Seven patients continued not to receive analgesics after four weeks.Conclusions: Ribavirin may have a direct antiviral property against Chikungunya leading to faster resolution of joint and soft tissue manifestations.
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