Arboviruses and the challenge to establish systemic and persistent infections in competent mosquito vectors : the interaction with the RNAi mechanism

Arboviruses are capable to establish long-term persistent infections in mosquitoes that do not affect significantly the physiology of the insect vectors. Arbovirus infections are controlled by the RNAi machinery via the production of viral siRNAs and the formation of RISC complexes targeting viral genomes and mRNAs. Engineered arboviruses that contain cellular gene sequences can therefore be transformed to "viral silencing vectors" for studies of gene function in reverse genetics approaches. More specifically, "ideal" viral silencing vectors must be competent to induce robust RNAi effects while other interactions with the host immune system should be kept at a minimum to reduce non-specific effects. Because of their inconspicuous nature, arboviruses may approach the "ideal" viral silencing vectors in insects and it is therefore worthwhile to study the mechanisms by which the interactions with the RNAi machinery occur. In this review, an analysis is presented of the antiviral RNAi response in mosquito vectors with respect to the major types of arboviruses (alphaviruses, flaviviruses, bunyaviruses, and others). With respect to antiviral defense, the exo-RNAi pathway constitutes the major mechanism while the contribution of both miRNAs and viral piRNAs remains a contentious issue. However, additional mechanisms exist in mosquitoes that are capable to enhance or restrict the efficiency of viral silencing vectors such as the amplification of RNAi effects by DNA forms, the existence of incorporated viral elements in the genome and the induction of a non-specific systemic response by Dicer-2. Of significance is the observation that no major "viral suppressors of RNAi" (VSRs) seem to be encoded by arboviral genomes, indicating that relatively tight control of the activity of the RNA-dependent RNA polymerase (RdRp) may be sufficient to maintain the persistent character of arbovirus infections. Major strategies for improvement of viral silencing vectors therefore are proposed to involve engineering of VSRs and modifying of the properties of the RdRp. Because of safety issues (pathogen status), however, arbovirus-based silencing vectors are not well suited for practical applications, such as RNAi-based mosquito control. In that case, related mosquito-specific viruses that also establish persistent infections and may cause similar RNAi responses may represent a valuable alternative solution

Viral delivery of dsRNA for control of insect agricultural pests and vectors of human disease : prospects and challenges

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediatemolecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed

Transcriptome analysis of Bombyx mori larval midgut during persistent and pathogenic cytoplasmic polyhedrosis virus infection

Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host's transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic /metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host's RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following pathogenic infection with CPV, while they also highlight the relative importance of RNAi as an antiviral mechanism

Anticholinergic burden for prediction of cognitive decline or neuropsychiatric symptoms in older adults with mild cognitive impairment or dementia

Background: Medications with anticholinergic properties are commonly prescribed to older adults with a pre‐existing diagnosis of dementia or cognitive impairment. The cumulative anticholinergic effect of all the medications a person takes is referred to as the anticholinergic burden because of its potential to cause adverse effects. It is possible that a high anticholinergic burden may be a risk factor for further cognitive decline or neuropsychiatric disturbances in people with dementia. Neuropsychiatric disturbances are the most frequent complication of dementia that require hospitalisation, accounting for almost half of admissions; hence, identification of modifiable prognostic factors for these outcomes is crucial. There are various scales available to measure anticholinergic burden but agreement between them is often poor. Objectives: Our primary objective was to assess whether anticholinergic burden, as defined at the level of each individual scale, was a prognostic factor for further cognitive decline or neuropsychiatric disturbances in older adults with pre‐existing diagnoses of dementia or cognitive impairment. Our secondary objective was to investigate whether anticholinergic burden was a prognostic factor for other adverse clinical outcomes, including mortality, impaired physical function, and institutionalisation. Search methods: We searched these databases from inception to 29 November 2021: MEDLINE OvidSP, Embase OvidSP, PsycINFO OvidSP, CINAHL EBSCOhost, and ISI Web of Science Core Collection on ISI Web of Science. Selection criteria: We included prospective and retrospective longitudinal cohort and case‐control observational studies, with a minimum of one‐month follow‐up, which examined the association between an anticholinergic burden measurement scale and the above stated adverse clinical outcomes, in older adults with pre‐existing diagnoses of dementia or cognitive impairment. Data collection and analysis: Two review authors independently assessed studies for inclusion, and undertook data extraction, risk of bias assessment, and GRADE assessment. We summarised risk associations between anticholinergic burden and all clinical outcomes in a narrative fashion. We also evaluated the risk association between anticholinergic burden and mortality using a random‐effects meta‐analysis. We established adjusted pooled rates for the anticholinergic cognitive burden (ACB) scale; then, as an exploratory analysis, established pooled rates on the prespecified association across scales. Main results: We identified 18 studies that met our inclusion criteria (102,684 older adults). Anticholinergic burden was measured using five distinct measurement scales: 12 studies used the ACB scale; 3 studies used the Anticholinergic Risk Scale (ARS); 1 study used the Anticholinergic Drug Scale (ADS); 1 study used the Anticholinergic Effect on Cognition (AEC) Scale; and 2 studies used a list developed by Tune and Egeli. Risk associations between anticholinergic burden and adverse clinical outcomes were highly heterogenous. Four out of 10 (40%) studies reported a significantly increased risk of greater long‐term cognitive decline for participants with an anticholinergic burden compared to participants with no or minimal anticholinergic burden. No studies investigated neuropsychiatric disturbance outcomes. One out of four studies (25%) reported a significant association with reduced physical function for participants with an anticholinergic burden versus participants with no or minimal anticholinergic burden. No study (out of one investigating study) reported a significant association between anticholinergic burden and risk of institutionalisation. Six out of 10 studies (60%) found a significantly increased risk of mortality for those with an anticholinergic burden compared to those with no or minimal anticholinergic burden. Pooled analysis of adjusted mortality hazard ratios (HR) measured anticholinergic burden with the ACB scale, and suggested a significantly increased risk of death for those with a high ACB score relative to those with no or minimal ACB scores (HR 1.153, 95% confidence interval (CI) 1.030 to 1.292; 4 studies, 48,663 participants). An exploratory pooled analysis of adjusted mortality HRs across anticholinergic burden scales also suggested a significantly increased risk of death for those with a high anticholinergic burden (HR 1.102, 95% CI 1.044 to 1.163; 6 studies, 68,381 participants). Overall GRADE evaluation of results found low‐ or very low‐certainty evidence for all outcomes. Authors' conclusions: There is low‐certainty evidence that older adults with dementia or cognitive impairment who have a significant anticholinergic burden may be at increased risk of death. No firm conclusions can be drawn for risk of accelerated cognitive decline, neuropsychiatric disturbances, decline in physical function, or institutionalisation

Molecular dissection of small RNA pathways in the silkworm (Bombyx mori): an in vitro and in vivo approach

The present PhD Thesis has focused on the study of the small RNA pathways in the silkworm Bombyx mori, highlighting the factors that affect the efficiency of the RNAi mechanism. The factors examined here, mostly, involved the expression of key-genes belonging to the RNA machinery and the presence of a viral infection (persistent or pathogenic), while the mechanisms of the innate immune response and the dsRNA uptake ability from the extracellular medium were also investigated. Analysis of the results yielded important findings and conclusions regarding the impact of the aforementioned factors on the efficiency of silkworm’s RNAi mechanism. Our findings reveal possible overlaps of the main RNAi pathways in the silkworm in vitro, during the gene-silencing process mediated by dsRNA (miRNA, siRNA and piRNA), or hairpin RNA (siRNA and piRNA). Interestingly, except for their implication in the dsRNA-mediated silencing, the genes BmDcr2 and BmAgo2 of the siRNA pathway have proved to operate as key factors in silkworm’ s midgut, both under normal and viral infection (BmCPV) conditions. Additionally, BmAgo3 of the piRNA pathway is an important component affecting the efficiency of the RNAi mechanism in the silkworm, while it can also, together with BmR2D2 and BmTranslin2, and likely BmAub as well, play a crucial role in the antiviral response (BmMLV and AcMNPV) in Lepidoptera. Furthermore, the presence of a persistent infection does not seem to negatively influence the efficiency of the RNAi mechanism (BmMLV) and the antiviral defense (BmCPV) of the silkworm. Finally, the classical pathways of the endogenous innate immunity of B. mori do not respond strongly during antiviral defense (BmCPV) and therefore they do not seem to significantly interact or crosstalk with the RNAi machinery, so as to affect its proficiency. However, the present research highlights new genes that presumably play strategic roles in the antiviral response of the silkworm in combination with the RNAi mechanism.Η παρούσα Διδακτορική Διατριβή εστιάζει στη μελέτη των μονοπατιών των μικρών μορίων RNA στο μεταξοσκώληκα Bombyx mori, υπό το πρίσμα των παραγόντων που επηρεάζουν την αποδοτικότητα του RNAi μηχανισμού. Οι παράγοντες που εξετάστηκαν αφορούσαν κυρίως στην έκφραση γονιδίων-κλειδιών του RNAi μηχανισμού και στην ύπαρξη ιϊκής μόλυνσης (εμμένουσας ή παθογόνου), ενώ ακόμα έγιναν αναφορές στους μηχανισμούς της ενδογενούς φυσικής ανοσίας και της ικανότητας πρόσληψης dsRNA από το εξωκυττάριο μέσο. Από την ανάλυση των αποτελεσμάτων προέκυψαν σημαντικά συμπεράσματα σχετικά με την επίδραση των προαναφερθέντων παραγόντων στην αποδοτικότητα του μηχανισμού του RNAi στο μεταξοσκώληκα. Τα ευρήματά μας αποκαλύπτουν πιθανή αλληλοεπικάλυψη των βασικών μονοπατιών του RNAi στο μεταξοσκώληκα in vitro, κατά την πρόκληση γονιδιακής αποσιώπησης μέσω dsRNA (miRNA, siRNA και piRNA), ή μέσω φουρκέτας RNA (siRNA και piRNA). Επιπλέον, πέραν της εμπλοκής τους στο μηχανισμό γονιδιακής αποσιώπησης μέσω dsRNA, τα γονίδια BmDcr2 και BmAgo2 του μονοπατιού siRNA αναδεικνύονται ως καίριοι παράγοντες για το μεσέντερο του μεταξοσκώληκα, τόσο υπό φυσιολογικές συνθήκες, όσο και υπό συνθήκες ιϊκής μόλυνσης (BmCPV). Όμως, και το γονίδιο BmAgo3 του piRNA μονοπατιού φαίνεται να συνιστά σημαντικό παράγοντα που επηρεάζει την αποδοτικότητα του RNAi μηχανισμού στο μεταξοσκώληκα, ενώ μπορεί επίσης μαζί με τους παράγοντες BmR2D2 και BmTranslin2, και ενδεχομένως και τον BmAub, να διαδραματίσει κρίσιμο ρόλο για την αντιϊκή απόκριση (BmMLV και AcMNPV) στα Λεπιδόπτερα. Ακόμη, η παρουσία μίας εμμένουσας μόλυνσης δε φαίνεται να επιδρά αρνητικά στην αποδοτικότητα του RNAi μηχανισμού (BmMLV) και στην αντιϊκή προστασία του μεταξοσκώληκα (BmCPV). Τέλος, τα κλασσικά μονοπάτια της ενδογενούς φυσικής ανοσίας του B. mori δεν αποκρίνονται έντονα κατά τη διάρκεια της αντιϊκής προστασίας (BmCPV), και κατά συνέπεια δε φαίνεται να αλληλεπιδρούν σημαντικά με τον RNAi μηχανισμό, ούτως ώστε να επηρεάζουν την αποδοτικότητά του. Ωστόσο, από την παρούσα έρευνα αναδεικνύονται νέα γονίδια που ενδεχομένως διαθέτουν στρατηγικής σημασίας ρόλους για την αντιϊκή απόκριση του μεταξοσκώληκα σε συνδυασμό με τη δράση του RNAi μηχανισμού

Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed

What Are the Functional Roles of Piwi Proteins and piRNAs in Insects?

Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered

PIWI pathway against viruses in insects

Piwi-interacting RNAs (piRNAs) are an animal-specific class of small non-coding RNAs that are generated via a biogenesis pathway distinct from small interfering RNAs (siRNAs) and microRNAs (miRNAs). There are variations in piRNA biogenesis that depend on several factors, such as the cell type (germline or soma), the organism, and the purpose for which they are being produced, such as transposon-targeting, viral-targeting, or gene-derived piRNAs. Interestingly, the genes involved in the PIWI/piRNA pathway are more rapidly evolving compared with other RNA interference (RNAi) genes. In this review, the role of the piRNA pathway in the antiviral response is reviewed based on recent findings in insect models such as Drosophila, mosquitoes, midges and the silkworm, Bombyx mori. We extensively discuss the special features that characterize host-virus piRNA responses with respect to the proteins and the genes involved, the viral piRNAs' sequence characteristics, the target strand orientation biases as well as the viral piRNA target hotspots across the viral genomes. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.status: publishe