Viral Infections In Insects Research Articles

The miR-184-3p promotes rice black-streaked dwarf virus infection by suppressing Ken in Laodelphax striatellus (Fallén).

MicroRNAs (miRNAs) play a key role in various biological processes by influencing the translation of target messenger RNAs (mRNAs) through post-transcriptional regulation. The miR-184-3p has been identified as an abundant conserved miRNA in insects. However, less is known about its functions in insect-plant virus interactions. The function of miR-184-3p in regulation of plant viral infection in insects was investigated using a rice black-streaked dwarf virus (RBSDV) and Laodelphax striatellus (Fallén) interaction system. We found that the expression of miR-184-3p increased in L. striatellus after RBSDV infection. Injection of miR-184-3p mimics increased RBSDV accumulation, while treatment with miR-184-3p antagomirs inhibits the viral accumulation in L. striatellus. Ken, a zinc finger protein, was identified as a target of miR-184-3p. Knockdown of Ken increased the virus accumulation and promoted RBSDV transmission by L. striatellus. This study demonstrates that RBSDV infection induces the expression of miR-184-3p in its insect vector L. striatellus. The miR-184-3p targets Ken to promote RBSDV accumulation and transmission. These findings provide a new insight into the function of the miRNAs in regulating plant viral infection in its insect vector. © 2023 Society of Chemical Industry.

Enoxacin Shows Broad-Spectrum Antiviral Activity against Diverse Viruses by Enhancing Antiviral RNA Interference in Insects.

RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we show that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived small interfering RNA (siRNA) into the RNA-induced silencing complex, thereby enhancing the antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited the replication of flaviviruses, including dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrate that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control the vector transmission of arboviruses or viral diseases in insect farming. IMPORTANCE RNAi has been widely recognized as one of the most broadly acting and robust antiviral mechanisms in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that was previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Here, we show that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into the Ago2-incorporated RNA-induced silencing complex and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin displayed effective RNAi-dependent antiviral effects against flaviviruses, such as dengue virus and Zika virus, in mosquito cells. This study is the first to demonstrate that enhancing RNAi by enoxacin elicits potent antiviral effects against diverse viruses in insects.

MicroRNA-315-5p promotes rice black-streaked dwarf virus infection by targeting a melatonin receptor in the small brown planthopper.

MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs, play key roles in various biological processes. Most plant viruses are transmitted by insect vectors. However, little is known about the function of miRNAs on plant virus-insect host interaction. We investigated the role of miR-315-5p in regulation of plant viral infection in insects using a rice black-streaked dwarf virus (RBSDV) and small brown planthopper (SBPH) interaction system. Our results showed that miR-315-5p had the highest expression level in 2nd-instar nymph, and was highly expressed in the salivary gland and midgut in SBPH. miR-315-5p was in response to and regulated RBSDV infection in SBPH. Injection of miR-315-5p mimic, agomir-315, significantly increased the RBSDV accumulation, whereas injection of miR-315-5p inhibitor, antagomir-315, reduced virus accumulation in SBPH. Furthermore, a melatonin receptor was identified as a target gene of miR-315-5p by the dual luciferase reporter assay. Knockdown of the melatonin receptor significantly increased the expression of RBSDV coat protein gene S10 and replication related genes, S5-1, S6, and S9-1. Furthermore, treatment with melatonin receptor antagonist luzindole and activator agomelatine significantly increased and reduced RBSDV accumulation in SBPH, respectively. Compared to the control, miR-315-5p did not affect the efficiency of RBSDV acquisition in SBPH. However, the efficiency of RBSDV transmission was significantly reduced after injecting antagomir-315. Taken together, our data reveal that miR-315-5p is beneficial for RBSDV infection in its insect vector by directly targeting a melatonin receptor. These findings provide a new insight to the function of miRNAs in virus-insect vector interaction. © 2021 Society of Chemical Industry.

Immunopathology and immune homeostasis during viral infection in insects.

Organisms clear infections by mounting an immune response that is normally turned off once the pathogens have been cleared. However, sometimes this immune response is not properly or timely arrested, resulting in the host damaging itself. This immune dysregulation may be referred to as immunopathology. While our knowledge of immune and metabolic pathways in insects, particularly in response to viral infections, is growing, little is known about the mechanisms that regulate this immune response and hence little is known about immunopathology in this important and diverse group of organisms. In this chapter we focus both on documenting the molecular mechanisms described involved in restoring immune homeostasis in insects after viral infections and on identifying potential mechanisms for future investigation. We argue that learning about the immunopathological consequences of an improperly regulated immune response in insects will benefit both insect and human health.

Sensing Viral Infections in Insects: A Dearth of Pathway Receptors.

Insects, the most diverse group of animals, can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses can be transmitted to humans, while bee and silkworm viruses cause important economic losses. Like all invertebrates, insects rely solely on innate immunity to counter viral infections. Protein-based mechanisms, involving restriction factors and evolutionarily conserved signaling pathways regulating transcription factors of the NF-kB and STAT families, participate in the control of viral infections in insects. In addition, RNA-based responses play a major role in the silencing of viral RNAs. We review here our current state of knowledge on insect antiviral defense mechanisms, which include conserved as well as adaptive, insect-specific strategies. Identification of the innate immunity receptors that sense viral infection in insects remains a major challenge for the field.

The insect reservoir of biodiversity for viruses and for antiviral mechanisms.

Insects are the most diverse group of animals. They can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses (arboviruses) can be transmitted to humans. High-throughput sequencing of small RNAs from insects provides insight into their virome, which may help understand the dynamics of vector borne infectious diseases. Furthermore, investigating the mechanisms that restrict viral infections in insects points to genetic innovations that may inspire novel antiviral strategies.

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.

Insects: An exceptional reservoir for viruses and antiviral genes

Insects are the most diverse group of animals. They can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses (arboviruses) can be transmitted to humans. High-throughput sequencing of small RNAs from insects provides insight on their virome, which may help understand the dynamics of vector borne infectious diseases. Furthermore, investigating the mechanisms that restrict viral infections in insects points to genetic innovations that may inspire novel antiviral strategies.

Dicer-2 Processes Diverse Viral RNA Species

RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.

P53-Mediated Rapid Induction of Apoptosis Conveys Resistance to Viral Infection in Drosophila melanogaster

Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress–responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

Modelling the within-host growth of viral infections in insects

Insects are infected by a variety of pathogens, including bacteria, fungi and viruses, which have been studied largely for their potential as biocontrol agents, but are also important in insect conservation (biodiversity) and as model systems for other diseases. Whilst the dynamics of host–pathogen interactions are well-studied at the population level, less attention has been paid to the critical within-host infection stage. Here, the reproductive rate of the pathogen is largely determined by how it exploits the host; the resources supplied by the host in terms of size and condition; competition with other pathogens; and the speed with which it kills the host (death being an inevitable outcome for obligate-killing pathogens). In this paper we aim to build upon recent developments in the literature by conducting single infection bioassays to obtain data on growth and fitness parameters for phenotypically different and similar strains of nucleopolyhedroviruses in the Lepdipoteran host Spodoptera exigua. Using these data, a simple mechanistic mathematical model (a coupled system of differential equations) is derived, fitted and parameter sensitivity predictions are made which support empirical findings. We unexpectedly found that initial growth of virus within the host occurs at a double-exponential rate, which contrasts with empirical findings for vertebrate host–pathogen systems. Moreover, these infection rates differ between strains, which has significant implications for the evolution of virulence and strain coexistence in the field, which are still relative unknowns. Furthermore, our model predicts that, counter to intuition, increased viral doses may lead to a decrease in viral yield, which is supported by other studies. We explain the mechanism for this phenomenon and discuss its implications for insect host–pathogen ecology.

Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spätzle-like Toll ligands (LvSpz1–3) from Litopenaeus vannamei

Toll-like receptor-mediated NF-κB pathways are essential for inducing immune related-gene expression in the defense against bacterial, fungal and viral infections in insects and mammals. Although a Toll receptor ( LvToll1) was cloned in Litopenaeus vannamei, relatively little is known about other types of Toll-like receptors and their endogenous cytokine-like ligand, Spätzle. Here, we report two novel Toll-like receptors (LvToll2 and LvToll3) and three Spätzle-like proteins (LvSpz1–3) from L. vannamei. LvToll2 has 1009 residues with an extracellular domain containing 18 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. LvToll3 is 1244 residues long with an extracellular domain containing 23 LRRs and a cytoplasmic TIR domain of 138 residues. The Spätzle-like proteins LvSpz1, LvSpz2 and LvSpz3 are 237, 245 and 275 residues in length, respectively, and all of them have a putative C-terminal cystine-knot domain. In Drosophila Schneider 2 (S2) cells, LvToll1 and LvToll3 were localized to the membrane and cytoplasm, and LvToll2 was confined to the cytoplasm. In Drosophila S2 cells, LvToll2 could significantly activate the promoters of NF-κB-pathway-controlled antimicrobial peptide genes, whereas LvToll1 and LvToll3 had no effect on them. LvSpz1 exerted some degree of inhibition on the promoter activities of Drosophila Attacin A and L. vannamei Penaeidin4. LvSpz3 also inhibited the Drosophila Attacin A promoter, but LvSpz2 could only slightly activate it. LvToll1, LvToll2 and LvToll3 were constitutive expressed in various tissues, while LvSpz1, LvSpz2 and LvSpz3 exhibited tissue-specific expression in the epithelium, eyestalk, intestine, gill and muscle. In the gill, after Vibrio alginolyticus challenge, LvToll1 was upregulated, but LvToll2 and LvToll3 showed no obvious changes. LvSpz1 and LvSpz3 were also strongly induced by V. alginolyticus challenge, but LvSpz2 only showed a slight downregulation. In the gill, after white spot syndrome virus (WSSV) challenge, LvToll1, LvToll2, LvToll3, LvSpz1 and LvSpz3 were upregulated, but LvSpz2 showed no obvious change, except for a slight downregulation at 12 h post-injection of WSSV. These findings might be valuable in understanding the innate immune signal pathways of shrimp and enabling future studies on the host–pathogen interactions in V. alginolyticus and WSSV infections.

Induction of reaper ortholog mx in mosquito midgut cells following baculovirus infection

Many vertebrate and insect viruses possess antiapoptotic genes that are required for their infectivity. This led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect antiviral defense because of the lack of adaptive immunity. However, the cellular mechanism that elicits apoptosis in response to viral infection in insects has not been determined. Using an in vivo infection system with the mosquito baculovirus CuniNPV (Culex nigripalpus nucleopolyhedrovirus), we demonstrated that michelob_x (mx), the mosquito ortholog of Drosophila proapoptotic gene reaper, is specifically induced in larval midgut cells following viral infection. Interestingly, the dynamics of mx induction corresponds with the outcome of the infection. In the permissive mosquito C. quinquefasciatus, a slow induction of mx failed to induce prompt apoptosis, and the infected cells eventually undergo necrosis with heavy loads of encapsulated viruses. In contrast, in the refractory mosquito Aedes aegypti, a rapid induction of mx within 30 min p.i. is followed by apoptosis within 2-6 h p.i., suggesting a possible role for apoptosis in limiting viral infection. When the execution of apoptosis was delayed by caspase inhibitors, viral gene expression became detectable in the A. aegypti larvae.

Eicosanoids mediate nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya megacephala

Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.