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PubMed2005

Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen.

Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genome-wide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.

Animals↗

PubMed2003

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines.

BACKGROUND: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. METHODS: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. RESULTS: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. CONCLUSIONS: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.

Antineoplastic Agents, Phytogenic↗

PubMed2005

RNA interference: from gene silencing to gene-specific therapeutics.

In the past 4 years, RNA interference (RNAi) has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. By harnessing an evolutionary conserved endogenous biological pathway, first identified in plants and lower organisms, double-stranded RNA (dsRNA) reagents are used to bind to and promote the degradation of target RNAs, resulting in knockdown of the expression of specific genes. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21-23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability and delivery in vivo, while minimising off-target and nonspecific effects, are developed.

Animals↗

PubMed2006

Effects of RNA interference of Atg4B on the limited proteolysis of LC3 in PC12 cells and expression of Atg4B in various rat tissues.

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.

Animals↗

PubMed2005

Computational design of antiviral RNA interference strategies that resist human immunodeficiency virus escape.

Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

Computational Biology↗

PubMed2006

Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization.

N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.

Actinin↗

PubMed2006

[Effects of expression silencing of MAGE3 by RNA interference on location and metastasis of lung carcinoma cells].

OBJECTIVE: To construct small interfering RNA (siRNA) expression vectors targeting human MAGE3 gene and to observe the effects of gene silencing of MAGE3 by RNA interference on location and metastasis of lung carcinoma cells. METHODS: MAGE3 mRNA targeted hairpin siRNA was devised and the oligonucleotide strands of DNA fragments encoding the above siRNA were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into pSUPERneoGFP, followed by amplification and DNA sequencing, then transfected into human lung carcinoma NCI-H446. The expression of MAGE3 gene mRNA and protein were examined by RT-PCR and Western blotting. Colony formation assay and Boyden chamber assay were performed to detect the effects of MAGE3 on colony formation and metastasis. RESULTS: The DNA fragments encoding MAGE3-targeted siRNA were cloned into the pSUPERneoGFP and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly decreased expression level of MAGE3. The lung carcinoma cells transfected by siRNA group was significantly lower than others, an effect on its colony formation and invasiveness. The colony formation of lung carcinoma cells transfected by siRNA in soft agar and the number of cells penetrating matrigel both reduced, there is significant difference compared with untransfected group and transfected empty vector. CONCLUSION: An siRNA vector targeting human MAGE3 gene has been successfully constructed. Expression silencing of MAGE3 by RNA interference could reduce location and metastasis of lung carcinoma cells effectively.

Antigens, Neoplasm↗

PubMed2004

RNA interference as a new biotechnological tool for the control of virus diseases in plants.

RNA silencing occurs in a wide variety of organisms, including protozoa, fungi, plants and animals and involves recognition of a target RNA and initiation of a sequence-specific RNA degradation pathway in the cytoplasm. In the last few years, there have been considerable advances in our understanding of post-transcriptional gene silencing (PTGS). This mechanism is conceived as a natural antiviral defense system in plants that is activated as a response to double-stranded RNA (dsRNA) formed during virus replication. To develop new approaches for plant protection against virus diseases based on PTGS we have expanded previous findings on RNA interference (RNAi) in animals by using dsRNA to specifically interfere with virus infection in plants. This approach differs from strategies based on transgenic expression of RNAs but still relies on PTGS as a means to achieve pathogen-derived resistance (PDR). Our findings suggest that exogenously supplied dsRNA could form the basis for the development of an environmentally safe, new biotechnological tool aimed at protecting crops against virus diseases, provided that some limitations of the current status of the approach could be overcome.

Biotechnology↗

PubMed2006

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Animals↗

PubMed2004

Gene silencing of HIV-1 by RNA interference.

Short interfering RNAs (siRNAs) are as effective at targeting and silencing genes by RNA interference (RNAi) as long double-stranded RNAs (dsRNAs). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. siRNAs are also promising reagents for developing gene-specific therapeutics. Specifically, the inhibition of HIV-1 replication is particularly well-suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on recent advances in siRNA-based silencing technologies.

Animals↗

PubMed2005

[Effects of RNA interference inhibiting SDF-1 expression in bone marrow stromal cells on the proliferation and apoptosis of co-cultured Jurkat cells].

OBJECTIVE: To study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells. METHOD: Inhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transferring SDF-1 specific short hairpin RNA (shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles, apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The un-transfected acute leukemic (group B) and normal (group C) bone marrow stromal cells were taken as controls. RESULTS: The content of SDF-1 protein in supernatant of group A\[(384 +/- 41) pg/ml] was significantly lower than that in group B[(2474 +/- 271) pg/ml] or group C[(1324 +/- 154) pg/ml]. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B: 29 h, group C: 33 h), and G(0)/G(1) stage cells increased [group A: (28.47 +/- 2.39)%, vs group B: (19.43 +/- 2.80)%, group C: (27.15 +/- 2.07)%], S stage cells decreased [group A: (25.57 +/- 1.90)%, vs group B: (74.48 +/- 3.23)%, group C: (60.99 +/- 2.33)%], G(2)/M stage cells increased [group A: (45.96 +/- 3.24)%, vs group B: (6.09 +/- 1.96)%, group C: (11.86 +/- 1.98)%], the apoptosis rate increased [group A: (15.2 +/- 0.8)%, vs group B: (5.4 +/- 0.7)%, group C: (9.5 +/- 0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased. CONCLUSION: The inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.

Apoptosis↗

PubMed2002

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

OBJECTIVES: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. METHODS: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. RESULTS: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. CONCLUSION: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

CCR5 Receptor Antagonists↗

PubMed2006

Stable silencing of SNAP-25 in PC12 cells by RNA interference.

BACKGROUND: SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation. RESULTS: A PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control. CONCLUSION: Knockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion.

Animals↗

PubMed2001

Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

5' Untranslated Regions↗

PubMed2003

Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells.

We examined whether suppression of Oct4 via RNA interference (RNAi) would affect embryonic stem (ES) cell lineage choice. Cells were transfected with plasmids containing an independently expressed reporter gene and an RNA polymerase type III promoter to constitutively express small stem-loop RNA transcripts corresponding to Oct4 mRNA. Cells transfected with Oct4 RNAi constructs demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal differentiation. These findings support the critical role of Oct4 in regulating stem cell identity and suggest that future experiments using RNAi in ES cells can elucidate the roles of other genes affecting lineage specification during differentiation.

Animals↗

PubMed2003

Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference.

The rice OsMADS16 gene is phylogenetically related to the angiosperm B-function MADS-box genes. To investigate if OsMADS16 functions as an AP3/DEF orthologue to regulate the development of lodicules and stamens in rice, we isolated its genomic sequences and characterized its functions in planta by RNA interference. The genomic sequence of the OsMADS16 gene shows that it shares high similarity in genomic structure and the deduced amino acid sequence with the maize B-class gene, Si1. Transgenic lines from the introduced gene expressing double-stranded RNA with the OsMADS16 cDNA fragment were male-sterile and displayed alternations of lodicules and stamens, occasionally depressed palea and overgrown glume. The two lodicules were converted into four palea/lemma-like organs and some stamens into carpels. Further investigations of the transcription of OsMADS16 gene in these transgenic lines by RT-PCR revealed that its transcript was significantly reduced. Transcription of a rice PI homologous gene, OsMADS4, was also reduced remarkably in the transgenic plants. Our results demonstrate that OsMADS16 is an AP3/DEF orthologue to specify the identities of lodicules and stamens in rice flower and also support that OsMADS4 is a PI orthologue. In addition, these results suggest that RNA interference is a useful tool for functional genomics in rice.

Amino Acid Sequence↗

PubMed2003

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.

Animals↗

PubMed2004

RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae.

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (dsbetagal; note that "ds" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsbetagal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsbetagal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

Alphavirus↗