siRNA Pathway Research Articles

Ras-related Protein in Brain 4A (Rab4A) is Downregulated by miR-496 to inhibit the Progression of Gastric Cancer.

Ras-related protein in brain 4A (Rab4A), as a member of the Rab family, is involved in the intracellular circulation of membrane receptors or endocytic substances and regulates the progression of multiple tumors. From our results, the knockdown of Rab4A inhibited the proliferation, migration and invasion in AGS cells. Importantly, the surface expression of epidermal growth factor receptor (EGFR) declined significantly in Rab4A knockdown cells. The downstream pathway of EGFR was also inhibited after the transfection of Rab4A-specific siRNA, including AKT and β-catenin pathways. In addition, miR-496 down-regulated the expression of Rab4A in AGS cells. The result of the luciferase reporter assay showed that miR-496 could bind to the 3'UTR of Rab4A. In conclusion, the expression of Rab4A is inhibited by miR-496, and the knockdown of Rab4A inhibits the proliferation, migration and invasion through down-regulating the surface expression of EGFR. Rab4A is a potential target in the treatment of gastric cancer.

The RNA interference response to alphanodavirus replication in Phlebotomus papatasi sand fly cells.

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi-derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo-siRNA) pathway, with minimal engagement from the PIWI-interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus-derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo-siRNA pathway.

Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

Functional characteristics of Dicer genes in Plutella xylostella.

Dicer is an endonuclease that belongs to the RNase III family and can specifically recognize and cleave double-stranded RNA (dsRNA). In most insects, there are two Dicer genes, Dicer-1 (Dcr-1) and Dicer-2 (Dcr-2), which are involved in the micro-RNA and small-interfering RNA pathways in many species, respectively. The function of Dicer in Plutella xylostella remains unknown. The full-length open reading frames of P. xylostella Dicer-1 (PxDcr-1) and Dicer-2 (PxDcr-2) were cloned and sequenced. Dcr-1andDcr-2 proteins shared similar structural domains with the Dicer-Partner Binding Domain (Dicer-PBD) and the double-strand RNA binding domain (dsRBD) present only in Dcr-1. The phylogenetic trees showed that lepidopteran Dcr-1s or Dcr-2s clustered in one branch, with PxDcr-1 in the basal position and PxDcr-2 closest to Plodia interpunctella Dicer. Two homozygous knockout lines, ΔPxDcr-1 and ΔPxDcr-2, were obtained by using the CRISPR-Cas9 technique. The ΔPxDcr-1 strain exhibited a high mortality rate, a low eclosion rate, a low egg-laying rate, a low hatching rate, and a shriveled ovariole without mature eggs. The ΔPxDcr-2 strain showed no significant difference from the wild-type in terms of survival, development and reproduction, but the RNA interference (RNAi) efficiency caused by dsRNA was significantly reduced. These findings demonstrate the involvement of PxDcr-1 in the development and reproduction of P. xylostella, specifically in the formation of ovarioles and eggs, and PxDcr-2 is indispensable for RNAi. These findings shed light on the function of Dcr-1 and Dcr-2 in Lepidoptera. © 2023 Society of Chemical Industry.

The costs of transgenerational immune priming for homologous and heterologous infections with different serotypes of dengue virus in Aedes aegypti mosquitoes.

The immune system is a network of molecules, signaling pathways, transcription, and effector modulation that controls, mitigates, or eradicates agents that may affect the integrity of the host. In mosquitoes, the innate immune system is highly efficient at combating foreign organisms but has the capacity to tolerate vector-borne diseases. These implications lead to replication, dissemination, and ultimately the transmission of pathogenic organisms when feeding on a host. In recent years, it has been discovered that the innate immune response of mosquitoes can trigger an enhanced immunity response to the stimulus of a previously encountered pathogen. This phenomenon, called immune priming, is characterized by a molecular response that prevents the replication of viruses, parasites, or bacteria in the body. It has been documented that immune priming can be stimulated through homologous organisms or molecules, although it has also been documented that closely related pathogens can generate an enhanced immune response to a second stimulus with a related organism. However, the cost involved in this immune response has not been characterized through the transmission of the immunological experience from parents to offspring by transgenerational immune priming (TGIP) in mosquitoes. Here, we address the impact on the rates of oviposition, hatching, development, and immune response in Aedes aegypti mosquitoes, the mothers of which were stimulated with dengue virus serotypes 2 and/or 4, having found a cost of TGIP on the development time of the progeny of mothers with heterologous infections, with respect to mothers with homologous infections. Our results showed a significant effect on the sex ratio, with females being more abundant than males. We found a decrease in transcripts of the siRNA pathway in daughters of mothers who had been exposed to an immune challenge with DV. Our research demonstrates that there are costs and benefits associated with TGIP in Aedes aegypti mosquitoes exposed to DV. Specifically, priming results in a lower viral load in the offspring of mothers who have previously been infected with the virus. Although some results from tests of two dengue virus serotypes show similarities, such as the percentage of pupae emergence, there are differences in the percentage of adult emergence, indicating differences in TGIP costs even within the same virus with different serotypes. This finding has crucial implications in the context of dengue virus transmission in endemic areas where multiple serotypes circulate simultaneously.

The role of estrogen receptors (ERs)-Notch pathway in thyroid toxicity induced by Di-2-ethylhexyl phthalate (DEHP) exposure: Population data and in vitro studies

BackgroundThis study aimed to assess the exposure level and risk of Di-2-ethylhexyl Phthalate (DEHP) among adults in Jilin Province, China, clarify the impact of DEHP on human thyroid function, and to explore the role of estrogen receptors (ERs)-Notch signaling pathway in the effect of DEHP metabolites on thyroid hormones based on population data and in vitro experiments. Methods312 adults participated in this study. Urinary DEHP metabolites were determined by high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). Two pharmacokinetic models were used to evaluate the estimated daily intake (EDI) and hazard quotient (HQ) of the adults. Multiple linear regression and mediating effect models were used to evaluate the target associations. In cell experiments, thyroid follicular epithelial (Nthy-ori3–1) cells were exposed to mono (2-ethylhexyl) phthalate (MEHP) for testing. The inhibitions of ERα and Notch pathway were conducted by siRNA and Notch pathway inhibitor DAPT. ResultsThe detection rate of five DEHP metabolites was 97.1∼100.0%. The HQ value of 0.3% of adults was higher than 1. The levels of urinary DEHP metabolites were significantly correlated with thyrotropin (TSH), thyrotropin-releasing hormone (TRH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3) and free thyroxine (FT4) and gene (estrogen receptor α (ERα), Notch1, Dll4) levels. The ERα-Notch pathway played a mediating role in the association between DEHP metabolite levels and FT4. The cell results showed, the levels of FT3 and FT4 in cell supernatant decreased after MEHP exposure, and the downward trend was reversed after ERα and notch pathways were inhibited, notch pathway genes also decreased after ERα inhibition. ConclusionAdults in the Jilin Province of China were widely exposed to DEHP. ERs-Notch pathway played an important role in the effect of DEHP metabolites on thyroid hormones.

IGF2BP3-EGFR-AKT axis promotes breast cancer MDA-MB-231 cell growth.

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an emerging prognostic indicator, and its elevated expression correlates with malignancy in a broad spectrum of cancers. However, its regulatory networks have not yet been reported. In this study, we identified the regulatory targets of IGF2BP3 in breast cancer MDA-MB-231 cells using RNA immunoprecipitation sequencing (RIP-seq) and high-throughput RNA-sequencing (RNA-seq). We discovered that these targets were enriched in the inflammatory response, endoplasmic reticulum stress, cell cycle, and cancer-related pathways, providing a new perspective for better understanding the functional mechanisms of IGF2BP3. Moreover, we identified that the epidermal growth factor receptor (EGFR), a downstream target, is regulated by IGF2BP3. IGF2BP3 binds to and protects EGFR mRNA from degradation and facilitates cell proliferation via the EGFR/AKT pathway in MDA-MB-231 cells. In addition, IGF2BP3 expression was robust and could not be altered by stimulation with EGF and anti-EGFR siRNA or EGFR signaling pathway inhibitors (gefitinib, LY294002 and SL-327). These results demonstrate that IGF2BP3, as a stubborn oncogene, promotes triple-negative breast cancer MDA-MB-231 cell proliferation by strengthening the role of the EGFR-AKT axis.

Hsp70 and Hsp90 Elaborately Regulate RNAi Efficiency in Plutella xylostella.

Heat-shock proteins (HSPs) serve as molecular chaperones in the RNA interference (RNAi) pathway of eukaryotic organisms. In model organisms, Hsp70 and Hsp90 facilitate the folding and remodeling of the client protein Argonaute (Ago). However, the specific function of HSPs in the RNAi pathway of Plutella xylostella (L.) (Lepidoptera: Plutellidae) remains unknown. In this study, we identified and analyzed the coding sequences of PxHsc70-4 and PxHsp83 (also known as PxHsp90). Both PxHsc70-4 and PxHsp83 exhibited three conserved domains that covered a massive portion of their respective regions. The knockdown or inhibition of PxHsc70-4 and PxHsp83 in vitro resulted in a significant increase in the gene expression of the dsRNA-silenced reporter gene PxmRPS18, leading to a decrease in its RNAi efficiency. Interestingly, the overexpression of PxHsc70-4 and PxHsp83 in DBM, Sf9, and S2 cells resulted in an increase in the bioluminescent activity of dsRNA-silenced luciferase, indicating a decrease in its RNAi efficiency via the overexpression of Hsp70/Hsp90. Furthermore, the inhibition of PxHsc70-4 and PxHsp83 in vivo resulted in a significant increase in the gene expression of PxmRPS18. These findings demonstrated the essential involvement of a specific quantity of Hsc70-4 and Hsp83 in the siRNA pathway in P. xylostella. Our study offers novel insights into the roles played by HSPs in the siRNA pathway in lepidopteran insects.

Maternally inherited siRNAs initiate piRNA cluster formation

PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. Here, we report spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. We discovered that maternally supplied small interfering RNAs (siRNAs) trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells

BackgroundMosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported.MethodsA mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed.ResultsA strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status.ConclusionsTaken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses.Graphical

Aedes aegypti Argonaute 2 controls arbovirus infection and host mortality

Ae. aegypti mosquitoes transmit some of the most important human viral diseases that are responsible for a significant public health burden worldwide. The small interfering RNA (siRNA) pathway is considered the major antiviral defense system in insects. Here we show that siRNA pathway disruption by CRISPR/Cas9-based Ago2 knockout impaired the mosquitoes’ ability to degrade arbovirus RNA leading to hyper-infection accompanied by cell lysis and tissue damage. Ago2 disruption impaired DNA repair mechanisms and the autophagy pathway by altering histone abundance. This compromised DNA repair and removal of damaged cellular organelles and dysfunctional aggregates promoted mosquito death. We also report that hyper-infection of Ago2 knockout mosquitoes stimulated a broad-spectrum antiviral immunity, including apoptosis, which may counteract infection. Taken together, our studies reveal novel roles for Ago2 in protecting mosquitoes from arbovirus infection and associated death.

Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti

Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing invivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.

Fibroblast Growth Factor 5 Promotes the Proliferation of Corneal Epithelium Through Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway Activation

The recovery of corneal epithelial wounds is a complex process involving several physiological and pathological mechanisms that require further investigation. Our study has unveiled that the primary expression of FGF5 and FGFR1 occurs in the basal epithelium of the limbus. In the early 48 hours of the corneal epithelial wound healing process, FGF5 expression gradually rose and subsequently returned to baseline levels. The mice corneal epithelial cells (TKE2) proliferation was promoted by rhFGF5. Furthermore, the stemness-related genes K14, K15, P63, and the proliferation marker Ki67 levels expression increased after treatment with rhFGF5. In vivo, rhFGF5 promoted corneal epithelial wound healing. The RNA sequencing analysis of rhFGF5 treated TKE2 cells revealed 1512 differentially expressed genes. Different genes related to the cell cycle, proliferation, and regulatory signaling pathways were enriched. Moreover, the activation of the phosphoinositide 3-kinase/protein kinase B (PI3K-AKT) signaling pathway due to FGF5 led to the amplification of corneal progenitor cells. When FGF5 expression was curbed using siRNA, both proliferation and PI3K-AKT signaling pathway activation decreased. Our investigation has demonstrated that FGF5 stimulates the expansion of corneal limbal stem cells by activating the PI3K-AKT signaling pathway. Therefore, FGF5 has the potential to be an efficacious therapeutic intervention for treating corneal epithelial injuries.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

A method for screening the suppressor genes of siRNA and piRNA pathways using cultured silkworm cells.

The BmN-4 cell line originates from the ovaries of silkworm, Bombyx mori , and possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. BmN-4 cells are latently infected with Bombyx mori latent virus (BmLV), an RNA virus whose replication is strictly controlled by both siRNA and piRNA pathways. Knockdown or knockout of the core factors of these two small RNA pathways increases BmLV RNA amount, which in turn inhibits cell growth. Here, we used the known RNAi suppressor CrPV-1A to assess whether the BmN-4 cell line can be used for screening the suppressors of siRNA and piRNA pathways.

Fusobacterium nucleatum triggers proinflammatory cell death via Z-DNA binding protein 1 in apical periodontitis

BackgroundZ-DNA binding protein 1 (ZBP1) is a vital innate immune sensor that regulates inflammation during pathogen invasion. ZBP1 may contribute to pyroptosis, apoptosis and necroptosis in infectious diseases. In this study, Fusobacterium nucleatum (F. nucleatum) infection caused periapical inflammation through proinflammatory cell death and ZBP1 was involved in regulating the inflammatory activities caused by F. nucleatum infection in apical periodontitis (AP).MethodsHuman periapical tissues were tested by fluorescent in situ hybridization, immunohistochemical staining, immunofluorescence staining, quantitative real-time PCR (qRT‒PCR) and western blotting. F. nucleatum-infected and F. nucleatum extracellular vesicles (F. nucleatum-EVs)-treated RAW264.7 cells were used to detect the expression of inflammatory cytokines and different cell death mechanisms by qRT‒PCR and western blotting. ZBP1 expression in F. nucleatum-infected tissues and RAW264.7 cells was detected by qRT‒PCR, western blotting, and immunohistochemical and immunofluorescence staining. Furthermore, the expression of ZBP1 was inhibited by siRNA and different cell death pathways, including pyroptosis, apoptosis, and necroptosis, and inflammatory cytokines were measured in F. nucleatum-infected RAW264.7 cells.ResultsF. nucleatum was detected in AP tissues. F. nucleatum-infected RAW264.7 cells polarized to the M1 phenotype, and this was accompanied by inflammatory cytokine production. High levels of ZBP1 and GSDME (gasdermin E)-mediated pyroptosis, caspase-3-mediated apoptosis and MLKL-mediated necroptosis (PANoptosis) were identified in F. nucleatum-infected tissues and RAW264.7 cells. ZBP1 inhibition reduced inflammatory cytokine secretion and the occurrence of PANoptosis.ConclusionThe present study identified a previously unknown role of ZBP1 in regulating F. nucleatum-induced proinflammatory cell death and inflammatory activation.1TCgPRxw1BA_EMvaPhvNWjVideo abstract

Culex Mosquito Piwi4 Is Antiviral against Two Negative-Sense RNA Viruses.

Culex spp. mosquitoes transmit several pathogens concerning public health, including West Nile virus and Saint Louis encephalitis virus. Understanding the antiviral immune system of Culex spp. mosquitoes is important for reducing the transmission of these viruses. Mosquitoes rely on RNA interference (RNAi) to control viral replication. While the siRNA pathway in mosquitoes is heavily studied, less is known about the piRNA pathway. The piRNA pathway in mosquitoes has recently been connected to mosquito antiviral immunity. In Aedes aegypti, Piwi4 has been implicated in antiviral responses. The antiviral role of the piRNA pathway in Culex spp. mosquitoes is understudied compared to Ae. aegypti. Here, we aimed to identify the role of PIWI genes and piRNAs in Culex quinquefasciatus and Culex tarsalis cells during virus infection. We examined the effect of PIWI gene silencing on virus replication of two arboviruses and three insect-specific viruses in Cx. quinquefasciatus derived cells (Hsu) and Cx. tarsalis derived (CT) cells. We show that Piwi4 is antiviral against the La Crosse orthobunyavirus (LACV) in Hsu and CT cells, and the insect-specific rhabdovirus Merida virus (MERDV) in Hsu cells. None of the silenced PIWI genes impacted replication of the two flaviviruses Usutu virus (USUV) and Calbertado virus, or the phasivirus Phasi-Charoen-like virus. We further used small RNA sequencing to determine that LACV-derived piRNAs, but not USUV-derived piRNAs were generated in Hsu cells and that PIWI gene silencing resulted in a small reduction in vpiRNAs. Finally, we determined that LACV-derived DNA was produced in Hsu cells during infection, but whether this viral DNA is required for vpiRNA production remains unclear. Overall, we expanded our knowledge on the piRNA pathway and how it relates to the antiviral response in Culex spp mosquitoes.

Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China

BackgroundAedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure.MethodsWe used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome.ResultsWe showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes.ConclusionsMosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses.

Inhibition of Venezuelan Equine Encephalitis Virus Using Small Interfering RNAs.

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.

The Aedes aegypti siRNA pathway mediates broad-spectrum defense against human pathogenic viruses and modulates antibacterial and antifungal defenses.

The mosquito’s innate immune system defends against a variety of pathogens, and the conserved siRNA pathway plays a central role in the control of viral infections. Here, we show that transgenic overexpression of Dicer2 (Dcr2) or R2d2 resulted in an accumulation of 21-nucleotide viral sequences that was accompanied by a significant suppression of dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) replication, thus indicating the broad-spectrum antiviral response mediated by the siRNA pathway that can be applied for the development of novel arbovirus control strategies. Interestingly, overexpression of Dcr2 or R2d2 regulated the mRNA abundance of a variety of antimicrobial immune genes, pointing to additional functions of DCR2 and R2D2 as well as cross-talk between the siRNA pathway and other immune pathways. Accordingly, transgenic overexpression of Dcr2 or R2d2 resulted in a lesser proliferation of the midgut microbiota and increased resistance to bacterial and fungal infections.