Double-stranded RNA Molecules Research Articles

Engineered Resistance to Tobamoviruses.

Tobacco mosaic virus (TMV) was the first virus to be studied in detail and, for many years, TMV and other tobamoviruses, particularly tomato mosaic virus (ToMV) and tobamoviruses infecting pepper (Capsicum spp.), were serious crop pathogens. By the end of the twentieth and for the first decade of the twenty-first century, tobamoviruses were under some degree of control due to introgression of resistance genes into commercial tomato and pepper lines. However, tobamoviruses remained important models for molecular biology, biotechnology and bio-nanotechnology. Recently, tobamoviruses have again become serious crop pathogens due to the advent of tomato brown rugose fruit virus, which overcomes tomato resistance against TMV and ToMV, and the slow but apparently inexorable worldwide spread of cucumber green mottle mosaic virus, which threatens all cucurbit crops. This review discusses a range of mainly molecular biology-based approaches for protecting crops against tobamoviruses. These include cross-protection (using mild tobamovirus strains to 'immunize' plants against severe strains), expressing viral gene products in transgenic plants to inhibit the viral infection cycle, inducing RNA silencing against tobamoviruses by expressing virus-derived RNA sequences in planta or by direct application of double-stranded RNA molecules to non-engineered plants, gene editing of host susceptibility factors, and the transfer and optimization of natural resistance genes.

Complete genome sequence and phylogenetic analysis of a newly discovered fusagra-like virus infecting Carica papaya in Ecuador.

A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae.

Phase 2 randomized study of rintatolimod following FOLFIRINOX in patients with locally advanced pancreatic adenocarcinoma.

TPS4214 Background: The immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) is a driver of its lethality and resistance to immunotherapy. Strategies which modulate the immunosuppressive milieu have the potential to improve the anti-tumor response and improve outcomes in patients with PDAC. Rintatolimod is a synthetic double-stranded RNA molecule that activates the toll-like receptor 3 (TLR3) present on dendritic cells, B-cells, natural killer cells and pancreatic epithelial cells. TLR3 stimulation may increase cytotoxic T-cell activity in PDAC through dendritic cell maturation, antigen priming and downstream proliferation of cytotoxic T-cells. Twenty-seven patients were treated with rintatolimod in a single center Named Patient Program at Erasmus University Medical Center in Rotterdam between January 2017 and February 2019. All had locally advanced or metastatic PDAC which had not progressed on FOLFIRINOX (median 8 cycles). Maintenance rintatolimod, administered intravenously twice weekly for up to 18 weeks, was generally well tolerated. The most common toxicities were grade 1-2 myalgia (30%), chills (52%) and fatigue (30%); no toxicities ≥ grade 3 were observed. Progression-free and overall survival (from cycle 1 FOLFIRINOX) were 13 and 19 months. Methods: This is a phase 2 randomized, open-label, multicenter clinical trial. The primary objective is to assess the efficacy of rintatolimod compared to observation in subjects with locally advanced PDAC which has not progressed after FOLFIRINOX. Eligible patients have completed at least 4 months of FOLFIRINOX, have no evidence of progressive disease, have measurable disease by RECIST v1.1, Karnofsky Performance Status ≥80 and have adequate organ function. Subjects with resected PDAC or metastatic disease are not eligible. Subjects are randomized 2:1 to rintatolimod vs. observation. Rintatolimod is administered intravenously twice weekly until disease progression in the experimental arm. Cross-sectional imaging is performed every 8 weeks in both arms. The primary endpoint is progression-free survival, as assessed by the investigator. Secondary endpoints include overall survival, objective response rate, duration of response and safety, as assessed by CTCAE v5.0. Exploratory endpoints include quality of life and changes in immune cell composition and T-cell response in the peripheral blood to characterize the immunomodulatory effects of rintatolimod. The planned sample size is 90 subjects. The final analysis will be performed after 64 events (disease progression or death) have been observed. The study is powered to detect a hazard ratio of 0.47 for median PFS with 80% power and significance level of 5%. Clinical trial information: NCT05494697 .

Analysis of the pharmacokinetics and efficacy of RBD1016 – A GalNAc-siRNA targeting Hepatitis B Virus X gene using semi-mechanistic PK/PD model

Small interference RNA (siRNA) is a class of short double-stranded RNA molecules that cause mRNA degradation through an RNA interference mechanism and is a promising therapeutic modality. RBD1016 is a siRNA drug in clinical development for the treatment of chronic Hepatitis B Virus (HBV) infection, which contains a conjugated with N-acetylglucosamine moiety that can facilitate its hepatic delivery. We aimed to construct a semi-mechanistic model of RBD1016 in pre-clinical animals, to elucidate the pharmacokinetic/pharmacodynamic (PK/PD) profiles in mice and PK profiles in monkeys, which can lay the foundation for potential future translation of RBD1016 PK and PD from the pre-clinical stage to the clinic stage. The proposed semi-mechanistic PK/PD model fitted PK and PD data in HBV transgenic mice well and described plasma and liver concentrations in the monkeys well. The simulation results showed that our model has a reasonable predictive ability for Hepatitis B surface antigen (HBsAg) levels after multiple dosing in mice. Further PK and PD data for RBD1016, including clinical data, will assist in refining the model presented here. Our current effort focused on model building for RBD1016, we anticipate that the model could apply to other GalNAc-siRNA drugs.

A removable and cosmopolitan dsRNA Toti-like virus causes asymptomatic but productive infection in a model diatom strain

Diatoms contribute to 20 % of global primary productivity. Although some diatom viruses have been identified, the molecular mechanisms underlying their interactions with the host remain poorly understood. In this study, we report the discovery of an RNA molecule in the DNA extracts of the Phaeodactylum tricornutum strain Pt1, which possesses a well-annotated genome and has been used as a diatom model system since 1956. We confirmed this molecule to be a double-stranded linear RNA molecule and, through sequencing, demonstrated it to be a virus in the Totiviridae family that is prevalent among marine stramenopiles. We also detected this virus in Phaeodactylum tricornutum strain Pt3, which was collected in 1930s from a similar geographic location to Pt1, suggesting its prevalence within the region. By employing various inhibitors of the viral RNA-dependent RNA polymerase, we successfully generated a virus-free line isogenic to Pt1, establishing a model system to investigate the impact of RNA viruses on diatom physiology. The virus-free lines did not display obvious growth advantages or defects, indicating a tendency of the virus towards asymptomatic infection. Furthermore, we generated a robust antibody against the coat protein of this virus. By performing immunoprecipitation coupled with mass spectrometry, we found that translation-related proteins are enriched as potential interacting partners of the coat protein. Our results suggest that potential viral impacts in molecular research should be considered when Pt1 and Pt3 are used for studying translation-related processes. Additionally, our study unveiled a mode of asymptomatic but productive infection between viruses and marine algal hosts that differs from the commonly-reported virulent, lytic infections.

A shorter inverted repeat length in a hairpin RNA results in a stronger silencing efficacy

RNA interference (RNAi), particularly hairpin RNA (hpRNA) constructs, is a potent genetic manipulation tool, influencing traits that are crucial for crop improvement. Typical hpRNA constructs consist of inverted repeat sequences derived from the mRNA of the target gene, separated by a non-complementary spacer. This configuration initiates the formation of a double-stranded RNA structure due to the complementary nature of sense and antisense sequences in the transcribed RNA. In this research, two hpRNA constructs, namely hp-TFL1-S and hp-TFL1-L, with distinct lengths of inverted repeats, were designed to target TFL1, a pivotal regulator of both flowering timing and inflorescence structure. hp-TFL1-L construct encompasses all the 150 nucleotide sequences of the hp-TFL1-S construct and an extra 135 nucleotides. These constructs included a chalcone synthase intron as a spacer, resulting in hairpin double-stranded RNA molecules of 150 and 285 base pair after transcription. The PCR and qRT-PCR analysis results confirmed the accurate design of both constructs and their successful suppression of TFL1 mRNA expression in transgenic Arabidopsis. Interestingly, Arabidopsis lines carrying hp-TFL1-S and hp-TFL1-L exhibited distinct phenotypic features. hp-TFL1-S induced more pronounced phenotypic changes, resulting in earlier flowering compared to the hp-TFL1-L and control group. Moreover, hp-TFL1-S had a significant impact on inflorescence architecture, distinguishing it from both hp-TFL1-L and control plants. This study provides an example where inverted repeat length alters the efficacy of silencing, and in this case, surprisingly the shorter construct appears to have a stronger silencing efficacy. This argues multiple sized hpRNAs should be tested in RNAi-based gene silencing experiments.

Improvement of economic and useful characters of wheat using RNA interference technology

Wheat is a strategic cereal crop of global importance and plays a leading role in the food supply of mankind. Despite the general trend to increase in its production, climatic changes leading to significant temperature changes, unpredictable precipitation or droughts and the appearance of new races of pathogens and pests significantly affect its yield. In order to prevent the negative impact of changes in climatic conditions on the productivity of this crop, it is necessary to develop innovative technologies for improving the resistance of wheat to environmental stresses. RNA interference (RNAi) represents a new potential tool for wheat breeding by introducing small non-coding RNA sequences with the ability to silence gene expression in a sequence-specific manner. A decrease in the expression of a certain gene determines the acquisition of a new characteristic through the elimination or accumulation of certain plant traits, which leads to biochemical or phenotypic changes that the original plants do not have. This literature review describes the progress achieved over the past decades in the application of RNAi to create wheat plants with improved economic and valuable traits. The main stages of the gene silencing mechanism mediated by short interfering RNA (siRNA) and microRNA (miRNA), features of their biogenesis, modes of action and distribution are presented. Examples of the use of various biotechnological approaches to wheat improvement using gene transformation, endogenous and exogenous double-stranded RNA molecules (dsRNA) are given. The possibility of using RNAi technology to increase the nutritional value and quality of grain, remove toxic compounds and allergens is highlighted. Considerable attention is paid to the practical results of various applications of RNAi to increase the resistance of wheat to biotic stress factors, in particular, viruses, bacteria, fungi, insect pests, and nematodes. Examples of the use of siRNA-mediated RNAi and the role of miRNA in improving wheat tolerance to abiotic stresses are summarized.

RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise.

Insect pests rank among the major limiting factors in agricultural production worldwide. In addition to direct effect on crops, some phytophagous insects are efficient vectors for plant disease transmission. Large amounts of conventional insecticides are required to secure food production worldwide, with a high impact on the economy and environment, particularly when beneficial insects are also affected by chemicals that frequently lack the desired specificity. RNA interference (RNAi) is a natural mechanism gene expression regulation and protection against exogenous and endogenous genetic elements present in most eukaryotes, including insects. Molecules of double-stranded RNA (dsRNA) or highly structured RNA are the substrates of cellular enzymes to produce several types of small RNAs (sRNAs), which play a crucial role in targeting sequences for transcriptional or post-transcriptional gene silencing. The relatively simple rules that underlie RNAi regulation, mainly based in Watson-Crick complementarity, have facilitated biotechnological applications based on these cellular mechanisms. This includes the promise of using engineered dsRNA molecules, either endogenously produced in crop plants or exogenously synthesized and applied onto crops, as a new generation of highly specific, sustainable, and environmentally friendly insecticides. Fueled on this expectation, this article reviews current knowledge about the RNAi pathways in insects, and some other applied questions such as production and delivery of recombinant RNA, which are critical to establish RNAi as a reliable technology for insect control in crop plants.

Isolation and Identification of dsRNA Mycovirus from Cultivated Mushroom Lentinula edodes

Viruses that infect the edible mushroom Lentinula edodes have been documented to exhibit either disease expression or remain asymptomatic. Nonetheless, our knowledge regarding viruses infecting L. edodes, as well as their diversity, remains relatively limited. This research aimed to isolate double-stranded RNA (dsRNA) molecules from 25 economically significant mushrooms using dsRNA extraction and gel electrophoresis techniques. The results revealed the presence of MSU16 dsRNA segments with a size of approximately 10,000 bp in a Lentinula sp. mushroom sample. This substantial dsRNA suggested the possible identification of the virus within the group of Lentinula edodes mycoviruses. Characterization of the MSU16 dsRNA molecule using a 2-step RT-PCR technique, DNA sequencing, and phylogenetic tree analysis based on partial RdRp nucleotide and amino acid sequences indicated a close relationship to other mushroom viruses found in Lentinula sp., with similarity ranging from 39.4 to 64.6 %. Molecular classification of the ITS gene sequence of the dsRNA-detected MSU16 showed a 92 % identity with L. edodes. However, the mushroom MSU16 could not be classified based on the limited LSU gene sequence available in GenBank. This study suggests that the MSU16 dsRNA molecule represents a Lentinula edodes dsRNA mycovirus isolate from Thailand. Our findings contribute valuable information about mycoviral presence in cultivated edible mushrooms. HIGHLIGHTS An approximately 10,000 bp dsRNA segment was successfully isolated from the Lentinula edodes mushroom isolate MSU16. This dsRNA was characterized through a 2-step RT-PCR, DNA sequencing, and phylogenetic analysis, focusing on partial RdRp nucleotide and amino acid sequences. The analysis indicated a close relationship between the isolated dsRNA and other viruses known to infect L. edodes mushrooms. Furthermore, the ITS gene sequence of MSU16 exhibited a high similarity to the corresponding ITS sequences in L. edodes. GRAPHICAL ABSTRACT

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules.

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells.

The crosstalk between miRNAs and signaling pathways in human cancers: Potential therapeutic implications.

MicroRNAs (miRNAs) are increasingly recognized as central players in the regulation of eukaryotic physiological processes. These small double stranded RNA molecules have emerged as pivotal regulators in the intricate network of cellular signaling pathways, playing significant roles in the development and progression of human cancers. The central theme in miRNA-mediated regulation of signaling pathways involves their ability to target and modulate the expression of pathway components. Aberrant expression of miRNAs can either promote or suppress key signaling events, influencing critical cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. For example, oncogenic miRNAs often promote cancer progression by targeting tumor suppressors or negative regulators of signaling pathways, thereby enhancing pathway activity. Conversely, tumor-suppressive miRNAs frequently inhibit oncogenic signaling by targeting key components within these pathways. This complex regulatory crosstalk underscores the significance of miRNAs as central players in shaping the signaling landscape of cancer cells. Furthermore, the therapeutic implications of targeting miRNAs in cancer are substantial. miRNAs can be manipulated to restore normal signaling pathway activity, offering a potential avenue for precision medicine. The development of miRNA-based therapeutics, including synthetic miRNA mimics and miRNA inhibitors, has shown promise in preclinical and clinical studies. These strategies aim to either enhance the activity of tumor-suppressive miRNAs or inhibit the function of oncogenic miRNAs, thereby restoring balanced signaling and impeding cancer progression. In conclusion, the crosstalk between miRNAs and signaling pathways in human cancers is a dynamic and influential aspect of cancer biology. Understanding this interplay provides valuable insights into cancer development and progression. Harnessing the therapeutic potential of miRNAs as regulators of signaling pathways opens up exciting opportunities for the development of innovative cancer treatments with the potential to improve patient outcomes. In this chapter, we provide an overview of the crosstalk between miRNAs and signaling pathways in the context of cancer and highlight the potential therapeutic implications of targeting this regulatory interplay.

Isolation of dsRNA from Plants by Cellulose Chromatography.

As the constitutive molecules of double-stranded RNA (dsRNA) virus genomes and replicative intermediates of single-stranded RNA (ssRNA) viruses, the high-molecular-weight dsRNAs are commonly found in RNA virus-infected plants. Therefore, the dsRNA is recognized as a hallmark of RNA virus infection and the profile of dsRNA has been applied as an efficient tool for diagnoses or characterization of unreported RNA viruses. Cellulose chromatography is one of the most useful procedures for the isolation of viral dsRNAs from total nucleic acids. Here, we describe rapid cellulose-based methods for purification of dsRNAs from plant tissue.

Characterisation and functionalisation of chitosan nanoparticles as carriers for double-stranded RNA (dsRNA) molecules towards sustainable crop protection.

The need to minimise the impact of phytosanitary treatments for disease control boosted researchers to implement techniques with less environmental impact. The development of technologies using molecular mechanisms based on the modulation of metabolism by short dsRNA sequences appears promising. The intrinsic fragility of polynucleotides and the high cost of these techniques can be circumvented by nanocarriers that protect the bioactive molecule enabling high efficiency delivery to the leaf surface and extending its half-life. In this work, a specific protocol was developed aiming to assess the best methodological conditions for the synthesis of low-size chitosan nanoparticles (NPs) to be loaded with nucleotides. In particular, NPs have been functionalised with partially purified Green Fluorescent Protein dsRNAs (GFP dsRNA) and their size, surface charge and nucleotide retention capacity were analysed. Final NPs were also stained with FITC and sprayed on Nicotiana benthamiana leaves to assess, by confocal microscopy, both a distribution protocol and the fate of NPs up to 6 days after application. Finally, to confirm the ability of NPs to increase the efficacy of dsRNA interference, specific tests were performed: by means of GFP dsRNA-functionalised NPs, the nucleotide permanence during time was assessed both in vitro on detached wild-type N. benthamiana leaves and in planta; lastly, the inhibition of Botrytis cinerea on single leaves was also evaluated, using a specific fungal sequence (Bc dsRNA) as the NPs' functionalising agent. The encouraging results obtained are promising in the perspective of long-lasting application of innovative treatments based on gene silencing.

Exogenous delivery of dsRNA for management of mungbean yellow mosaic virus on blackgram.

Exogenous application of dsRNA molecules targeting MYMV genes offers a promising approach to effectively mitigate yellow mosaic disease in blackgram, demonstrating potential for sustainable plant viral disease management. The exogenous application of double-stranded RNA (dsRNA) molecules to control plant viral diseases is gaining traction due to its advantages over conventional methods, such as target specificity, non-polluting nature, and absence of residue formation. Furthermore, this approach does not involve genome modification. In this study, dsRNA molecules targeting the coat protein gene (dsCP) and replication initiator protein gene (dsRep) of mungbean yellow mosaic virus (MYMV) were synthesised using an in vitro transcription method. To evaluate the effectiveness of dsRNA treatment, blackgram plants exhibiting MYMV symptoms at the first trifoliate stage were subjected to exogenous application of dsRNA. Second, third, and fourth trifoliate leaves, which emerged at 7, 15, and 21days after dsRNA application, respectively, were monitored for MYMV symptoms. Remarkably, a significant reduction in yellow mosaic disease (YMD) symptoms was observed in the newly emerged trifoliate leaves of MYMV-infected blackgram plants after treatment with dsRNA targeting both gene regions. This reduction was evident as a decrease in the intensity of yellow mosaic coverage on the leaf lamina compared to control. dsCP effectively reduced the MYMV titre in the treated plants for up to 15days. However, dsRep demonstrated greater efficiency in conferring resistance to MYMV at 15days post-application. These findings were supported by quantitative real-time PCR analysis, where the observed Ct values for DNA extracted from dsRep-treated plants were significantly higher compared to the Ct values of DNA from dsCP-treated plants at 15days post-application. Similarly, higher viral copy numbers were observed in dsCP-treated plants 15days after dsRNA treatment, in contrast to plants treated with dsRep.

An RNA interference (RNAi) target with potential to control Dothistroma needle blight

Spray-induced gene silencing (SIGS) involves exogenous spray applications of double-stranded RNA (dsRNA) molecules targeting specific genes, such as those essential to pathogens. This technique has shown great potential for controlling fungal pathogens of horticultural and agricultural plants, but few studies have evaluated its application to forest pathogens. Here we report the first demonstration of exogenous application of dsRNA molecules targeting a gene required for virulence in the foliar pine pathogen Dothistroma septosporum, the causal agent of Dothistroma needle blight (DNB). Constructs expressing sense and antisense dsRNAs were generated targeting a 509 bp fragment of the dothistromin toxin regulatory gene (DsAflR) in D. septosporum. DsAflR is required for the production of dothistromin, a phytotoxin essential for full pathogen virulence. We found that exogenous dsRNA is taken up and the RNA silencing mechanism is functional in D. septosporum through a series of in vitro and in planta trials, such as monitoring the uptake of fluorescently labelled DsAflR-dsRNA into the cell by confocal microscopy and quantifying the extent of gene silencing by quantitative Polymerase Chain Reaction (qPCR). In vitro applications of DsAflR-dsRNA to D. septosporum reduced the accumulation of DsAflR mRNA compared to controls and, in some cases, were found to be statistically significant, despite a high level of variability in the results. Our study also demonstrated a reduction in DNB symptoms on infected pine needles sprayed with DsAflR-dsRNA, which was further supported by a significant reduction in fungal biomass. This preliminary study highlights the potential for SIGS as a future management scheme for controlling the forest pathogen D. septosporum, as well as other forest pathogens.

Advances in transdermal siRNAs delivery: A review of current research progress.

Small interfering RNA (siRNAs) is a double-stranded RNA molecule which can hybridize with a specific mRNA sequence and block the translation of numerous genes to regulate endogenous genes and to defend the genome from invasive nucleic acids. The use of siRNAs has been studied as a treatment option for various skin conditions. One of the main obstacles in the dermal or transdermal delivery of this compound is low skin permeability, and application is limited by its negative charge, high polarity, susceptibility to degradation by nucleases, and difficulty in penetrating the skin barrier. Effective delivery of therapeutic biomolecules to their target is a challenging issue, which can be solved by innovations in drug delivery systems and lead to improvement of the efficiency of many new biopharmaceuticals. Designing of novel transdermal delivery systems garnered tremendous attention in both cosmeceutical and pharmaceutical research and industries, which offers a number of advantages. Developing safe and efficient siRNAs delivery vectors is essential for effective treatment of skin diseases. In recent years, significant progress has been made in the creation of delivery systems using lipids, polymers, cell-penetrating peptides, nanoparticles and other biologically active agents. In this review we will focus on the recent advancements in transdermal siRNAs delivery vectors, such as liposomes, dendrimers, cell-penetrating peptides, and spherical nucleic acid nanoparticles.

Possibilities of using mussels (Mytilus galloprovincialis) to predict rotavirus contamination in Albania.

Rotaviruses are non-enveloped viruses that each consist of 11 double-stranded RNA molecules. These viruses are able to persist in the environment, and therefore play a fundamental role in the epidemiology of gastroenteritis and severe diarrhoea in children worldwide. While mussels have been primarily used as indicators of chemical pollution, they can also be used to monitor viral contamination. The purpose of this study was to demonstrate that the Mytilus galloprovincialis mussel can also be used to detect microbial contamination, owing to its tendency to naturally concentrate viruses and other pathogens. A total of 102 Mytilus galloprovincialis mussel samples from Albania were collected over a three-year period: 37 samples off the Cape of Stillo in 2015, 39 samples from Butrinti Lake in 2019 and 26 samples from Butrinti Lake in 2021. The presence of rotavirus in the Cape of Stillo samples in 2015 was noted in 47% of samples from site 1, 33% from site 2, and 52% from site 3. In Butrinti Lake the percentage of infected individuals in 2019 was 33% from site 1, 41% from site 2, and 33% from site 3, whereas in 2021, it was 50% from site 1, 19% from site 2, and 0% from site 3. In total the percentage of infected individuals off the Cape of Stillo in 2015 was 44%, in Butrinti Lake in 2019 it was 36%, and in Butrinti Lake in 2021 it was 23 %. These results indicate the presence of rotavirus in the shellfish specimens tested, and further analysis is needed to assess the potential health risks associated with consuming these shellfish. This study also indicates that mussels can be used in marine virological surveillance programmes.

Infection Defects of RNA and DNA Viruses Induced by Antiviral RNA Interference.

Immune recognition of viral genome-derived double-stranded RNA (dsRNA) molecules and their subsequent processing into small interfering RNAs (siRNAs) in plants, invertebrates, and mammals trigger specific antiviral immunity known as antiviral RNA interference (RNAi). Immune sensing of viral dsRNA is sequence-independent, and most regions of viral RNAs are targeted by virus-derived siRNAs which extensively overlap in sequence. Thus, the high mutation rates of viruses do not drive immune escape from antiviral RNAi, in contrast to other mechanisms involving specific virus recognition by host immune proteins such as antibodies and resistance (R) proteins in mammals and plants, respectively. Instead, viruses actively suppress antiviral RNAi at various key steps with a group of proteins known as viral suppressors of RNAi (VSRs). Some VSRs are so effective in virus counter-defense that potent inhibition of virus infection by antiviral RNAi is undetectable unless the cognate VSR is rendered nonexpressing or nonfunctional. Since viral proteins are often multifunctional, resistance phenotypes of antiviral RNAi are accurately defined by those infection defects of VSR-deletion mutant viruses that are efficiently rescued by host deficiency in antiviral RNAi. Here, we review and discuss in vivo infection defects of VSR-deficient RNA and DNA viruses resulting from the actions of host antiviral RNAi in model systems.

Topical application of coat protein specific dsRNA molecules confers resistance against cognate Potato virus Y (PVY) infecting potato of North-East India

Sustainable agriculture relies on techniques and technology that combine efficiency with the smallest possible ecological and environmental impact. RNA interference (RNAi), a post-transcriptional eukaryotic pathway in eukaryotes can be co-opted for the control of plant pathogens in the topical application system. RNAi is the biological mechanism of mRNA degradation triggered by endogenous or exogenous double-stranded small interfering RNAs (dsRNA/siRNA) and regulates the target gene expression. Topically applied double-stranded RNA (dsRNA) molecules on plants imitate the viral RNA intermediate which is involved in plant virus replication and thereby triggers post-transcriptional gene silencing via., RNAi pathway and reducing viral replication. In this study, a non-transgenic-based RNAi approach was adopted to induce resistance against PVY in potato crop. dsRNA was successfully produced through in vitro transcription and hybridization. To test the effect of dsRNA molecules, in vitro synthesized dsRNA were applied on potato leaves along with PVY. dsRNA-treated plants showed higher resistance to viral disease and exhibited normal growth compared with PVY-treated plants. Therefore, the topical application of dsRNA molecules from the PVY CP gene could serve as a promising method for suppressing PVY infection and ultimately enhancing potato yield.

MicroRNAs as Possible Biomarkers for Diagnosis and Therapy of Alzheimer’s Disease by Regulating the Abnormal Expression of Genes Related to Tau

Alzheimer’s disease (AD), the most common dementia with the symptom of deterioration of memory and cognitive functions, becomes one of the prevalent threats globally. Tau hyperphosphorylation is one of major risk factors of AD. At present, there is no effective treatment or quick diagnostic methods for the pre-clinical stage of this disease. MicroRNAs (miRNAs) are small (20-25bp) non-coding, double stranded RNA molecule. They mainly regulate gene expression during the post-transcription by binding to the 3-’UTR of the mRNA then stop translation. Unlike other biomarkers using for Alzheimer’s disease, miRNAs are stable and widely found in body fluids such as serum, tissues, and Cerebrospinal fluid (CSF) and one miRNA can regulate multiple genes. As a result, they are potentially used as diagnosis or therapeutic biomarkers for many diseases including Alzheimer’s disease. Currently, this field that using miRNAs as biomarkers for diagnosis and treatment has been rapidly developed. To examine the mechanism and function of miRNAs and potential in AD, this review summarizes current diagnostic and therapeutic techniques and comparing several microRNAs that especially regulating tau toxicity as practicable diagnostic biomarkers and treatment agents. It is likely to detect different stages of Alzheimer’s disease and reduce Tau hyperphosphorylation if this field of miRNA as biomarkers carries forward.