RESUMO
While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis. Reducing the length of the direct repeat from 700-bp to 200-bp reduces, but does not eliminate, SSA activity. In contrast, increasing direct repeat length to 1.5-kb does not increase SSA rates, suggesting diminishing returns above a certain threshold size. Finally, we show that while the homing endonuclease Y2-I-AniI triggered both SSA and NHEJ at significantly higher rates than I-SceI at one genomic locus (P5-EGFP), repair events are heavily skewed towards NHEJ at another locus (kmo), suggesting the nuclease used and the genomic region targeted have a substantial influence on repair outcomes. Taken together, this work establishes the feasibility of engineering temporary transgenes in disease vector mosquitoes, while providing critical details concerning important operational parameters.
Assuntos
Aedes , Endonucleases , Transgenes , Aedes/genética , Aedes/enzimologia , Animais , Endonucleases/metabolismo , Endonucleases/genética , Animais Geneticamente Modificados , Mosquitos Vetores/genéticaRESUMO
We demonstrate here that single strand annealing (SSA) repair can be co-opted for the precise autocatalytic excision of a drive element. Although SSA is not the predominant form of DNA repair in eukaryotic organisms, we increased the likelihood of its use by engineering direct repeats at sites flanking the drive allele, and then introducing a double-strand DNA break (DSB) at a second endonuclease target site encoded within the drive allele. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). Incorporation of ReMEDE into the previously described mutagenic chain reaction (MCR) gene drive, targeting the yellow gene of Drosophila melanogaster, replaced drive alleles with wild-type alleles demonstrating proof-of-principle. Although the ReMEDE system requires further research and development, the technology has a number of attractive features as a gene drive mitigation strategy, chief among these the potential to restore a wild-type population without releasing additional transgenic organisms or large-scale environmental engineering efforts.
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OBJECTIVE: The MGDrivE (MGDrivE 1 and MGDrivE 2) modeling framework provides a flexible and expansive environment for testing the efficacy of novel gene-drive constructs for the control of mosquito-borne diseases. However, the existing model framework did not previously support several features necessary to simulate some types of intervention strategies. Namely, current MGDrivE versions do not permit modeling of small molecule inducible systems for controlling gene expression in gene drive designs or the inheritance patterns of self-eliminating gene drive mechanisms. RESULTS: Here, we demonstrate a new MGDrivE 2 module that permits the simulation of gene drive strategies incorporating small molecule-inducible systems and self-eliminating gene drive mechanisms. Additionally, we also implemented novel sparsity-aware sampling algorithms for improved computational efficiency in MGDrivE 2 and supplied an analysis and plotting function applicable to the outputs of MGDrivE 1 and MGDrivE 2.
Assuntos
Tecnologia de Impulso Genético , Doenças Transmitidas por Vetores , Animais , Simulação por Computador , Controle de MosquitosRESUMO
To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.
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While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.
Assuntos
Aedes , Flavivirus , Febre Amarela , Animais , Humanos , Interferência de RNA , Febre Amarela/genética , Flavivirus/genética , Antivirais , RNA Interferente Pequeno/genéticaRESUMO
CRISPR-based autonomous homing gene drives are a potentially transformative technology with the power to reduce the prevalence of, or even eliminate, vector-borne diseases, agricultural pests, and invasive species. However, there are a number of regulatory, ethical, environmental, and sociopolitical concerns surrounding the potential use of gene drives, particularly regarding the possibility for any unintended outcomes that might result from such a powerful technology. Therefore, there is an imminent need for countermeasures or technologies capable of exerting precise spatiotemporal control of gene drives, if their transformative potential is ever to be fully realized. This review summarizes the current state of the art in the development of technologies to prevent the uncontrolled spread of CRISPR-based autonomous homing gene drives.
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Promising genetics-based approaches are being developed to reduce or prevent the transmission of mosquito-vectored diseases. Less clear is how such transgenes can be removed from the environment, a concern that is particularly relevant for highly invasive gene drive transgenes. Here, we lay the groundwork for a transgene removal system based on single-strand annealing (SSA), a eukaryotic DNA repair mechanism. An SSA-based rescuer strain (kmoRG ) was engineered to have direct repeat sequences (DRs) in the Aedes aegypti kynurenine 3-monooxygenase (kmo) gene flanking the intervening transgenic cargo genes, DsRED and EGFP. Targeted induction of DNA double-strand breaks (DSBs) in the DsRED transgene successfully triggered complete elimination of the entire cargo from the kmoRG strain, restoring the wild-type kmo gene, and thereby, normal eye pigmentation. Our work establishes the framework for strategies to remove transgene sequences during the evaluation and testing of modified strains for genetics-based mosquito control.
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Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of ß2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-ß2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-ß2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.
Assuntos
Adenosina Trifosfatases/genética , Enzimas Reparadoras do DNA/genética , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Fosfopiruvato Hidratase/genética , Sequências Reguladoras de Ácido Nucleico , Tribolium/genética , Tubulina (Proteína)/genética , Adenosina Trifosfatases/metabolismo , Animais , Animais Geneticamente Modificados , Enzimas Reparadoras do DNA/metabolismo , Ordem dos Genes , Marcação de Genes , Genes Reporter , Loci Gênicos , Vetores Genéticos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Modelos Biológicos , Fenótipo , Fosfopiruvato Hidratase/metabolismo , Recombinação Genética , Transformação Genética , Tubulina (Proteína)/metabolismoRESUMO
Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.
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Regiões 3' não Traduzidas , Aedes/metabolismo , Animais Geneticamente Modificados/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Transgenes , Aedes/genética , Animais , Animais Geneticamente Modificados/genética , Proteínas de Fluorescência Verde/genética , Luciferases/genéticaRESUMO
As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.
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Aedes/genética , Dispepsia/genética , Proteínas de Insetos/genética , Proteínas de Membrana Transportadoras/genética , Aedes/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Dispepsia/metabolismo , Inativação Gênica , Aptidão Genética , Proteínas de Insetos/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Análise de Sequência de RNA , Zinco/metabolismoRESUMO
Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strnΔ35/Δ41) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strnΔ35/Δ41 mosquitoes to fly restricted their escape from aquatic rearing media following eclosion, and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L4 larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.
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Aedes , Proteínas de Drosophila , Voo Animal , Quinase de Cadeia Leve de Miosina , Aedes/genética , Aedes/crescimento & desenvolvimento , Aedes/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamento de Genes , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Controle de Mosquitos/métodos , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Doenças Transmitidas por Vetores/transmissãoRESUMO
Gene drive systems have long been sought to modify mosquito populations and thus combat malaria and dengue. Powerful gene drive systems have been developed in laboratory experiments, but may never be used in practice unless they can be shown to be acceptable through rigorous field-based testing. Such testing is complicated by the anticipated difficulty in removing gene drive transgenes from nature. Here, we consider the inclusion of self-elimination mechanisms into the design of homing-based gene drive transgenes. This approach not only caused the excision of the gene drive transgene, but also generates a transgene-free allele resistant to further action by the gene drive. Strikingly, our models suggest that this mechanism, acting at a modest rate (10%) as part of a single-component system, would be sufficient to cause the rapid reversion of even the most robust homing-based gene drive transgenes, without the need for further remediation. Modelling also suggests that unlike gene drive transgenes themselves, self-eliminating transgene approaches are expected to tolerate substantial rates of failure. Thus, self-elimination technology may permit rigorous field-based testing of gene drives by establishing strict time limits on the existence of gene drive transgenes in nature, rendering them essentially biodegradable. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.
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Culicidae/genética , Tecnologia de Impulso Genético/métodos , Mosquitos Vetores/genética , Transgenes , Animais , Dengue/prevenção & controle , Tecnologia de Impulso Genético/instrumentação , Malária/prevenção & controleRESUMO
Transcriptomic studies of Tribolium castaneum have led to significant advances in our understanding of co-regulation and differential expression of genes in development. However, previously used microarray approaches have covered only a subset of known genes. The aim of this study was to investigate gene expression patterns of beetle embryo, germ-line and somatic tissues. We identified 12,302 expressed genes and determined differentially expressed up and down-regulated genes among all samples. For example, 1624 and 3639 genes were differentially increased in expression greater than or equal to twofold change (FDR < 0.01) in testis vs. ovary (virgin female) and ovary vs. embryo (0-5 hr), respectively. Of these, many developmental, somatic and germ-line differentially expressed genes were identified. Furthermore, many maternally deposited transcripts were identified, whose expression either decreased rapidly or persisted during embryogenesis. Genes with the largest change in expression were predominantly decreased during early embryogenesis as compared to ovary or were increased in testis compared to embryo. We also identify zygotic genes induced after fertilization. The genome wide variation in transcript regulation in maternal and zygotic genes could provide additional information on how the anterior posterior axis formation is established in Tribolium embryos as compared to Drosophila Together, our data will facilitate studies of comparative developmental biology as well as help identify candidate genes for identifying cis-elements to drive transgenic constructs.
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Embrião não Mamífero , Regulação da Expressão Gênica , Genes de Insetos , Células Germinativas/metabolismo , Tribolium/genética , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Masculino , Anotação de Sequência Molecular , TranscriptomaRESUMO
Mosquito-borne pathogens continue to be a significant burden within human populations, with Aedes aegypti continuing to spread dengue, chikungunya, and Zika virus throughout the world. Using data from a previously conducted study, a linear regression model was constructed to predict the aquatic development rates based on the average temperature, temperature fluctuation range, and larval density. Additional experiments were conducted with different parameters of average temperature and larval density to validate the model. Using a paired t-test, the model predictions were compared to experimental data and showed that the prediction models were not significantly different for average pupation rate, adult emergence rate, and juvenile mortality rate. The models developed will be useful for modeling and estimating the upper limit of the number of Aedes aegypti in the environment under different temperature, diurnal temperature variations, and larval densities.
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Aedes/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Mosquitos Vetores/crescimento & desenvolvimento , Água/química , Aedes/virologia , Animais , Febre de Chikungunya/transmissão , Febre de Chikungunya/virologia , Vírus Chikungunya/isolamento & purificação , Dengue/transmissão , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Humanos , Larva/virologia , Mosquitos Vetores/virologia , Zika virus/isolamento & purificação , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
Several medically important flaviviruses that are transmitted by mosquitoes have been shown to bind to the C-type lectin fold that is present in either vertebrate or invertebrate proteins. While in some cases this interaction is part of a neutralizing anti-viral immune response, many reports have implicated this as critical for successful virus entry. Despite the establishment of mosquito C-type lectin domain containing proteins (CTLDcps) as known host factors in assisting the infectious process for flaviviruses, little is known about the structural characteristics of these proteins and their relationships to each other. In this report, we describe the manual annotation and structural characterization of 52 Aedes aegypti CTLDcps. Using existing RNAseq data, we establish that these genes can be subdivided into two classes: those highly conserved with expression primarily in development (embryo/early larvae) and those with no clear orthologs with expression primarily in late larvae/pupae or adults. The latter group contained all CTLDcps that are regulated by the Toll/Imd immune pathways, all known microbiome-regulating CTLDcps, and almost all CTLDcps that are implicated as flavivirus host factors in A. aegypti. Finally, we attempt to synthesize results from multiple conflicting gene expression profiling experiments in terms of how flavivirus infection changes steady-state levels of mRNA encoding CTLDcps.
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Aedes/genética , Aedes/virologia , Infecções por Arbovirus/transmissão , Infecções por Arbovirus/virologia , Flavivirus/fisiologia , Lectinas Tipo C/genética , Família Multigênica , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Genoma de Inseto , Interações Hospedeiro-Patógeno , Humanos , Anotação de Sequência Molecular , FilogeniaRESUMO
Mosquito-transmitted viral pathogens, such as dengue and Zika, afflict tens of thousands of people every year. These viruses are transmitted during the blood-feeding process that is required for mosquito reproduction, the most important vector being Aedes aegypti. While vertebrate blood is rich in protein, its high iron content is potentially toxic to mosquitoes. Although iron transport and sequestration are essential in the reproduction of vector mosquitoes, we discovered that culicine mosquitoes lack homologs of the common iron transporter NRAMP. Using a novel cell-based screen, we identified two ZIP and one ZnT genes as candidate iron transporters in the mosquito A. aegypti, the vector of dengue, Zika, and chikungunya. We determined the organ-specific expression pattern of these genes at critical time points in early reproduction. The result indicates modulation of these genes upon blood feeding, especially a ZIP13 homolog that is highly up-regulated after blood feeding, suggesting its importance in iron mobilization during blood digestion and reproduction. Gene silencing resulted in differential iron accumulation in the midgut and ovaries. This study sets a foundation for further investigation of iron transport and control strategies of this viral vector.
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The increasing range of Aedes aegypti, vector for Zika, dengue, chikungunya, and other viruses, has brought attention to the need to understand the population and transmission dynamics of this mosquito. It is well understood that environmental factors and breeding site characteristics play a role in organismal development and the potential to transmit pathogens. In this study, we observe the impact of larval density in combination with diurnal temperature on the time to pupation, emergence, and mortality of Aedes aegypti. Experiments were conducted at two diurnal temperature ranges based on 10 years of historical temperatures of Houston, Texas (21-32°C and 26.5-37.5°C). Experiments at constant temperatures (26.5°C, 32°C) were also conducted for comparison. At each temperature setting, five larval densities were observed (0.2, 1, 2, 4, 5 larvae per mL of water). Data collected shows significant differences in time to first pupation, time of first emergence, maximum rate of pupation, time of maximum rate of pupation, maximum rate of emergence, time of maximum rate of emergence, final average proportion of adult emergence, and average proportion of larval mortality. Further, data indicates a significant interactive effect between temperature fluctuation and larval density on these measures. Thus, wild population estimates should account for temperature fluctuations, larval density, and their interaction in low-volume containers.
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Aedes/crescimento & desenvolvimento , Temperatura , Animais , Insetos Vetores/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Pupa/crescimento & desenvolvimento , Fatores de Tempo , ÁguaRESUMO
More than 100 pathogens, spanning multiple virus families, broadly termed 'arthropod-borne viruses (arboviruses)' have been associated with human and/or animal diseases. These viruses persist in nature through transmission cycles that involve alternating replication in susceptible vertebrate and invertebrate hosts. Collectively, these viruses are among the greatest burdens to global health, due to their widespread prevalence, and the severe morbidity and mortality they cause in human and animal hosts. Specific examples of mosquito-borne pathogens include Zika virus (ZIKV), West Nile virus (WNV), dengue virus serotypes 1-4 (DENV 1-4), Japanese encephalitis virus (JEV), yellow fever virus (YFV), chikungunya virus (CHIKV), and Rift Valley fever virus (RVFV). Interactions between arboviruses and the immune pathways of vertebrate hosts have been extensively reviewed. In this review we focus on the antiviral immune pathways present in mosquitoes. We also discuss mechanisms by which mosquito-borne viruses may antagonize antiviral pathways in disease vectors. Finally, we elaborate on the possibility that mosquito-borne viruses may be engaged in an evolutionary arms race with their invertebrate vector hosts, and the possible implications of this for understanding the transmission of mosquito-borne viruses.
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Antivirais/antagonistas & inibidores , Antivirais/imunologia , Infecções por Arbovirus/imunologia , Infecções por Arbovirus/veterinária , Arbovírus/imunologia , Culicidae/imunologia , Mosquitos Vetores/imunologia , Imunidade Adaptativa , Animais , Infecções por Arbovirus/transmissão , Vírus Chikungunya , Culicidae/virologia , Vírus da Dengue , Vírus da Encefalite Japonesa (Espécie) , Interações Hospedeiro-Patógeno/imunologia , Humanos , MicroRNAs/metabolismo , Mosquitos Vetores/virologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Vírus da Febre do Vale do Rift , Replicação Viral/imunologia , Vírus da Febre Amarela , Zika virusRESUMO
Numerous arthropod species represent potential targets for gene-drive-based population suppression or replacement, including those that transmit diseases, damage crops, or act as deleterious invasive species. Containment measures for gene drive research in arthropods have been discussed in the literature, but the importance of developing safe and effective standard operating procedures (SOPs) for these types of experiments has not been adequately addressed. Concisely written SOPs link safe work practices, containment measures, institutional training, and research-specific protocols. Here we discuss information to be considered by principal investigators, biosafety officers, and institutional biosafety committees as they work together to develop SOPs for experiments involving gene drive in arthropods, and describe various courses of action that can be used to maintain the effectiveness of SOPs through evaluation and revision. The information provided herein will be especially useful to investigators and regulatory personnel who may lack extensive experience working with arthropods under containment conditions.