Chapter 1 RNAi: An Antiviral Defense System in Insects
Bertsy Goic and Maria-Carla Saleh
Multicellular organisms evolved sophisticated defense systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations (Baulcombe, 2004; Dorner and Radbruch, 2007; Roitt et al., 2001; Voinnet, 2005). Insects rely on multiple immune responses to combat infection; one of them is RNA interference (RNAi). Here we review the current knowledge on the general mechanism of RNAi and summarize what is known about the antiviral role of RNAi in the insect model Drosophila melanogaster. We also discuss the strategies evolved by viruses to suppress the RNAi response. Finally, we briefly describe the RNAi mechanism in other insects of economical and/or health relevance.

Chapter 2 RNA Silencing in Plants and the Role of Viral Suppressors
Ana Giner, Juan José López-Moya and Lorant Lakatos
The term RNA silencing refers to several pathways present in eukaryotic organisms that lead to the sequence specific elimination or functional blocking of RNAs with homology to double stranded RNAs (dsRNAs) that have previously triggered the mechanism. Besides playing important roles in developmental control, RNA silencing forms part of the defence against viruses in plants, acting as a potent antiviral mechanism. To escape from the RNA silencing-based defence, most plant viruses make use of different strategies, the most common relying in the action of viral proteins with the capacity to suppress RNA silencing. The characterization of these viral suppressors is providing useful insights to understand how RNA silencing works, revealing components and steps in the silencing pathways.

Chapter 3 The Properties and Roles of Virus-encoded MicroRNAs
Mélanie Tanguy and Sébastien Pfeffer
The discovery that viruses could encode micro (mi)RNAs, similarly to the eukaryotic organisms they infect, has opened new perspectives in the study of host-virus interactions. These small regulatory RNAs, which are critically involved in an ever-increasing number of biological processes, have revolutionized the way we used to see gene regulation. Some mammalian viruses, mainly from the herpesvirus and polyomavirus families, have hijacked this mechanism in order to help them achieve the infection of their host. In this chapter, we will present the diversity of known viral miRNAs, their specific properties, their viral and cellular targets and the roles they play during the course of infection. We will see that more and more it appears that virally encoded miRNAs seem to be critically involved in every step of the virus life cycle

Chapter 4 Virus-encoded Suppressors of RNA Silencing and the Role of Cellular miRNAs in Mammalian Antiviral Immune Responses
Joost Haasnoot and Ben Berkhout
Small RNA-directed silencing mechanisms play important roles in the regulation of eukaryotic gene expression. In plants, insects, nematodes and fungi RNA silencing mechanisms are also involved in innate antiviral defence responses. To counter antiviral RNA silencing, viruses from plants, insects and fungi encode RNA silencing suppressors (RSSs). Recent studies suggest that RNA silencing in mammals, or RNA interference (RNAi), is also involved in antiviral responses. In particular, there is increasing evidence that cellular regulatory microRNAs (miRNAs) have a function in restricting virus replication in mammalian cells. Similar to plant and insect viruses, several mammalian viruses encode RSS factors that inhibit the RNAi mechanism. Several of these suppressors are multifunctional proteins that were previously shown to block innate antiviral immune responses involving the interferon (IFN) pathway. Here we summarize the current data on mammalian virus-encoded RSS factors. In addition, different aspects of antiviral RNAi and the role of cellular miRNAs in restricting virus replication in mammalian cells are discussed.

Chapter 5 HCV and the Interaction with miR-122 in the Liver
Gabriele Fuchs and Cara T. Pager
MicroRNAs (miRNA) are endogenous non-coding RNA molecules, typically 21 nucleotides in length that can inhibit target gene expression when bound to 3í untranslated regions in target mRNAs. In this review we summarize how miRNAs are generated, how miRNA-target interactions occur and the different outcomes of these interactions. In detail, we describe the unusual interaction between the liver-specific miRNA miR-122 and Hepatitis C virus (HCV) RNA, and review what is known about the mechanism by which miR-122 modulates HCV gene expression. Furthermore, we discuss the role of miR-122 in the liver, and how therapeutic approaches that alter miR-122 levels can influence HCV replication and hepatocarcinogenesis.

Chapter 6 Viral Escape From RNAi in Mammalian Cells
Maria Nevot and Miguel Angel MartÌnez
Fire, Mello, and coworkers first discovered that introducing long double-stranded RNA (dsRNA) into the nematode Caenorhabditis elegans led to the targeted degradation of homologous messenger (m)RNA. This discovery revealed the existence of a fundamental mechanism for gene expression regulation, now known as RNA interference (RNAi) (Fire et al., 1998). Later, Elbashir et al. showed that RNAi also occurs in mammalians cells (Elbashir et al., 2001). Importantly, they performed the extraordinary demonstration by cell transfection that synthetic, short, 21 base-pair (bp) duplexes of interfering RNA (siRNA) could mediate RNAi in a sequence-specific manner. This finding enabled the specific regulation of gene expression in a variety of biological systems, including diseased cells. Recent studies regarding the utility of RNAi to specifically inhibit virus replication have opened new possibilities for the development of novel therapies against viral infection. However, viruses appear to be capable of escaping RNAi inhibition in mammalian cells. Viral mechanisms for escaping RNAi may include suppression of RNAi, mutational escape from RNAi, and modulation of the cell's microRNA (miRNA) /RNAi profile. Here, we summarize these viral mechanisms and discuss potential strategies for neutralizing viral escape from RNAi.

Chapter 7 RNAi Gene Therapy to Control HIV-1 Infection
Ben Berkhout and Olivier ter Brake
RNA interference (RNAi) was discovered as cellular gene regulation mechanism in 1998, but several RNAi-based applications for gene silencing have already made it into clinical trials. We will discuss RNAi approaches to target pathogenic human viruses causing acute or chronic infections, with a focus on persistent HIV-1 infection that would most likely require an RNAi-based gene therapy. Viruses like HIV-1 are particularly difficult targets for RNAi-attack because they are escape-prone, which requires combinatorial RNAi strategies to prevent viral escape. The future of antiviral RNAi therapeutics is very promising, but it remains of critical importance to include many controls in pre-clinical test models to unequivocally demonstrate sequence-specific action of the RNAi inducers.

Chapter 8 Advances in the Use of RNAi to Treat Chronic Hepatitis B Virus Infection
Abdullah Ely and Patrick Arbuthnot
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the worldís population and carriers of the virus are at risk for complicating hepatocellular carcinoma (HCC) and cirrhosis. Although effective vaccination is available, it is prophylactic and of little use to individuals who are already infected with the virus. Furthermore, current treatment options have limited efficacy and chronic HBV infection is likely to be a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. Numerous studies have reported highly successful suppression of viral replication, which bodes well for employing this approach to counter HBV infection. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realised. Improvement of delivery efficiency, dose regulation, limiting of off target effects and preventing reactivation of viral replication are some of the hurdles that need to be overcome. Nevertheless, the vast potential of RNAi-based therapeutics will continue to drive innovative research, and this promises to surmount the obstacles that face this exciting field.

Chapter 9 Hepatitis C: New Insights and Therapeutics by RNAi
Qiuwei Pan and Luc J.W. van der Laan
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis and its sequels, end-stage liver disease and hepatocellular carcinoma, are the most common indications for liver transplantation. The current standard treatment, pegylated-interferon-alpha in combination with ribavirin, represents a milestone in therapy, but only eradicates the virus in half of the patients, leaving tens of millions without hope for a cure. Therefore, it is urgent to explore novel and more effective therapeutic options. Such advancements could be derived from a better understanding of HCV biology. Progress in this area has accelerated with the application of RNA interference (RNAi) as a discovery tool to investigate viral and host cell factors involved in viral infection. Furthermore, RNAi has high potential as a therapeutic avenue for treatment of HCV. In this chapter, we aim to provide a comprehensive overview of the current developments and the potential application of RNAi in research and therapy of HCV infection.

Chapter 10 RNAi Applications to Defeat Respiratory Viral Infections
Sailen Barik
Pathogenic respiratory viruses, exemplified by respiratory syncytial virus (RSV), influenza (Flu) and parainfluenza virus (PIV), are major disease agents that kill millions of humans worldwide. Other respiratory viruses that are also potential agents of bioterrorism include highly pathogenic avian flu virus, SARS coronavirus, and the henipaviruses. Respiratory infection by RSV and PIV is the most prevalent cause of pediatric hospitalization in industrialized Western nations. In the US alone, it leads to about 100,000 admissions per year at a cost of about 300 M. The lack of a reliable vaccine or antiviral against these viruses, in part due to the high mutation rate of the viral RNA genomes, has led to the adoption of the novel RNA interference (RNAi) strategy. This review summarizes significant recent progress in the development of RNAi therapeutics to defeat these viral respiratory diseases.

Chapter 11 RNAi With Viral Vectors That Deliver Small Interference RNAs
Jovanna González-Rojas, Xabier Abad and Puri Fortes
RNA interference (RNAi) has been successfully applied as a technology to inhibit gene expression for functional studies and offers great promise in therapeutic applications. The inhibitors that mediate interference are called siRNAs, small RNA molecules that can be expressed from viral vectors. Depending on the viral vector used and the route of administration, the inhibitors can be delivered in specific organs, for shorter or longer terms and with varying efficacy. In this chapter, we review the characteristics of the most relevant viral vectors for RNAi technology and the different cassettes that allow expression of the small RNA inhibitors. Further, we summarize the application of viral vectors delivering siRNAs in preclinical models of relevant diseases, such as cancer, neurological disorders and viral infections. Finally, we discuss several concerns that should be taken into account when considering viruses as vectors to mediate RNAi. We believe that the information provided in this chapter will help the successful design of experiments aimed to silence the expression of specific genes, both for gene function studies or for therapeutic purposes.