H3K4me3 histone modification in baculovirus-infected silkworm cells.

Baculovirus infection modulates the chromatin states and gene expression of host insect cells. Here we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of H3 trimethylated at Lys4 (H3K4me3) histone modification in Bombyx mori nucleopolyhedrovirus-infected Bombyx mori cells. The ChIP-seq data revealed the changes of the genome-wide distribution and accumulation of euchromatic histone marks in host insect cells during the progression of baculovirus infection.

Antiviral Treatment Reveals a Cooperative Pathogenicity of Baculovirus and Iflavirus in Spodoptera exigua, a Lepidopteran Insect.

NPVThe beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Doublestranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

Characterization of the Gene Expression Patterns in the Murine Liver Following Intramuscular Administration of Baculovirus.

Intramuscular administration of wild-type baculovirus is able to both protect against Plasmodium sporozoite challenge and eliminate liver-stage parasites via a Toll-like receptor 9-independent pathway. To investigate its effector mechanism(s), the gene expression profile in the liver of baculovirus-administered mice was characterized by cDNA microarray analysis. The ingenuity pathway analysis gene ontology module revealed that the major gene subsets induced by baculovirus were immune-related signaling, such as interferon signaling. A total of 40 genes commonly upregulated in a Toll-like receptor 9-independent manner were included as possible candidates for parasite elimination. This gene subset consisted of NT5C3, LOC105246895, BTC, APOL9a/b, G3BP3, SLC6A6, USP25, TRIM14, and PSMB8 as the top 10 candidates according to the special unit. These findings provide new insight into effector molecules responsible for liver-stage parasite killing and, possibly, the development of a new baculovirus-mediated prophylactic and therapeutic biopharmaceutical for malaria.

High-resolution analysis of baculovirus-induced host manipulation in the domestic silkworm, Bombyx mori.

Many parasites manipulate host behaviour to enhance their transmission. Baculoviruses induce enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. However, the mechanisms underlying host manipulation system are largely unknown. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points, which are unlikely to characterize ELA precisely, as ELA typically persists for several hours. In this study, we modified a recently developed method using time-lapse recording to characterize locomotion of Bombyx mori larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) for 24 h at 3 s resolution. Our data showed that the locomotion of the mock-infected larvae was restricted to a small area, whereas the BmNPV-infected larvae exhibited a large locomotory area. These results indicate that BmNPV dysregulates the locomotory pattern of host larvae. Furthermore, both the mock- and BmNPV-infected larvae showed periodic cycles of movement and stationary behavior with a similar frequency, suggesting the physiological mechanisms that induce locomotion are unaffected by BmNPV infection. In contrast, the BmNPV-infected larvae exhibited fast and long-lasting locomotion compared with mock-infected larvae, which indicates that locomotory speed and duration are manipulated by BmNPV.

Entomopathogenic Viruses in the Neotropics: Current Status and Recently Discovered Species.

The market for biological control of insect pests in the world and in Brazil has grown in recent years due to the unwanted ecological and human health impacts of chemical insecticides. Therefore, research on biological control agents for pest management has also increased. For instance, insect viruses have been used to protect crops and forests around the world for decades. Among insect viruses, the baculoviruses are the most studied and used viral biocontrol agent. More than 700 species of insects have been found to be naturally infected by baculoviruses, with 90% isolated from lepidopteran insects. In this review, some basic aspects of baculovirus infection in vivo and in vitro infection, gene content, viral replication will be discussed. Furthermore, we provide examples of the use of insect viruses for biological pest control and recently characterized baculoviruses in Brazil.

An Empirical Test of the Role of Small-Scale Transmission in Large-Scale Disease Dynamics.

A key assumption of epidemiological models is that population-scale disease spread is driven by close contact between hosts and pathogens. At larger scales, however, mechanisms such as spatial structure in host and pathogen populations and environmental heterogeneity could alter disease spread. The assumption that small-scale transmission mechanisms are sufficient to explain large-scale infection rates, however, is rarely tested. Here, we provide a rigorous test using an insect-baculovirus system. We fit a mathematical model to data from forest-wide epizootics while constraining the model parameters with data from branch-scale experiments, a difference in spatial scale of four orders of magnitude. This experimentally constrained model fits the epizootic data well, supporting the role of small-scale transmission, but variability is high. We then compare this model's performance to an unconstrained model that ignores the experimental data, which serves as a proxy for models with additional mechanisms. The unconstrained model has a superior fit, revealing a higher transmission rate across forests compared with branch-scale estimates. Our study suggests that small-scale transmission is insufficient to explain baculovirus epizootics. Further research is needed to identify the mechanisms that contribute to disease spread across large spatial scales, and synthesizing models and multiscale data are key to understanding these dynamics.

Application of the Scorpion Neurotoxin AaIT against Insect Pests.

Androctonus australis Hector insect toxin (AaIT), an insect-selective toxin, was identified in the venom of the scorpion Androctonus australis. The exclusive and specific target of the toxin is the voltage-gated sodium channels of the insect, resulting in fast excitatory paralysis and even death. Because of its strict toxic selectivity and high bioactivity, AaIT has been widely used in experiments exploring pest bio-control. Recombinant expression of AaIT in a baculovirus or a fungus can increase their virulence to insect pests and diseases vectors. Likewise, transgenic plants expressing AaIT have notable anti-insect activity. AaIT is an efficient toxin and has great potential to be used in the development of commercial insecticides.

Where the baculoviruses lead, the caterpillars follow: baculovirus-induced alterations in caterpillar behaviour.

Baculoviruses are well-known for altering the behaviour of their caterpillar hosts by inducing hyperactivity (enhanced locomotion) and/or tree-top disease (climbing to elevated positions before death). These features, along with the genomic small size of baculoviruses compared to non-viral parasites and the at hand techniques for producing mutants, imply that baculoviruses are excellent tools for unravelling the molecular mechanisms underlying parasitic alteration of host behaviour. Baculoviruses can be easily mutated, allowing an optimal experimental setup in comparative studies, where for instance host gene expression can be compared between insects infected with wild-type viruses or with mutant viruses lacking genes involved in behavioural manipulation. Recent studies have revealed the first insight into the underlying molecular pathways that lead to the typical behaviour of baculovirus-infected caterpillars and into the role of light therein. Since host behaviour in general is mediated through the host's central nervous system (CNS), a promising future step will be to study how baculoviruses regulate the neuronal activity of the host.

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus.

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine. The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.

Pathogen-Mediated Tritrophic Interactions: Baculovirus-Challenged Caterpillars Induce Higher Plant Defenses than Healthy Caterpillars.

Although the tritrophic interactions of plants, insect herbivores and their natural enemies have been intensely studied for several decades, the roles of entomopathogens in their indirect modulation of plant-insect relationships is still unclear. Here, we employed a sublethal dose of a baculovirus with a relatively broad host range (AcMNPV) to explore if feeding by baculovirus-challenged Helicoverpa zea caterpillars induces direct defenses in the tomato plant. We examined induction of plant defenses following feeding by H. zea, including tomato plants fed on by healthy caterpillars, AcMNPV-challenged caterpillars, or undamaged controls, and subsequently compared the transcript levels of defense related proteins (i.e., trypsin proteinase inhibitors, peroxidase and polyphenol oxidase) and other defense genes (i.e., proteinase inhibitor II and cysteine proteinase inhibitor) from these plants, in addition to comparing caterpillar relative growth rates. As a result, AcMNPV-challenged caterpillars induced the highest plant anti-herbivore defenses. We examined several elicitors and effectors in the secretions of these caterpillars (i.e., glucose oxidase, phospholipase C, and ATPase hydrolysis), which surprisingly did not differ between treatments. Hence, we suggest that the greater induction of plant defenses by the virus-challenged caterpillars may be due to differences in the amount of these secretions deposited during feeding or to some other unknown factor(s).

The baculovirus Ac108 protein is a per os infectivity factor and a component of the ODV entry complex.

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.

Baculovirus Per Os Infectivity Factor Complex: Components and Assembly.

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae).

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

Timely trigger of caterpillar zombie behaviour: temporal requirements for light in baculovirus-induced tree-top disease.

Host behavioural manipulation is a common strategy used by parasites to enhance their survival and/or transmission. Baculoviruses induce hyperactivity and tree-top disease (pre-death climbing behaviour) in their caterpillar hosts. However, little is known about the underlying mechanisms of this behavioural manipulation. A previous study showed that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induced tree-top disease at 3 days post infection in third instar S. exigua larvae and that light plays a key role in triggering this behaviour. Here we investigated the temporal requirements for the presence of light to trigger this behaviour and found that light from above was needed between 43 and 50 h post infection to induce tree-top disease. Infected larvae that were not exposed to light from above in this period finally died at low positions. Exposure to light prior to this period did not affect the final positions where larvae died. Overall we conclude that light in a particular time frame is needed to trigger SeMNPV-induced tree-top disease in S. exigua larvae.

Host miRNAs are involved in hormonal regulation of HaSNPV-triggered climbing behaviour in Helicoverpa armigera.

Baculoviruses manipulate host climbing behaviour to ensure that the hosts die at elevated positions on host plants to facilitate virus proliferation and transmission, which is a process referred to as tree-top disease. However, the detailed molecular mechanism underlying tree-top disease has not been elucidated. Using transcriptome analysis, we showed that two hormone signals, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are key components involved in HaSNPV-induced tree-top disease in Helicoverpa armigera larvae. RNAi-mediated knockdown and exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced tree-top disease, while JH mediates tree-top disease behaviour. Knockdown of BrZ2, a downstream signal of JH and 20E, promoted HaSNPV-induced tree-top disease. We also found that two miRNAs target BrZ2 and are involved in the cross-talk regulation between 20E and JH manipulating HaSNPV replication, time to death and HaSNPV-induced tree-top disease.

Per os infectivity factors: a complicated and evolutionarily conserved entry machinery of baculovirus.

Baculoviruses are a family of arthropod-specific large DNA viruses that infect insect species belonging to the orders Lepidoptera, Hymenoptera and Diptera. In nature, occlusion-derived viruses (ODVs) initiate baculovirus primary infection in the midgut epithelium of insect hosts, and this process is largely dependent on a number of ODV envelope proteins designated as per os infectivity factors (PIFs). Interestingly, PIF homologs are also present in other invertebrate large DNA viruses, which is indicative that per os infection is an ancient and phylogenetically conserved entry mechanism shared by these viruses. Here, we review the advances in the knowledge of the functions of individual PIFs and recent discoveries about the PIF complex, and discuss the evolutionary implications of PIF homologs in invertebrate DNA viruses. Furthermore, future research highlights on the per os infection mechanism are also prospected.

Microbial Pest Control Agents: Are they a Specific And Safe Tool for Insect Pest Management?

Microorganisms (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA). They are used as alternative strategies to chemical insecticides to counteract the development of resistances and to reduce adverse effects on both environment and human health. These natural entomopathogenic agents, which have specific modes of action, are generally considered safer as compared to conventional chemical insecticides. Baculoviruses are the only viruses being used as the safest biological control agents. They infect insects and have narrow host ranges. Bacillus thuringiensis (Bt) is the most widely and successfully used bioinsecticide in the integrated pest management programs in the world. Bt mainly produces crystal delta-endotoxins and secreted toxins. However, the Bt toxins are not stable for a very long time and are highly sensitive to solar UV. So genetically modified plants that express toxins have been developed and represent a large part of the phytosanitary biological products. Finally, entomopathogenic fungi and particularly, Beauveria bassiana and Metarhizium anisopliae, are also used for their insecticidal properties. Most studies on various aspects of the safety of MPCA to human, non-target organisms and environment have only reported acute but not chronic toxicity. This paper reviews the modes of action of MPCA, their toxicological risks to human health and ecotoxicological profiles together with their environmental persistence. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

Genotype-by-genotype interactions between an insect and its pathogen.

Genotype-by-genotype (G×G) interactions are an essential requirement for the coevolution of hosts and parasites, but have only been documented in a small number of animal model systems. G×G effects arise from interactions between host and pathogen genotypes, such that some pathogen strains are more infectious in certain hosts and some hosts are more susceptible to certain pathogen strains. We tested for G×G interactions in the gypsy moth (Lymantria dispar) and its baculovirus. We infected 21 full-sib families of gypsy moths with each of 16 isolates of baculovirus and measured the between-isolate correlations of infection rate across host families for all pairwise combinations of isolates. Mean infectiousness varied among isolates and disease susceptibility varied among host families. Between-isolate correlations of infection rate were generally less than one, indicating nonadditive effects of host and pathogen type consistent with G×G interactions. Our results support the presence of G×G effects in the gypsy moth-baculovirus interaction and provide empirical evidence that correlations in infection rates between field-collected isolates are consistent with values that mathematical models have previously shown to increase the likelihood of pathogen polymorphism.

[Varieties and Mechanisms of Synergistic Factors Enhancing Baculovirus Infectivity].

Baculoviruses are a diverse group of viruses with double-stranded circular DNA genomes. They have certain advantageous properties for biotechnology applications, including high host specificity and environmental friendliness.Baculoviruses could play more important roles in sustainable agriculture as potential microbial insecticides. However, the popularization and application of baculovirus-based insecticides were seriously restricted due to deficiencies such as low virulence and slow rates of action. The infectivity of baculoviruses can be improved by synergistic factors. The present review summarizes characteristics of seven types of synergistic factors including baculovirus enhancin, entomopoxvirus fusolin and calcofluor. The mechanisms of these seven synergistic factors were analyzed. The information presented in this review can serve as a reference to aid in the development and application of baculovirus-based insecticides.

Effects of pathogen exposure on life-history variation in the gypsy moth (Lymantria dispar).

Investment in host defences against pathogens may lead to trade-offs with host fecundity. When such trade-offs arise from genetic correlations, rates of phenotypic change by natural selection may be affected. However, genetic correlations between host survival and fecundity are rarely quantified. To understand trade-offs between immune responses to baculovirus exposure and fecundity in the gypsy moth (Lymantria dispar), we estimated genetic correlations between survival probability and traits related to fecundity, such as pupal weight. In addition, we tested whether different virus isolates have different effects on male and female pupal weight. To estimate genetic correlations, we exposed individuals of known relatedness to a single baculovirus isolate. To then evaluate the effect of virus isolate on pupal weight, we exposed a single gypsy moth strain to 16 baculovirus isolates. We found a negative genetic correlation between survival and pupal weight. In addition, virus exposure caused late-pupating females to be identical in weight to males, whereas unexposed females were 2-3 times as large as unexposed males. Finally, we found that female pupal weight is a quadratic function of host mortality across virus isolates, which is likely due to trade-offs and compensatory growth processes acting at high and low mortality levels, respectively. Overall, our results suggest that fecundity costs may strongly affect the response to selection for disease resistance. In nature, baculoviruses contribute to the regulation of gypsy moth outbreaks, as pathogens often do in forest-defoliating insects. We therefore argue that trade-offs between host life-history traits may help explain outbreak dynamics.