A (RP)UHPLC/UV analytical method to quantify dsRNA during the mRNA vaccine manufacturing process.

dsRNA is a product related impurity produced during the mRNA manufacturing process. The established immuno-based detection methods lack the flexibility and speed required to be applied throughout the manufacturing process. The RP-HPLC method developed outperforms these in terms of precision, broader detection range, LOD and LOQ, as well as in output variance. Using this method, dsRNA can be quantified in under 30 min for a single sample.

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

A Quadrupolar Bis-Triarylborane Chromophore as a Fluorimetric and Chirooptic Probe for Simultaneous and Selective Sensing of DNA, RNA and Proteins.

A water-soluble tetracationic quadrupolar bis-triarylborane chromophore showed strong binding to ds-DNA, ds-RNA, ss-RNA, as well as to the naturally most abundant protein, BSA. The novel dye can distinguish between DNA/RNA and BSA by fluorescence emission separated by Δ ν ˜ =3600 cm-1 , allowing for the simultaneous quantification of DNA/RNA and protein (BSA) in a mixture. The applicability of such fluorimetric differentiation in vitro was demonstrated, strongly supporting a protein-like target as a dominant binding site of 1 in cells. Moreover, our dye also bound strongly to ss-RNA, with the unusual rod-like structure of the dye, decorated by four positive charges at its termini and having a hydrophobic core, acting as a spindle for wrapping A, C and U ss-RNAs, but not poly G, the latter preserving its secondary structure. To the best of our knowledge, such unmatched, multifaceted binding activity of a small molecule toward DNA, RNA, and proteins and the selectivity of its fluorimetric and chirooptic response makes the quadrupolar bis-triarylborane a novel chromophore/fluorophore moiety for biochemical applications.

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC.

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA. We have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications. The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA.

Oligonucleotide Primers with G8AE-Clamp Modifications for RT-qPCR Detection of the Low-Copy dsRNA.

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G8AE-clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA⁻protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

Development of a homogeneous time-resolved fluorescence assay for detection of viral double-stranded RNA.

The group of positive-sense single-stranded RNA ((+) ssRNA) viruses includes many important human pathogens. However, specific antiviral agents are not currently available for many RNA viruses. For screening of antiviral agents, methods that are simple, rapid, and compatible with high-throughput are required. Here, we describe a novel method for measurement of double-stranded RNA using a homogeneous time-resolved fluorescence assay. This method allowed detection of human rhinovirus (HRV), enterovirus, coxsackievirus, and murine norovirus. Furthermore, this method detected antiviral activity of a HRV 3C protease inhibitor. The assay may be useful for discovery of antiviral agents against (+) ssRNA viruses.

Molecular Analysis for Characterizing Transgenic Events.

To develop a commercial trait product, a large number of transgenic events are often produced to obtain the event with desired level of expression. It is crucial to develop efficient and sensitive molecular characterization methods to advance events with stable transgene expression, free of vector backbone sequences and without major changes to the native genome caused by transgene insertion. Here, we discuss a variety of analytical tools, including quantitative PCR (qPCR), Southern blot analysis, and various sequencing technologies, which have been widely used to determine the insert copy number, presence/absence of vector backbone sequences, integrity of the T-DNA, and genomic location of the T-DNA insertion. Moreover, since the discovery of RNA interference in 1998 (Fire et al., Nature 391:806-811, 1998), RNAi has emerged as another powerful tool in in the development of a new transgenic trait for insect control. RNAi creates a double-stranded RNA duplex as the active molecule which forms a strong secondary structure, resulting in challenges for detection. In addition to molecular analysis at the DNA level, this chapter describes detection methods of the active molecules (i.e., double-stranded RNA) for RNAi-based traits.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography.

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important 'fingerprint' for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.

Identification of the role of Rh protein in ammonia excretion of the swimming crab Portunus trituberculatus.

In Portunus trituberculatus, a full-length cDNA of Rhesus-like glycoprotein (Rh protein), encoding the entire 478 amino acid protein, has been identified in gills, and plays an essential role in ammonia (NH3/NH4+) excretion. Phylogenetic analysis of Rh-like proteins from crabs was clustered, showing high conservation of the ammonium transporter domain and transmembrane segments essential to the function of Rh protein. Rh protein of P. trituberculatus (PtRh) was detected in all tested tissues, and showed the highest expression in the gills. To further characterize the role of PtRh in ammonia metabolism and excretion, double-stranded RNA-mediated RNA interference of PtRh was employed. Knockdown of PtRh upregulated mRNA expression of ammonia excretion-related genes encoding aquaporin (AQP), K+ channels and vesicle-associated membrane protein (VAMP), increased the activity of Na+/K+-ATPase (NKA) and V-type H+-ATPase (V-ATPase), and initially reduced then elevated the expression of the Na+/H+-exchanger (NHE). dsRNA-mediated reduction in PtRh significantly reduced ammonia excretion rate and increased ammonia and glutamine (Gln) levels in the hemolymph, together with an increase of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activity, indicating a central role for PtRh in ammonia excretion and detoxification mechanisms. Taken together, we conclude that Rh protein is a primary contributor to ammonia excretion of P. trituberculatus, which may be the basis of their ability to inhabit benthic water with high ammonia levels.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells.

An important step in the initiation of the innate immune response to virus infection is the recognition of non-self, viral RNA, including double-stranded RNA (dsRNA), by cytoplasmic pattern recognition receptors (PRRs). For many positive-sense RNA viruses and DNA viruses, the production of viral dsRNA, and the interaction of viral dsRNA and PRRs are well characterized. However, for negative-sense RNA viruses, viral dsRNA was thought to be produced at low to undetectable levels and PRR recognition of viral dsRNA is still largely unclear. In the case of arenaviruses, the nucleocaspid protein (NP) has been identified to contain an exoribonuclease activity that preferentially degrades dsRNA in biochemical studies. Nevertheless, pathogenic New World (NW) arenavirus infections readily induce an interferon (IFN) response in a RIG-I dependent manner, and also activate the dsRNA-dependent Protein Kinase R (PKR). To better understand the innate immune response to pathogenic arenavirus infection, we used a newly identified dsRNA-specific antibody that efficiently detects viral dsRNA in negative-sense RNA virus infected cells. dsRNA was detected in NW arenavirus infected cells colocalizing with virus NP in immunofluorescence assay. Importantly, the dsRNA signals also colocalized with cytoplasmic PRRs, namely, PKR, RIG-I and MDA-5, as well as with the phosphorylated, activated form of PKR in infected cells. Our data clearly demonstrate the PRR recognition of dsRNA and their activation in NW arenavirus infected cells. These findings provide new insights into the interaction between NW arenaviruses and the host innate immune response.

Adventitious viruses persistently infect three commonly used mosquito cell lines.

Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.

Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry.

RATIONALE: Recent developments in RNA interference (RNAi) have created a need for cost-effective and large-scale synthesis of double-stranded RNA (dsRNA), in conjunction with high-throughput analytical techniques to fully characterise and accurately quantify dsRNA prior to downstream RNAi applications. METHODS: Stable isotope labeled dsRNA was synthesised both in vivo (15 N) and in vitro (13 C,15 N-guanosine-containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from E. coli engineered to express dsRNA. RNase mass mapping approaches were subsequently performed using liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) for both the identification and absolute quantification of the dsRNA using the ratios of the light and heavy oligonucleotide pairs. RESULTS: Absolute quantification was performed based on the resulting light and heavy oligoribonucleotides identified using MS. Using this approach we determined that 624.6 ng/µL and 466.5 ng/µL of dsRNA was present in 80 µL total RNA extracted from 108 E. coli cells expressing 765 bp and 401 bp dsRNAs, respectively. CONCLUSIONS: Stable isotope labeling of dsRNA in conjunction with MS enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast.

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

Red-emissive triplex-forming PNA probes carrying cyanine base surrogates for fluorescence sensing of double-stranded RNA.

Red-emissive fluorescent probes have been developed by integration of quinoline blue or thiazole red as the base surrogate into triplex-forming PNAs, allowing selective sensing of a sequence of double-stranded RNA.

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA.

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.

Analysis of Cyclin E1 Functions in Porcine Preimplantation Embryonic Development by Fluorescence Microscopy.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.