Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

Background: Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB).Results: The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies.Conclusion: We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications

Repertoire of Bovine miRNA and miRNA-Like Small Regulatory RNAs Expressed upon Viral Infection

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals

Detection and profiling of circular RNAs in uninfected and maize Iranian mosaic virus-infected maize

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that are usually derived from exonic regions of genes, but can also arise from intronic and intergenic regions. Studies of circRNAs in humans, animals and several plant species have shown an altered population of circRNAs in response to abiotic and biotic stress. Recently it was shown that circRNAs also occur in maize, but it is unknown if maize circRNAs are responsive to stress. Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. In this study, we used data from RNA-Seq of MIMV-infected maize and uninfected controls to identify differentially expressed circRNAs. Such circRNAs were confirmed by two-dimensional polyacrylamide gel electrophoresis, northern blot, RT-qPCR and sequencing. A total of 1443 circRNAs were identified in MIMV-infected maize and 1165 circRNAs in uninfected maize. Two hundred and one circRNAs were in common between MIMV-infected and uninfected samples. Of these, 155 circRNAs were up-regulated and 5 down-regulated in MIMV infected plants, compared to the uninfected control. This study for the first time identified and profiled circRNA expression in maize in response to virus infection. Moreover, we predict that 33 circRNAs may bind 23 maize miRNAs, possibly affecting plant metabolism and development. Our data suggest a role for circRNAs in plant cell regulation and response to biotic stress such as virus infection, and give new insights into the complexity of plant-microbe interactions

Transcriptome-wide identification of host genes targeted by tomato spotted wilt virus-derived small interfering RNAs

RNA silencing mechanism functions as a major defense against invading viruses. The caveat in the RNA silencing mechanism is that the effector small interfering RNAs (siRNAs) act on any RNA transcripts with sequence complementarity irrespective of target's origin. A subset of highly expressed viral small interfering RNAs (vsiRNAs) derived from the tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) genome was analyzed for their propensity to downregulate the tomato transcriptome. A total of 11898 putative target sites on tomato transcripts were found to exhibit a propensity for down regulation by TSWV-derived vsiRNAs. In total, 2450 unique vsiRNAs were found to have potential cross-reacting capability with the tomato transcriptome. VsiRNAs were found to potentially target a gamut of host genes involved in basal cellular activities including enzymes, transcription factors, membrane transporters, and cytoskeletal proteins. KEGG pathway annotation of targets revealed that the vsiRNAs were mapped to secondary metabolite biosynthesis, amino acids, starch and sucrose metabolism, and carbon and purine metabolism. Transcripts for protein processing, hormone signalling, and plant-pathogen interactions were the most likely targets from the genetic, environmental information processing, and organismal systems, respectively. qRT-PCR validation of target gene expression showed that none of the selected transcripts from tomato cv. Marglobe showed up regulation, and all were down regulated even upto 20 folds (high affinity glucose transporter). However, the expression levels of transcripts from cv. Red Defender revealed differential regulation as three among the target transcripts showed up regulation (Cc-nbs-lrr, resistance protein, AP2-like ethylene-responsive transcription factor, and heat stress transcription factor A3). Accumulation of tomato target mRNAs of corresponding length was proved in both tomato cultivars using 5′ RACE analysis. The TSWV-tomato interaction at the sRNA interface points to the ability of tomato cultivars to overcome vsiRNA-mediated targeting of NBS-LRR class R genes. These results suggest the prevalence of vsiRNA-induced RNA silencing of host transcriptome, and the interactome scenario is the first report on the interaction between tospovirus genome-derived siRNAs and tomato transcripts, and provide a deeper understanding of the role of vsiRNAs in pathogenicity and in perturbing host machinery

The use of cell and larval assays to identify target genes for RNA interference-meditated control of the Australian sheep blowfly (Lucilia cuprina)

BACKGROUND Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. RESULTS A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. CONCLUSION This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 Society of Chemical Industry

Shaping nanoparticles with hydrophilic compositions and hydrophobic properties as nanocarriers for antibiotic delivery

Inspired by the lotus effect in nature, surface roughness engineering has led to novel materials and applications in many fields. Despite the rapid progress in superhydrophobic and superoleophobic materials, this concept of Mother Nature’s choice is yet to be applied in the design of advanced nanocarriers for drug delivery. Pioneering work has emerged in the development of nanoparticles with rough surfaces for gene delivery; however, the preparation of nanoparticles with hydrophilic compositions but with enhanced hydrophobic property at the nanoscale level employing surface topology engineering remains a challenge. Herein we report for the first time the unique properties of mesoporous hollow silica (MHS) nanospheres with controlled surface roughness. Compared to MHS with a smooth surface, rough mesoporous hollow silica (RMHS) nanoparticles with the same hydrophilic composition show unusual hydrophobicity, leading to higher adsorption of a range of hydrophobic molecules and controlled release of hydrophilic molecules. RMHS loaded with vancomycin exhibits an enhanced antibacterial effect. Our strategy provides a new pathway in the design of novel nanocarriers for diverse bioapplications

Characterization of the Biodistribution of a Silica Vesicle Nanovaccine Carrying a Rhipicephalus (Boophilus) microplus Protective Antigen With in vivo Live Animal Imaging

Development of veterinary subunit vaccines comes with a spectrum of challenges, such as the choice of adjuvant, antigen delivery vehicle, and optimization of dosing strategy. Over the years, our laboratory has largely focused on investigating silica vesicles (SVs) for developing effective veterinary vaccines for multiple targets. Rhipicephalus microplus (cattle tick) are known to have a high impact on cattle health and the livestock industry in the tropical and subtropical regions. Development of vaccine using Bm86 antigen against R. microplus has emerged as an attractive alternative to control ticks. In this study, we have investigated the biodistribution of SV in a live animal model, as well as further explored the SV ability for vaccine development. Rhodamine-labeled SV-140-C18 (Rho-SV-140-C18) vesicles were used to adsorb the Cy5-labeled R. microplus Bm86 antigen (Cy5-Bm86) to enable detection and characterization of the biodistribution of SV as well as antigen in vivo in a small animal model for up to 28 days using optical fluorescence imaging. We tracked the in vivo biodistribution of SVs and Bm86 antigen at different timepoints (days 3, 8, 13, and 28) in BALB/c mice. The biodistribution analysis by live imaging as well as by measuring the fluorescent intensity of harvested organs over the duration of the experiment (28 days) showed greater accumulation of SVs at the site of injection. The Bm86 antigen biodistribution was traced in lymph nodes, kidney, and liver, contributing to our understanding how this delivery platform successfully elicits antibody responses in the groups administered antigen in combination with SV. Selected tissues (skin, lymph nodes, spleen, kidney, liver, and lungs) were examined for any cellular abnormalities by histological analysis. No adverse effect or any other abnormalities were observed in the tissues

Pristine mesoporous carbon hollow spheres as safe adjuvants induce excellent Th2-biased immune response

The development of a safe and effective adjuvant that amplifies the immune response to an antigen is important for vaccine delivery. In this study, we developed pristine mesoporous carbon hollow spheres as high-capacity vaccine protein nanocarriers and safe adjuvants for boosting the immune response. Mono-dispersed invaginated mesostructured hollow carbon spheres (IMHCSs) have an average particle size of ∼200 nm, large pore size of 15 nm, and high pore volume of 2.85 cm·g. IMHCSs exhibited a very high loading capacity (1,040 μg·mg) towards ovalbumin (OVA, a model antigen), controlled OVA release behavior, excellent safety profile to normal cells, and high antigen delivery efficacy towards macrophages. In vivo immunization studies in mice demonstrated that OVA-loaded IMHCSs induced a 3-fold higher IgG response compared to a traditional adjuvant QuilA used in veterinary vaccine research. OVA delivered by IMHCSs induced a higher IgG1 concentration than IgG2a, indicating a T-helper 2 (Th2)-polarized response. Interferon-γ and interleukin-4 concentration analysis revealed both T-helper 1 (Th1) and Th2 immune responses induced by OVA-loaded IMHCSs. IMHCSs are safer adjuvants than QuilA. Our study revealed that pure IMHCSs without further functionalization can be used as a safe adjuvant for promoting Th2-biased immune responses for vaccine delivery

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems