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Insect Viruses: Nonoccluded

Autographa californica NPV 1000 Adoxophyes honmai NPV

1000

Cydia pomonella GV Baculoviridae

999 Neodiprion lecontei NPV 1000

728

Neodiprion sertifer NPV Culex nigripalpus NPV

978

Melanoplus sanguinipes entomopoxvirus 1000

Poxviridae

Amsacta moorei entomopoxvirus Gryllus bimaculatus virus 1000 Oryctes rhinoceros virus Nudiviridae

999 Helicoverpa zea virus 1 1000 Helicoverpa zea virus 2 White spot syndrome virus

Nimaviridae

0.1 Figure 4 A dendogram showing phylogenetic relationships.

have the potential to serve both as model to study the nature of viral persistence and as a tool for altering the genetic make-up of insects. See also: Baculoviruses: Molecular Biology of Nucleopolyhedroviruses; Herpesviruses: Latency; Insect Pest Control by Viruses; Oryctes Rhinocerous Virus; Persistent and Latent Viral Infection.

Further Reading Burand JP (1998) Nudiviruses. In: Miller LK and Ball A (eds.) The Insect Viruses, 69pp. New York: Plenum. Burand JP, Kawanishi CY, and Huang Y-S (1986) Persistent baculovirus infections. In: Granados RR and Federici BA (eds.) The Biology of Baculoviruses, vol. 1, pp. 159–177. Boca Raton, FL: CRC Press.

Burand JP and LU H (1997) Replication of a gonad-specific insect virus in TN-368 cells in culture. Journal of Invertebrate Pathology 70: 88–95. Burand JP, Rallis CP, and Tan W (2004) Horizontal transmission of Hz-2V by virus infected Helicoverpa zea moths. Journal of Invertebrate Pathology 85: 128–131. Hamm JJ, Carpenter JE, and Styer EL (1996) Oviposition day effect on incidence of agonadal progeny of Helicoverpa zea (Lepidoptera: Noctuidae) infected with a virus. Annals of the Entomological Society of America 89: 266–275. Huger AM (2005) The Oryctes virus: Its detection, identification, and implementation in biological control of the coconut palm rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae). Journal of Invertebrate Pathology 89: 78–84. Huger AM and Kreig A (1991) Baculoviridae: Nonoccluded baculoviruses. In: Adams JR and Bonami JR (eds.) Atlas of Invertebrate Viruses, pp. 287–319. Boca Raton, FL: CRC Press. Wang Y, van Oers MM, Crawford AM, Vlak JM, and Jehle JA (2007) Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1. Archives of Virology 152(3): 519–531.

Interfering RNAs K E Olson, K M Keene, and C D Blair, Colorado State University, Fort Collins, CO, USA ã 2008 Elsevier Ltd. All rights reserved.

Glossary dsRNA Double-stranded RNA, the trigger for RNAi and intermediate in RNA virus replication. miRNA MicroRNA; small RNAs, about 21–23 bases in length, that post-transcriptionally regulate

the expression of genes by binding to the 30 untranslated regions (30 UTR) of specific mRNAs. RISC RNA-induced silencing complex; a multiprotein complex that brings the guide strand of the siRNA duplex and the cellular mRNA together and cleaves

Interfering RNAs

the mRNA with associated endonuclease activity. mRNA is then degraded. RNAi An evolutionarily conserved, sequence-specific antiviral pathway triggered by double-stranded RNA (dsRNA) that leads to degradation of both the dsRNA and mRNA with homologous sequence. siRNA Small interfering RNAs; 21–25 bp duplexes that provide guides for sequence-specific cleavage of mRNA. siRNAs are considered the hallmark of RNAi.

Introduction Antiviral innate immune responses in vertebrate hosts restrict viral invasion until humoral and cell-mediated acquired immune responses specifically clear virus infection. In mammals, innate immune responses are induced when infected cells recognize viral components such as nucleic acids through host pattern-recognition receptors. Toll-like receptors are important pattern-recognition sensors that recognize viral components (e.g., doublestranded RNA (dsRNA)) and signal induction of type I interferons and inflammatory cytokines that lead to an antiviral state. Organisms such as insects probably depend entirely on antiviral innate immune responses to overcome viral infection, since acquired immune responses have not been identified in invertebrates. Interferon-like molecules also have not been found in insects, making it unclear how insects cope with viral infections. In 1998, researchers first described an intracellular response in the worm Caenorhabditis elegans that could be triggered by dsRNA and efficiently silenced the expression of genes having sequence identity with the dsRNA trigger. This response, termed RNA interference or RNAi, is now known to be an ancient antiviral innate immune response in eukaryotic organisms including worms, plants, insects, and mammals and is a common evolutionary link among these organisms in their fight against virus invasion.

RNA Interference RNAi, post-transcriptional gene silencing (PTGS), quelling, and sense suppression are terms for related pathways that have been described in different organisms. All are RNAi responses triggered by dsRNA that result in degradation of both dsRNA and mRNA with cognate sequence. These pathways are highly conserved evolutionarily and exist in many organisms including plants, fungi, and animals. RNAi and related pathways have several functions, including regulation of development, silencing and regulation of gene expression, and defense against viruses and transposable elements. In this article, we focus on RNAi as an antiviral, innate immune pathway.

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RNAi Mechanism of Action The RNAi pathway is divided into an initiator phase and an effector phase. The initiator phase consists of the recognition and processing of long dsRNA molecules by the RNaseIII enzyme Dicer into small interfering RNAs (siRNAs) of 21–25 bp. siRNAs, considered the hallmark of the RNAi response, are duplexes with 30 overhangs of 2 nt. Each strand has a 50 -PO4 and 30 -OH. In the fruitfly Drosophila melanogaster (drosophila), the siRNAs are unwound and incorporated into the RNA-induced silencing complex, or RISC, with the assistance of Dicer-2 and R2D2, to start the effector phase of the pathway. In the effector phase, one strand of the siRNA duplex acts a as guide sequence to target the RISC to complementary mRNAs and determine the cleavage site on the mRNA. The RISC is known to include products of the following genes: Argonaute2 (Ago2), Vasa intronic gene (VIG), fragile X mental retardation (FXR), and Tudor Staphylococcal nuclease (Tudor-SN). Other genes in drosophila encode proteins having RNA helicase activity associated with RNAi and include spn-E, Rm62, and armi. The latter gene products have been implicated with heterochromatin and transposon silencing.

RNAi Components and Function: Dicers, the Sensors of dsRNA and Initiators of siRNA Production The majority of biochemical information related to Dicer proteins has been elucidated from studies in drosophila, showing that Dicer enzymes are intracellular sensors of dsRNA that initiate RNAi in drosophila cultured cell and embryo lysates. Candidate genes from three families encoding RNase III motifs were expressed in drosophila S2 cells, immunoprecipitated, and tested in vitro for their ability to transform long dsRNA molecules (>30 bp) into small RNAs of 21 nt (Figure 1). The enzyme capable of producing siRNAs was termed Dicer and contained a helicase domain as well as two RNase III domains. The production of siRNAs by Dicer-2 in drosophila was ATP-dependent and the enzyme was inactive in degrading single stranded RNAs. Dicer depletion by immunoprecipitation from drosophila cell lysates resulted in decreased siRNA production. Dicer is a 200 kDa protein with an N-terminal RNA helicase domain, a PAZ (PIWI/ Argonaute/Zwille) domain, a conserved domain of unknown function (DUF283), two C-terminal RNase III domains, and an RNA binding motif. In its active form, Dicer is a homodimer. Dicer is evolutionarily conserved, and is found in drosophila, C. elegans, humans, mice, trypanosomes, zebrafish, the fungi Magnaporthe oryzae and Neurospora crassa, budding yeast (Schizosaccharomyces pombe), plants, and many other organisms. The number

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Dicer

Dicer acts on long dsRNA, dicer generates 21–23 nt dsRNA R2D2

Dicer

Dicer/R2D2/siRNA duplex: R2D2 is important in loading of Dicer/siRNA onto RISC

R2D2

AGO2

ATP

Holo-RISC

Unwind siRNA duplex and activate RISC

Dicer R2D2

mRNA

Activated RISC with siRNA guide strand acts on target RNA (AGO2 of RISC displaces R2D2 and one of the siRNA strands)

Degradation of target mRNA identified by the guide sequence (AGO2 has catalytic activity for siRNA-directed silencing)

Figure 1 General scheme for RNAi leading to degradation of a specific mRNA.

of dicer (dcr) genes varies in different organisms. In drosophila there are two dcr genes (dcr1 and dcr2); in the plant Arabidopsis thaliana there are four, each of which processes siRNAs from different dsRNA sources; however, in the genomes of humans and C. elegans, there is only one dcr gene.

Dicers and miRNA Biogenesis The RNAi pathway has two branches leading to production of either siRNA or microRNA (miRNA). The miRNAs lin-4 and let-7 were first discovered in C. elegans and play a crucial role in development. Subsequently, hundreds of different miRNAs have been found in most eukaryotic organisms and exploring their expression and function is now an important topic of research in a wide range of invertebrates and vertebrates. miRNAs are produced from endogenous pre-miRNA transcripts that form stem–loop precursors. Silencing of genes by the miRNA pathway occurs not by degradation of the mRNA, but rather by translational arrest during protein synthesis. Also, unlike the siRNA pathway, miRNA silencing does not require complete base pairing between the miRNA and the target sequence to be silenced. Distinct RISCs process small RNAs for the effector phase of the two gene-silencing mechanisms. It is not known how the distinction is made between siRNA production and miRNA production with a single Dicer enzyme in C. elegans. In humans, interferon and other innate immune responses are induced by long dsRNA, so Dicer-like activity generating siRNAs may not be as crucial. In organisms such as drosophila that encode two Dicer proteins, Dicer-1 is the enzyme that produces miRNAs from endogenous transcripts.

R2D2 Protein and RNAi R2D2 is a 36 kDa protein with two dsRNA binding domains that bridges the initiator and effector stages of the RNAi pathway. R2D2 co-purifies with Dicer-2 from an siRNA-generating extract of drosophila S2 cells. R2D2 association does not affect the enzymatic activity of Dicer-2, but is required to load the newly formed siRNAs into RISC. Dicer-2 forms a heterodimer complex with R2D2 and the siRNA duplex that appears to detect the thermodynamic asymmetry of the siRNA duplex. The passenger strand of the siRNA duplex is separated from the guide strand, which interacts at its 50 and 30 ends with the PIWI (possessing RNase H-like activity) and PAZ domains of Argonaute-2 (AGO2), respectively. Strand selection (passenger strand vs. guide strand) depends on the thermodynamic stability of the first four nucleotides of the 50 terminus of an siRNA duplex. The siRNA strand whose 50 end has lower base-pairing stability becomes the guide strand, leaving the more stable strand as the passenger strand. R2D2 binds to the thermodynamically more stable end of an siRNA whereas Dicer-2 binds on the opposite end. Release of the degraded passenger strand is believed to be an ATP-dependent reaction. The guide siRNA strand remains associated with RISC and guides AGO2 to the target mRNA containing the complementary sequence. After hybridization, AGO2 cleaves the phosphodiester backbone of the target mRNA. Target RNA cleavage occurs between the 10th and 11th nucleotides of the guide siRNA measured from its 50 phosphate group. The phosphate group of the 50 end of the guide siRNA influences the fidelity of the cleavage position as well as the stability of RISC. Recently it was confirmed that the RNAi pathway of drosophila functions as an antiviral

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immunity mechanism. Drosophila with null mutations for genes encoding Dicer-2, Argonaute-2, or R2D2 were highly susceptible to RNA viruses such as Flock house virus (FHV) (Nodaviridae), Drosophila C virus (Dicistroviridae), or Sindbis virus (Togaviridae) which in some cases were lethal for the flies.

number of other proteins have now been associated with the RNAi pathway, including Vig, Tudor-SN, and Fmr-1, all associated with RISC.

Argonaute Proteins and RNAi

In C. elegans and plants, an amplification of the RNAi response occurs. In these systems, the original siRNAs act as primers for synthesis of new intracellular dsRNA by the endogenous RNA-dependent RNA polymerase (RdRP). These newly produced dsRNAs are processed by Dicer and generate an additional and potentially more diverse pool of siRNAs. This phenomenon is termed ‘transitive RNAi’ and allows for the degradation of a full-length mRNA even when the initial trigger sequence represents only a portion of the gene or genome. Amplification of the siRNA signal is bidirectional along the transcript in plants; however, in C. elegans the signal can only spread 30 –50 along the transcript. Transitive RNAi does not occur in species such as D. melanogaster, where RdRP genes are absent from the genome. Gene knockdown experiments in organisms lacking transitive RNAi require design of dsRNA for specific disruption of gene expression. One unique aspect of PTGS in plants is the ability of the silencing signal to spread throughout the organism. The siRNAs, complexed with host proteins, are part of a silencing complex that can move to other tissues in the plant through phloem tissues. The distance that the siRNAs travel is dependent on their exact length and the Dicer enzyme by which they were generated. The exact mechanism for long-distance movement has yet to be elucidated. RNAi in C. elegans can spread from the point of induction, especially if the dsRNA is introduced into the intestine either by injection or by feeding. RNAi has become an important reverse genetics tool for studying gene function. Systematic knockdown of all genes has been accomplished in both drosophila and C. elegans with great success, identifying previously unknown functions of genes in various biological pathways and revealing differences in the RNAi pathways of each organism. In drosophila, the siRNA signal appears not to be amplified (transitive RNAi) and does not spread as seen in C. elegans and plants. This means that RNAi activity is most likely confined to those cells in which the dsRNA trigger is detected by Dicer. RNA silencing has been used in many other invertebrates, including mosquito disease vectors. Several studies have shown that RNAi can be used to knock down endogenous and exogenous gene expression in mosquitoes that transmit medically important pathogens to animals and humans. Studies of RNAi in mosquitoes have also shown that it is possible to silence RNAi complex genes using RNAi. As an example, the ability to silence the RNAi pathway in a hemocyte

Another family of genes that has already been discussed in this review as important to RNAi is the argonaute gene family. In drosophila, there are five argonaute genes. Two of the genes, argonaute1 (ago1) and argonaute2 (ago2), have been implicated in RNAi. Argonaute-2 is an important component of the RISC complex. AGO2 has been termed ‘Slicer’ and is the only component of human RISC that is required for the degradation of mRNA molecules, and its PIWI domain may contain the endonuclease activity. Drosophila embryo mutants lacking ago2 are unable to perform siRNA-directed mRNA cleavage, although miRNA-directed cleavage is still possible. The embryos also lack the capacity to load siRNAs into RISC. Drosophila Argonaute-1 (AGO1) is not involved in siRNA-directed cleavage as is AGO2. AGO1 is believed to function downstream of the production of the siRNAs and not as a component of RISC. These studies also demonstrated that Drosophila mutants lacking AGO1 are embryonic lethals, implicating AGO1 in Drosophila development. AGO1 is required for miRNA-directed cleavage and dispensable for siRNA-directed cleavage, showing divergent roles for different Argonaute proteins. This functional differentiation of the Argonaute proteins has also been noted in plants. Argonaute proteins are characterized by the PAZ domain and the PIWI domain. The crystal structures of the PAZ domain of AGO2 from Drosophila and the thermophile Pyrococcus furiosus were determined and shown to have structural properties similar to proteins that bind single-stranded nucleic acid. The PAZ domain recognizes the 30 overhangs of the siRNA duplexes. Interestingly, this is the same region that is recognized by certain viral suppressors of RNAi. In addition to its endonuclease motif, the PIWI domain is involved with protein–protein interactions between Argonaute and Dicer and may play a role in siRNA loading onto RISC. Recently, researchers have shown that both human AGO1 and AGO2 proteins reside in intracellular structures known as ‘cytoplasmic bodies’. These areas of the cell are believed to be sites of regulation of cellular mRNA turnover. Several other AGO proteins from drosophila are associated with the RNAi pathway. These proteins include Piwi, implicated in transposon silencing, Aubergine, associated with germline gene repression, and AGO3, an argonaute-like protein of unknown function. As already mentioned, a

Unique Properties of RNAi in Plants and Animals

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Interfering RNAs

cell line of Anopheles gambiae was tested by transfecting dsRNA derived from exon sequences of the A. gambiae dcr1 and dcr2 and argonaute 1–5 (ago1–5) genes. RNAi in A. gambiae cells required expression of Dicer-2, AGO2, and AGO3 proteins. This study also demonstrated that RNAi in the mosquito, as in drosophila, does not spread from the target cells, suggesting that RdRP-mediated transitive amplification is absent in the mosquito.

Viruses and RNAi Since the discovery of RNA silencing, a number of researchers have hypothesized that RNAi plays an important role in antiviral defense. RNA viruses with positivesense genomes, in particular, form dsRNA intermediates as they replicate in host cells. Whether this is the general viral trigger of RNAi in infected cells is still controversial, since viruses with negative-strand genomes generate little detectable dsRNA during their replication. Studies to elucidate a mechanism termed pathogen-derived resistance (PDR) in plants were among the first to show that viruses could be targeted by RNA silencing. Transgenic plants were engineered to express a portion of the tobacco etch virus (TEV) coat protein. Upon challenge with TEV, the transgenic plants were found to be resistant to the virus. No overt symptoms were seen, and no virus could be recovered from the leaf tissue. PDR was shown to be virus specific, as the transgenic plants could be infected with a genetically unrelated virus; however, any RNA that was introduced into plant cells that shared homology with the RNA in the transgene was degraded. Plant virologists also observed that the delivery of a fragment of viral RNA to plant cells by a heterologous virus expression vector made the cells resistant to challenge by the first virus, and termed this related phenomenon virus-induced gene silencing. Soon after the discovery that plants used an RNA-mediated defense mechanism against viruses, it was shown that many plant viruses encoded protein suppressors of RNA silencing. This would be expected as the virus and host develop a biological arms race to express countermeasures that limit the ability of one to overcome the other.

Viral Encoded Suppressors of RNAi Several suppressors have been found that interrupt the RNA-silencing pathway at different steps, indicating that evasion of RNA silencing has evolved more than once. One protein, helper component-proteinase, or HC-Pro, from potyviruses of plants is thought to be the most potent suppressor of RNA silencing found to date. HC-Pro functions at a step that prevents the accumulation of siRNAs by interacting with the RNase III enzyme

Dicer. HC-Pro also has the capability to reverse established silencing of a transgene, suggesting that the protein inhibits a mechanism required for the maintenance of silencing. The 19 kDa protein (p19) from tombusviruses acts as a suppressor of PTGS in plants in a different manner from HC-Pro. p19 does not block production of the 21–25 nt siRNAs; rather it binds them via the 2-nt 30 overhangs. Notably, the protein will bind only doublestranded 21 nt sequences; single-stranded RNAs of the same length are not recognized by p19. In binding the siRNAs, p19 forms a homodimer and sequesters the guide sequences that are required for RISC incorporation and targeting mRNA degradation. Another virus, cucumber mosaic virus, encodes a protein 2b that interferes with the spread of siRNA signal in the plant host, allowing systemic spread of the virus. In addition to the PTGS suppressors encoded by plant viruses, the insect virus Flock house virus encodes a protein, B2, which can suppress RNAi activity in both drosophila S2 and plant cells. In transgenic plants containing a green fluorescent protein (GFP) gene, in which transient expression of siRNAs targeting GFP mRNA had silenced its expression, the presence of B2 protein reversed silencing of GFP. siRNAs were still detected in the tissues, indicating that the suppression of gene silencing did not occur before the production of siRNAs. It is likely that this protein sequesters the siRNAs from the RNAi machinery, possibly in a manner similar to p19. Finally, two other animal viruses encode suppressors of interferon induction that also have apparent RNAi suppressor activity. These are the influenza virus NS1 protein and the vaccinia virus E3L protein. Viruses may employ other strategies to evade the RNAi pathway. These strategies include (1) sequestration of the viral dsRNA replicative intermediates in viral cores or in double-membrane structures in the host cell formed during viral replication; (2) viral replication and spread outpacing the RNAi pathway; and (3) replication in tissues that are resistant to RNAi.

miRNAs and Viruses In the last couple of years, a number of viral-encoded miRNAs have been discovered. The functions of most viral-derived miRNAs are unknown; however, DNA viruses such as polyomaviruses and herpesviruses transcribe miRNAs in infected vertebrate cells that appear to regulate expression of critical viral and host genes during infection. The location of miRNAs within different virus genomes are not conserved, suggesting that miRNAs are likely to be recent acquisitions in viral genomes that help adapt the virus to the host during the virus lifecycle. For instance, a viral miRNA in SV40 virus was recently discovered that regulates the viral T antigen. The SV40

Interfering RNAs

miRNA accumulates in late stages of infection and targets the early T antigen mRNA for degradation, thus reducing its expression. Studies of many families of RNA viruses have failed to identify miRNAs in RNA genomes and this finding is consistent with the prominent role of the cellular DNA-dependent RNA polymerase II in the biogenesis of miRNA precursors.

Use of RNAi for Disease Control Virus diseases of plants cause extensive economic losses. Control of plant virus diseases has usually been associated with control of insects or nematodes that transmit viruses, or through sanitation and quarantine of infected plants. The transformation of plants with effector genes designed to transcribe inverted-repeat RNA (dsRNAs) that target plant viruses can provide novel virus-resistant varieties. Transgenic plants expressing inverted repeats of viral sequences exhibit varying degrees of resistance to the virus or viruses with genome sequences closely related to the source of the transgene. This resistance is due to PTGS wherein viral mRNA is degraded in the cytoplasm soon after synthesis. In a similar genetic approach to that described previously in plants, we have genetically modified Aedes aegypti mosquitoes to exhibit impaired vector competence for dengue type 2 virus (DENV-2) transmission. DENVs are normally transmitted by A. aegypti mosquitoes to humans during epidemic outbreaks of dengue diseases. If a DENV-derived dsRNA trigger is expressed in the cell prior to viral translation and replication, an antiviral state can be induced in the mosquito that blocks virus infection. To do this, mosquitoes were genetically modified to express an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA. The IR RNA formed a 560 bp dsRNA in infected midgut epithelial cells of the mosquitoes to induce the RNAi pathway. A transgenic family, Carb77, was selected that expressed IR RNA in the midgut after a blood meal. Carb77 mosquitoes ingesting an artificial blood meal with 107 pfu ml–1 of DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. Transmission of virus by the Carb77 line was significantly diminished when compared to control mosquitoes. As evidence that the resistance was RNAi mediated, DENV-2-derived siRNAs were readily detected in RNA extracts from midguts following ingestion of a blood meal with no virus. In addition, loss of the resistance phenotype was observed when the RNAi pathway was interrupted by injecting ago2 dsRNA 2 days prior to induction of IR-RNA transgene, confirming that DENV-2 resistance was caused by an RNAi response. Targeting of replicating animal viruses using RNAi has prompted discussion about whether RNAi can be used as

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an antiviral therapy. In mammalian cells, siRNAs, rather than long dsRNAs, are required to induce RNAi as an antiviral therapy because these cells possess interferon and other antiviral innate immune pathways that are triggered by dsRNA >30 bp in length. Numerous studies in cell culture have shown that HIV replication can be halted when cells are treated with siRNAs that target the viral genome. West Nile virus (WNV) replication also can be reduced in cultured cells by treatment with siRNAs targeting the virus RNA. These studies showed a significant reduction in levels of WNV RNA if cells were pretreated with siRNAs; however, the cells that were treated subsequent to the establishment of viral replication did not show the same reduction in viral RNA, suggesting that the RNA may be sequestered from the RNAi machinery after replication is established in the cell. Studies investigating RNAi as a therapy for hepatitis C virus (HCV) infection have used siRNAs to effectively target HCVreplicon RNAs in cultured human cells as well as in a mouse model.

Model Systems for Studying Role of RNA in Virus Infections While the genetics and biochemistry of RNAi in C. elegans and D. melanogaster have been investigated in detail, there have been no virus infection models of these animals until very recently. RNAi-based innate immunity has been detected in C. elegans-derived cultured cells infected with vesicular stomatitis virus (VSV; Rhaboviridae). FHV replication in C. elegans triggered potent antiviral silencing that required RDE-1, an argonaute protein essential for RNAi mediated by siRNAs. This antiviral innate immunity was capable of rapid virus clearance in C. elegans in the absence of FHV RNAi suppressor protein B2. Two recent papers have shown that successful infection and killing of Drosophila by FHV was strictly dependent on expression of the viral suppressor protein B2. Drosophila with a knockout mutation in the gene encoding Dicer-2 showed enhanced susceptibility to infection by FHV, cricket paralysis, and Drosophila C viruses (Dicistroviridae) and Sindbis virus (Alphavirus; Togaviridae). These data demonstrate the importance of RNAi for controlling virus replication in vivo and establish dcr2 as a drosophila susceptibility locus for virus infections. C. elegans and drosophila are important models for studying virus–RNAi interactions. The drosophila model system allows advanced genetic approaches such as generation of null mutants of RNAi components in a system that has few other dsRNAtriggered defensive responses to complicate mechanistic studies. The availability of the annotated drosophila genome sequence and established genetic approaches is critical for understanding RNAi mechanisms and continues to make the drosophila model important to our understanding of innate immune responses to viruses.

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Interfering RNAs

Mosquitoes, RNAi, and Arboviruses Mosquitoes and arboviruses provide an important naturally occurring insect–virus system to study the potential role of RNAi in host defense. Arboviruses are RNA viruses that must replicate in their arthropod vector for amplification before they can be transmitted to a vertebrate host, such as humans. Obviously, arboviruses must somehow evade the RNAi pathway to successfully replicate in the mosquito prior to transmission. There are several advantages to studying RNAi in mosquitoes. First, the complete genome sequence is now available for at least two medically important vectors, A. gambiae and A. aegypti. Second, the RNAi pathway of mosquitoes is similar in structure and function to the pathway in drosophila and many of the component genes of RNAi have been identified. Third, new genetic approaches are allowing researchers to manipulate genes that affect innate immune responses in the mosquito. Fourth, infectious cDNA clones of arbovirus genomes from at least three virus families are available to allow manipulation of the viral genes and identify determinants of RNAi modulation. Fifth, RNAi–virus interactions studies can occur in systems directly relating to medically important pathogens. Mosquitoes, like drosophila, do not have responses comparable to interferon induction, so they can be injected with long dsRNAs (300–500 bp) to trigger the RNAi pathway and efficiently silence specific vector genes that may participate in innate immune pathways, including RNAi. As an example, to determine whether RNAi conditions the vector competence of A. gambiae for O’nyong-nyong virus (ONNV; Alphavirus), a genetically modified ONNV expressing GFP (eGFP) was developed to readily track virus infection. After intrathoracic injection, ONNVeGFP slowly spread to other A. gambiae tissues over a 9 day period. Mosquitoes were co-injected with virus and dsRNA derived from the ONNV nsP3 gene. Treatment with nsP3 dsRNA inhibited virus spread significantly, as determined by GFP expression patterns. ONNV-GFP titers from mosquitoes co-injected with nsP3 dsRNA also were significantly lower at 3 and 6 days after injection than in mosquitoes co-injected with non-virus-related b-galactosidase (b-gal) dsRNA. However, mosquitoes coinjected with ONNV-GFP and dsRNA derived from the A. gambiae ago2 gene displayed widespread GFP expression and virus titers 16-fold higher than b-gal dsRNA controls at 3 or 6 days after injection. These observations provided direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae and suggest that this innate immune response plays a role in conditioning vector competence. These types of experiments could be vital to understanding why some mosquito species are excellent vectors of disease and others are not.

The Biological Arms Race between Viruses and Hosts RNA virus–host interactions are often characterized as an escalating arms race between two mortal enemies. The host evolves innate and acquired immune pathways to counter the destructive effects of virus invasion and the virus adapts to these defense measures by rapidly evolving new ways of evading the host’s attempts at pathogen control. RNA–virus interactions with the host’s RNAi pathway exemplify this struggle for dominance. Many RNA viruses evolve rapidly because their RNA-directed RNA polymerases are error prone, providing significant variation in virus genome populations needed to probe weaknesses in the host’s defense and allow selection of new virus variants that have an advantage in their replication. As we have described earlier, a number of families of plant RNA viruses have evolved RNAi suppressors. Each family has developed a different strategy to downregulate RNAi, as shown by the fact that their suppressors attack different steps in the pathway. Still other viruses may have adapted to host RNAi by sequestering their replicative intermediate dsRNA triggers in double-membrane structures derived from host endoplasmic reticulum. Rapid evolution of RNA viruses also produces significant challenges to developing therapeutic strategies for humans using siRNAs to target and destroy viruses. Strategies that use siRNAs to trigger RNAi can be thwarted by point mutations in the target sequence. This has prompted development of siRNAs that target multiple regions of the viral RNA, highly conserved regions of the viral genome, or host genes essential for virus infection. However, RNA viruses are constrained in the amount of genetic variation they can tolerate and remain genetically fit for cell entry, replication, packaging, and egress. Viruses that have complex life cycles, such as arthropod-borne viruses that must replicate in both vertebrate and invertebrate cells, are further constrained in their evolutionary potential. Finally, there is evidence that the host can evolve to counter the threat posed by RNA viruses. A recent finding emphasized the critical role of RNAi as an innate immune mechanism in Drosophila when researchers showed that RNAi pathway genes (dcr2, ago2, r2d2) are among the 3% fastest evolving genes among drosophila species, confirming the biological arms race between viruses and insects. The antiviral role of RNAi has been studied for less than a decade and is only now being exploited as a mechanism to fight viral diseases. Knowledge gained since the discovery of RNAi in 1998 should allow researchers to fully exploit RNAi as a means of controlling a number of infectious agents that cause disease in plants, animals, and humans.

Iridoviruses of Vertebrates

Acknowledgments Our research is funded by NIH grants AI34014 and AI48740 and the Grand Challenges in Global Health through the foundation for NIH. See also: Satellite Nucleic Acids and Viruses.

Further Reading Akira S, Uematsu S, and Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124: 783–801. Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363. Franz AWE, Sanchez-Vargas I, Adelman ZN, et al. (2006) Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proceedings of the National Academy of Sciences, USA 103: 4198–4203.

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Galiana-Arnoux D, Dostert C, Schneemann A, Hoffmann JA, and Imler JL (2006) Essential function in vivo for dicer-2 in host defense against RNA viruses in Drosophila. Nature Immunology 7: 590–597. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, and Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146–1150. Kavi HH, Fernandez HR, Xie W, and Birchler JA (2005) RNA silencing in Drosophila. FEBS Letters 579: 5940–5949. Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, and Olson KE (2004) RNA interference as a natural antiviral response to O’nyongnyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae. Proceedings of the National Academy of Sciences, USA 101: 17240–17245. Leonard JN and Schaffer DV (2006) Antiviral RNAi therapy: Emerging approaches for hitting a moving target. Gene Therapy 13: 532–540. Li WX, Li H, Lu R, et al. (2004) Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proceedings of the National Academy of Sciences, USA 101(5): 1350–1355. Voinnet O (2005) Induction and suppression of RNA silencing: Insights from viral infections. Nature Reviews Genetics 6: 206–220. Zamore PD (2002) Ancient pathways programmed by small RNAs. Science 296: 1265–1269.

Iridoviruses of Vertebrates A D Hyatt, Australian Animal Health Laboratory, Geelong, VIC, Australia V G Chinchar, University of Mississippi Medical Center, Jackson, MS, USA ã 2008 Elsevier Ltd. All rights reserved.

Glossary Anemia Reduced number (below normal) of erythrocytes (red blood cells). Ectothemic Animals whose temperature varies with the surrounding environment. Also known as poikliotherms. Epitheliotropic Having a special affinity for epithelial cells. Hyperplasia Abnormal increase in the volume of a tissue or organ caused by an increase in the number of normal cells. Karyolysis Dissolution of a cell nucleus. Karyorrhexis Rupture of the cell nucleus in which the chromatin disintegrates and is extruded from the cell. Pyknosis Degeneration of a cell in which the nucleus shrinks in size and the chromatin appears as a solid, structureless feature. Urodeles Amphibians belonging to the order Caudata, including the salamanders and newts, in which the larval tail persists in adult life.

Introduction The family Iridoviridae encompasses five recognized genera, two of which infect invertebrates (Iridovirus, Chloriridovirus), and three of which infect ectothermic vertebrates (Ranavirus, Lymphocystivirus, and Megalocytivirus). In addition, two other viruses that infect cold-blooded vertebrates (Erthrocytic necrosis virus and White sturgeon iridovirus (WSIV) remain unassigned members of the family. Iridoviruses that infect ‘cold-blooded’ vertebrates (fish, amphibians, and reptiles) have become the focus of recent interest. These viruses are being identified and isolated with increasing frequency and their importance is being measured in terms of their impact on farmed production and trade in fish and amphibians. There are also significant impacts of iridoviruses on biodiversity, most notably the decline of local amphibian populations. The history of research into these viruses extends back to the nineteenth century for lymphocystis virus, the 1940s for Frog virus 3 (FV3, genus Ranavirus) and 1980s for the first description of a highly infectious fresh water piscine ranavirus, Epizootic haematopoietic necrosis virus (EHNV, genus Ranavirus) and saltwater megalocytiviruses