Identification and Profiling of a Novel Bombyx mori latent virus Variant Acutely Infecting Helicoverpa armigera and Trichoplusia ni.

Insect cell expression systems are increasingly being used in the medical industry to develop vaccines against diseases such as COVID-19. However, viral infections are common in these systems, making it necessary to thoroughly characterize the viruses present. One such virus is Bombyx mori latent virus (BmLV), which is known to be specific to Bombyx mori and to have low pathogenicity. However, there has been little research on the tropism and virulence of BmLV. In this study, we examined the genomic diversity of BmLV and identified a variant that persistently infects Trichoplusia ni-derived High Five cells. We also assessed the pathogenicity of this variant and its effects on host responses using both in vivo and in vitro systems. Our results showed that this BmLV variant causes acute infections with strong cytopathic effects in both systems. Furthermore, we characterized the RNAi-based immune response in the T. ni cell line and in Helicoverpa armigera animals by assessing the regulation of RNAi-related genes and profiling the generated viral small RNAs. Overall, our findings shed light on the prevalence and infectious properties of BmLV. We also discuss the potential impact of virus genomic diversity on experimental outcomes, which can help interpret past and future research results.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies.

RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world's poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Insights into RNAi-based antiviral immunity in Lepidoptera: acute and persistent infections in Bombyx mori and Trichoplusia ni cell lines.

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.

RNA Interference in Insects: Protecting Beneficials and Controlling Pests.

Insects constitute the largest and most diverse group of animals on Earth with an equally diverse virome. The main antiviral immune system of these animals is the post-transcriptional gene-silencing mechanism known as RNA(i) interference. Furthermore, this process can be artificially triggered via delivery of gene-specific double-stranded RNA molecules, leading to specific endogenous gene silencing. This is called RNAi technology and has important applications in several fields. In this paper, we review RNAi mechanisms in insects as well as the potential of RNAi technology to contribute to species-specific insecticidal strategies. Regarding this aspect, we cover the range of strategies considered and investigated so far, as well as their limitations and the most promising approaches to overcome them. Additionally, we discuss patterns of viral infection, specifically persistent and acute insect viral infections. In the latter case, we focus on infections affecting economically relevant species. Within this scope, we review the use of insect-specific viruses as bio-insecticides. Last, we discuss RNAi-based strategies to protect beneficial insects from harmful viral infections and their potential practical application. As a whole, this manuscript stresses the impact of insect viruses and RNAi technology in human life, highlighting clear lines of investigation within an exciting and promising field of research.

Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids.

High throughput sequencing techniques are poorly adapted for in vivo studies of parasites, which require prior in vitro culturing and purification. Trypanosomatids, a group of kinetoplastid protozoans, possess a distinctive feature in their transcriptional mechanism whereby a specific Spliced Leader (SL) sequence is added to the 5'end of each mRNA by trans-splicing. This allows to discriminate Trypansomatid RNA from mammalian RNA and forms the basis of our new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq. We provided a proof-of-concept of SL-seq in Leishmania donovani, the main causative agent of visceral leishmaniasis in humans, and successfully applied the method to sequence Leishmania mRNA directly from infected macrophages and from highly diluted mixes with human RNA. mRNA profiles obtained with SL-seq corresponded largely to those obtained from conventional poly-A tail purification methods, indicating both enumerate the same mRNA pool. However, SL-seq offers additional advantages, including lower sequencing depth requirements, fast and simple library prep and high resolution splice site detection. SL-seq is therefore ideal for fast and massive parallel sequencing of parasite transcriptomes directly from host tissues. Since SLs are also present in Nematodes, Cnidaria and primitive chordates, this method could also have high potential for transcriptomics studies in other organisms.