ABSTRACT
Over the past 25 years, the field of evolutionary developmental biology (evo-devo) has used genomics and genetics to gain insight on the developmental mechanisms underlying the evolution of morphological diversity of animals. Evo-devo exploits the key insight that conserved toolkits of development (e.g. Hox genes) are used in animals to produce genetic novelties that provide adaptation to a new environment. Like development, immunity is forged by interactions with the environment, namely the microbial world. Yet, when it comes to the study of immune defence mechanisms in invertebrates, interest primarily focuses on evolutionarily conserved molecules also present in humans. Here, focusing on antiviral immunity, we argue that immune genes not conserved in humans represent an unexplored resource for the discovery of new antiviral strategies. We review recent findings on the cGAS-STING pathway and explain how cyclic dinucleotides produced by cGAS-like receptors may be used to investigate the portfolio of antiviral genes in a broad range of species. This will set the stage for evo-immuno approaches, exploiting the investment in antiviral defences made by metazoans over hundreds of millions of years of evolution. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.
Subject(s)
Nucleotidyltransferases , Animals , Humans , Nucleotidyltransferases/metabolismABSTRACT
Viruses represent a major threat to all animals, which defend themselves through induction of a large set of virus-stimulated genes that collectively control the infection. In vertebrates, these genes include interferons that play a critical role in the amplification of the response to infection. Virus- and interferon-stimulated genes include restriction factors targeting the different steps of the viral replication cycle, in addition to molecules associated with inflammation and adaptive immunity. Predictably, antiviral genes evolve dynamically in response to viral pressure. As a result, each animal has a unique arsenal of antiviral genes. Here, we exploit the capacity to experimentally activate the evolutionarily conserved stimulator of IFN genes (STING) signaling pathway by injection of the cyclic dinucleotide 2'3'-cyclic guanosine monophosphate-adenosine monophosphate into flies to define the repertoire of STING-regulated genes in 10 Drosophila species, spanning 40 million years of evolution. Our data reveal a set of conserved STING-regulated factors, including STING itself, a cGAS-like-receptor, the restriction factor pastel, and the antiviral protein Vago, but also 2 key components of the antiviral RNA interference pathway, Dicer-2, and Argonaute2. In addition, we identify unknown species- or lineage-specific genes that have not been previously associated with resistance to viruses. Our data provide insight into the core antiviral response in Drosophila flies and pave the way for the characterization of previously unknown antiviral effectors.
Subject(s)
Drosophila , Immunity, Innate , Animals , Nucleotides, Cyclic , Antiviral Agents/pharmacologyABSTRACT
In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.
Subject(s)
Antiviral Agents , Drosophila , Animals , Drosophila melanogaster , Cyclic GMP , MammalsABSTRACT
In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP. This CDN binds to and activates the protein STING to trigger immunity. We recently discovered in the model organism Drosophila melanogaster two cGAS-like receptors (cGLRs) that activate STING-dependent antiviral immunity and can produce 3'2'-cGAMP, in addition to 2'3'-cGAMP. Here we explore CDN-mediated immunity in 14 different Drosophila species covering 50 million years of evolution and report that 2'3'-cGAMP and 3'2'-cGAMP fail to control infection by Drosophila C virus in D. serrata, D. sechellia and D. mojavensis . Using an accurate and sensitive mass spectrometry method, we discover an unexpected diversity of CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster , including a novel CDN, 2'3'-c-di-GMP. We show that 2'3'-c-di-GMP is the most potent STING agonist identified so far in D. melanogaster and that this molecule also activates a strong antiviral transcriptional response in D. serrata . Our results shed light on the evolution of cGLRs in flies and provide a basis for the understanding of the function and regulation of this emerging family of PRRs in animal innate immunity.
ABSTRACT
Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.
Subject(s)
Leishmania , Leishmaniasis, Cutaneous , Phlebotomus , Psychodidae , Animals , Humans , Phlebotomus/parasitology , Psychodidae/parasitology , Leishmania/genetics , GenomicsABSTRACT
Characterization of double-stranded (ds)RNAs is relevant to the understanding of viral replication and immune sensing. Here, we provide a protocol describing the use of anti-dsRNA antibodies for immunofluorescence and immunoblotting in virus-infected insect cells, which can also be applied to tissues and other organisms. We describe the procedures to prepare insect cells for viral infection, followed by RNA extraction and in vitro production of synthetic dsRNA controls. We then detail the steps for dsRNA detection by immunoblotting and immunofluorescence. For complete details on the use and execution of this protocol, please refer to de Faria et al. (2022).1.
Subject(s)
Insect Viruses , Insecta , RNA, Double-Stranded , Insecta/cytology , Insecta/virology , Insect Viruses/genetics , Fluorescent Antibody Technique , ImmunoblottingABSTRACT
Aedes aegypti and A. albopictus mosquitoes are the main vectors for dengue virus (DENV) and other arboviruses, including Zika virus (ZIKV). Understanding the factors that affect transmission of arboviruses from mosquitoes to humans is a priority because it could inform public health and targeted interventions. Reasoning that interactions among viruses in the vector insect might affect transmission, we analysed the viromes of 815 urban Aedes mosquitoes collected from 12 countries worldwide. Two mosquito-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most abundant in A. aegypti worldwide. Spatiotemporal analyses of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both HTV and PCLV were present. Using a mouse model in the laboratory, we showed that the presence of HTV and PCLV increased the ability of mosquitoes to transmit DENV and ZIKV to a vertebrate host. By transcriptomic analysis, we found that in DENV-infected mosquitoes, HTV and PCLV block the downregulation of histone H4, which we identify as an important proviral host factor in vivo.
Subject(s)
Aedes , Arboviruses , Dengue Virus , Dengue , Insect Viruses , RNA Viruses , Zika Virus Infection , Zika Virus , Animals , Humans , Zika Virus/genetics , Insect Viruses/physiology , Dengue Virus/genetics , Mosquito Vectors , Arboviruses/geneticsABSTRACT
dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. Here, we show that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, our results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.
Subject(s)
DNA, Viral , Drosophila , Animals , Antiviral Agents , DNA Viruses/genetics , Drosophila/metabolism , Iridovirus , RNA Interference , RNA Polymerase II/metabolism , RNA, Double-Stranded/genetics , RNA, Small Interfering/metabolism , RNA, Viral/metabolismABSTRACT
The cGAS-STING pathway plays a central role in the detection of DNA in the cytosol of mammalian cells and activation of immunity. Although the early evolutionary origin of this pathway in animals has been noted, its ancestral functions have remained elusive so far. We review here new findings in invertebrates establishing a role in sensing and signaling infection, triggering potent transcriptional responses, in addition to autophagy. Results from flies and moths/butterflies point to the importance of STING signaling in antiviral immunity in insects. The recent characterization of cGAS-like receptors in Drosophila reveals the plasticity of this family of pattern-recognition receptors, able to accommodate ligands different from DNA and to produce cyclic dinucleotides beyond 2'3'-cGAMP.
Subject(s)
Butterflies , Membrane Proteins , Nucleotidyltransferases , Animals , Butterflies/immunology , Humans , Immunity, Innate , Membrane Proteins/immunology , Nucleotidyltransferases/immunologyABSTRACT
The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, -2 and -3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.
Subject(s)
Immunity, Innate , Nucleic Acids/chemistry , Nucleic Acids/immunology , Viral Proteins/chemistry , Viral Proteins/immunology , Animals , Antiviral Agents , Drosophila melanogaster , Evolution, Molecular , Humans , Mice , Protein Serine-Threonine Kinases , Proteomics , RNA Interference , RNA, Double-Stranded , Species Specificity , THP-1 CellsABSTRACT
Stimulator of interferon genes (STING) functions in the cytosolic DNA-sensing pathway of innate immunity in mammals. It is activated upon binding the cyclic dinucleotide 2'3'-cGAMP, a second messenger produced by the enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), which acts as the receptor for DNA in this pathway, and triggers the expression of interferons and other viral stress-induced genes. The ancient origin of STING in the evolution of animals had been noted, but its primitive function was speculative. We review here recent advances in the remarkable history of cGAS-STING signaling, which establish that cGAS is a member of the family of cGAS/DncV-like nucleotidyltransferases (CD-NTases). In bacteria, CD-NTases synthesize a wide range of cyclic oligonucleotide second messengers in response to bacteriophage infections, which in turn activate a variety of effector proteins to abort phage infection. Among these effectors, some are related to STING, revealing an ancestral function for the cGAS-STING cassette in antiviral host defense. Study of STING signaling in invertebrate animals is consistent with an early acquisition in the history of metazoans of CD-NTase- and STING-encoding genes to counter the universal threat of viruses. In particular, STING-dependent immunity appears to play a previously unsuspected important role in some insects. These discoveries open up interesting perspectives for the use of model organisms to decipher emerging aspects of cGAS-STING biology in mammals, such as the activation of interferon-independent responses or the function and regulation of cGAS in the nucleus.
ABSTRACT
Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.
Subject(s)
Drosophila melanogaster/metabolism , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/metabolism , RNA, Double-Stranded/metabolism , Receptors, Pattern Recognition/metabolism , Second Messenger Systems , Amino Acid Sequence , Animals , Crystallography, X-Ray , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster/immunology , Drosophila melanogaster/virology , Female , Humans , Immunity, Innate , Male , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/immunology , RNA, Double-Stranded/analysis , RNA, Double-Stranded/immunology , Receptors, Pattern Recognition/chemistry , Receptors, Pattern Recognition/immunology , Viruses/immunologyABSTRACT
In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.
Subject(s)
Drosophila melanogaster/immunology , Nucleotidyltransferases/immunology , Receptors, Pattern Recognition/metabolism , Viruses/immunology , Amino Acid Sequence , Animals , Cell Line , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/virology , Female , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Ligands , Male , Membrane Proteins/metabolism , Models, Molecular , NF-kappa B/metabolism , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/classification , Nucleotidyltransferases/deficiency , Nucleotidyltransferases/metabolism , RNA, Double-Stranded/analysis , RNA, Double-Stranded/immunology , RNA, Double-Stranded/metabolism , Receptors, Pattern Recognition/classification , Receptors, Pattern Recognition/deficiency , Receptors, Pattern Recognition/immunologyABSTRACT
Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.
Subject(s)
Aedes/cytology , Aedes/virology , Dengue Virus/physiology , Hemocytes/immunology , Hemocytes/virology , Zika Virus/physiology , Aedes/anatomy & histology , Animals , Female , Hemocytes/physiology , Mosquito Vectors , Phagocytes/virology , PhagocytosisABSTRACT
Insects appeared more than 400 million years ago and they represent the richest and most diverse taxonomic group with several million species. Yet, under the combined effect of the loss of natural habitats, the intensification of agriculture with massive use of pesticides, global warming and biological invasions, insects show alarming signs of decline. Although difficult to quantify, species extinction and population reductions are confirmed for many ecosystems. This results in a loss of services such as the pollination of plants, including food crops, the recycling of organic matter, the supply of goods such as honey and the stability of food webs. It is therefore urgent to halt the decline of Insects. We recommend implementing long-term monitoring of populations, tackling the causes of insect decline by reducing the use of synthetic insecticides, preserving natural habitats, and reinventing a positive relationship between humans and insects.
Apparus il y a plus de 400 millions d'années, les Insectes représentent le groupe taxonomique le plus riche et diversifié, avec plusieurs millions d'espèces. Sous l'effet de la disparition des habitats, de l'intensification de l'agriculture avec l'usage massif des pesticides, du réchauffement climatique et des invasions biologiques, les Insectes montrent des signes alarmants de déclin. Bien que difficiles à quantifier, la disparition des espèces et la réduction de leurs populations sont avérées et communes à de nombreux écosystèmes. Elles se traduisent par une perte des services rendus, comme la pollinisation des plantes vivrières, le recyclage de la matière organique, la fourniture de biens comme le miel, et l'équilibre des réseaux trophiques. Il est donc urgent de freiner le déclin des Insectes. Pour cela, il faut mettre en Åuvre des suivis à long terme des populations, réduire l'usage des insecticides de synthèse, préserver les habitats naturels, et réinventer la relation de l'Homme à l'Insecte en revalorisant son image et ses usages.
Subject(s)
Biodiversity , Ecosystem , Agriculture , Animals , Extinction, Biological , Humans , Insecta , PollinationABSTRACT
The fruitfly Drosophila melanogaster is a valuable model to unravel mechanisms of innate immunity, in particular in the context of viral infections. RNA interference, and more specifically the small interfering RNA pathway, is a major component of antiviral immunity in drosophila. In addition, the contribution of inducible transcriptional responses to the control of viruses in drosophila and other invertebrates is increasingly recognized. In particular, the recent discovery of a STING-IKKß-Relish signalling cassette in drosophila has confirmed that NF-κB transcription factors play an important role in the control of viral infections, in addition to bacterial and fungal infections. Here, we review recent developments in the field, which begin to shed light on the mechanisms involved in sensing of viral infections and in signalling leading to production of antiviral effectors.
Subject(s)
Drosophila Proteins/immunology , Drosophila melanogaster/immunology , Immunity, Innate/immunology , RNA Interference/immunology , Signal Transduction/immunology , Viruses/immunology , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/virology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , I-kappa B Kinase/metabolism , Immunity, Innate/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Membrane Proteins/metabolism , RNA, Viral/genetics , RNA, Viral/immunology , RNA, Viral/metabolism , Signal Transduction/genetics , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , Viruses/geneticsABSTRACT
We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.
Subject(s)
Drosophila Proteins/metabolism , Membrane Proteins/metabolism , NF-kappa B/metabolism , Nucleotides, Cyclic/metabolism , Transcription Factors/metabolism , Viruses/metabolism , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster , Membrane Proteins/genetics , Mutation , NF-kappa B/genetics , Nucleotides, Cyclic/genetics , Transcription Factors/genetics , Viruses/geneticsABSTRACT
Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.