RNA interference in mosquito: understanding immune responses, double‐stranded RNA delivery systems and potential applications in vector control

Pillai, Agieshkumar Balakrishna; Nagarajan, Usha; Mitra, Arindam; Krishnan, Umamaheswari; Rajendran, Senthilkumar; Hoti, S.L.; Mishra, Rakesh

doi:10.1111/imb.12282

Cited by 27 publications

(23 citation statements)

References 105 publications

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“…Nevertheless, siRNAs are not exclusive for viral clearance, and other immune mechanisms could act during oral infections, as was reported in Drosophila infected with different viruses 15 . Currently, our knowledge about the antiviral immune response in insects remains limited 20,[28][29][30][31] .…”

Section: Discussionmentioning

confidence: 99%

Immune priming with inactive dengue virus during the larval stage of Aedes aegypti protects against the infection in adult mosquitoes

Vargas

Cime-Castillo

Lanz‐Mendoza

2020

Sci Rep

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Several studies have observed that the immune response in insects can be conserved, a phenomenon known as immune priming, which has been mostly tested in adult stages. However, it is unknown if induction of immune priming in larval stages protects against dengue virus (DenV) infections in adult mosquitoes. In this work, we primed larval instar 3 rd of Aedes aegypti with inactive dengue virus, producing adult mosquitoes with i) an enhanced antiviral-immune response; ii) a reduction in the load and replication of RNA of dengue virus (DENV); iii) a decline in viral infective particles production. Adult mosquitoes previously primed during larval stages over-expressed RNA interference (RNAi) markers Argonaute-2 (AGO-2) and Dicer-2 (DCR-2). We also observed inter-individual variations of DENV infection in adult mosquitoes, indicating a heterogeneous response to DENV infection in the same mosquito strain. However, mosquitoes primed during larval stages appear to control the infection, reducing the viral load. The over-expression of interferon-like factors (VAGO) and AGO-2 in the pupa stage suggests a fast activation of antiviral mechanisms after immune priming in larvae, creating a condition in which adult mosquitoes are resistant to the pathogen in the posterior exposure. Aedes aegypti is the primary vector of dengue virus (DENV). It is responsible for an estimated 390 million human infections each year 1,2. The vaccine lacking to prevent the infection in humans, and the mosquito resistance to different insecticides, make it urgent to develop new strategies to interrupt the transmission by this mosquito 3,4. Immune priming has become an important strategy to induce a protective condition against different pathogens in insects, occurring when a primary infectious exposure leads to an immune response that is enhanced upon re-exposure. This phenomenon has been described in various invertebrate taxa, including mosquitoes 3,4. Recent work by our group has analyzed the ability of Ae. aegypti to mount enhanced antiviral responses upon secondary oral DENV immune challenges. Following priming with a live or inactive virus, secondary DENV infections were shown to diminish in midguts and carcasses 5. Also, higher de novo midgut DNA synthesis was detected in challenged tissues, as determined by the incorporation of 5-bromo-2-deoxyuridine (BrDU) in DNA. Endoreplication, a type of de novo DNA synthesis, was shown to correlate with the activation of the Notch pathway, where Hindsight, Delta, and Notch were overexpressed after a secondary challenge with active DENV, in primed Ae. aegypti mosquitoes 6. These findings open a new perspective on the mechanisms underlying the vector's antiviral immune response and the effector molecules involved. Similar results have been observed in Anopheles albimanus 7 , in the LSB-AA695BB cell line (An. albimanus) and trained monocytes 8. The induction of this immune response could be difficult to carry out in the field. Therefore, it is essential to determine if we can activate the immune response in ...

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Section: Discussionmentioning

confidence: 99%

Immune priming with inactive dengue virus during the larval stage of Aedes aegypti protects against the infection in adult mosquitoes

Vargas

Cime-Castillo

Lanz‐Mendoza

2020

Sci Rep

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“…RNAi mediated gene silencing is a very common practice in mosquito gene function studies. Recently, application versions have been developed for mosquito vector control [4,5]. In these application cases, dsRNA is used to trigger RNAi machinery.…”

Section: Discussionmentioning

confidence: 99%

“…The last is perhaps the most immediately impactful, as, with increasing insecticide resistance, the array of options for vector control is shrinking and in dire need of replenishment. RNA interference (RNAi) based approaches have been widely used to identify genes that are relevant to vector competence, and RNAi-based effectors for mosquito control have been developed [1][2][3][4][5]. As an alternative or complement to RNAi-based tools, CRISPR-Cas9 based genome editing tools have been developed for gene function study in mosquito research field [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

Kulkarni¹,

Yu²,

Moon³

et al. 2020

J. Genomics

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In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the αand δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.

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“…Therefore, binding of the siRNA to these sequences induces epigenetic silencing of the targeted gene. Although, TGS has been least explored for gene silencing by RNAi, both pathways have been the heart for the development of powerful RNAi therapeutic agents [66, 67]. RNAi can be achieved by targeting conserved viral mRNAs, particularly the ones involved in viral entry, reverse transcription and integration as well as genes that encode structural and enzymatic proteins needed for viral assembly and infection (Table 2).…”

Section: Anti-hiv-1 Rna Interference (Rnai)-based Therapeutic Landscapementioning

confidence: 99%

The therapeutic landscape of HIV-1 via genome editing

2017

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Current treatment for HIV-1 largely relies on chemotherapy through the administration of antiretroviral drugs. While the search for anti-HIV-1 vaccine remain elusive, the use of highly active antiretroviral therapies (HAART) have been far-reaching and has changed HIV-1 into a manageable chronic infection. There is compelling evidence, including several side-effects of ARTs, suggesting that eradication of HIV-1 cannot depend solely on antiretrovirals. Gene therapy, an expanding treatment strategy, using RNA interference (RNAi) and programmable nucleases such as meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR–Cas9) are transforming the therapeutic landscape of HIV-1. TALENS and ZFNS are structurally similar modular systems, which consist of a FokI endonuclease fused to custom-designed effector proteins but have been largely limited, particularly ZFNs, due to their complexity and cost of protein engineering. However, the newly developed CRISPR–Cas9 system, consists of a single guide RNA (sgRNA), which directs a Cas9 endonuclease to complementary target sites, and serves as a superior alternative to the previous protein-based systems. The techniques have been successfully applied to the development of better HIV-1 models, generation of protective mutations in endogenous/host cells, disruption of HIV-1 genomes and even reactivating latent viruses for better detection and clearance by host immune response. Here, we focus on gene editing-based HIV-1 treatment and research in addition to providing perspectives for refining these techniques.

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RNA interference in mosquito: understanding immune responses, double‐stranded RNA delivery systems and potential applications in vector control

Cited by 27 publications

References 105 publications

Immune priming with inactive dengue virus during the larval stage of Aedes aegypti protects against the infection in adult mosquitoes

Immune priming with inactive dengue virus during the larval stage of Aedes aegypti protects against the infection in adult mosquitoes

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

The therapeutic landscape of HIV-1 via genome editing

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