WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection.

The most powerful approach to detect distant homologues of a protein is based on structure prediction and comparison. Yet this approach is still inapplicable to many viral proteins. Therefore, we applied a powerful sequence-based procedure to identify distant homologues of viral proteins. It relies on three principles: (1) traces of sequence similarity can persist beyond the significance cutoff of homology detection programmes; (2) candidate homologues can be identified among proteins with weak sequence similarity to the query by using 'contextual' information, e.g. taxonomy or type of host infected; (3) these candidate homologues can be validated using highly sensitive profile-profile comparison. As a test case, this approach was applied to a protein without known homologues, encoded by ORF4 of Lake Sinai viruses (which infect bees). We discovered that the ORF4 protein contains a domain that has homologues in proteins from >20 taxa of viruses infecting arthropods. We called this domain 'widespread, intriguing, versatile' (WIV), because it is found in proteins with a wide variety of functions and within varied domain contexts. For example, WIV is found in the NSs protein of tospoviruses, a global threat to food security, which infect plants as well as their arthropod vectors; in the RNA2 ORF1-encoded protein of chronic bee paralysis virus, a widespread virus of bees; and in various proteins of cypoviruses, which infect the silkworm Bombyx mori. Structural modelling with AlphaFold indicated that the WIV domain has a previously unknown fold, and bibliographical evidence suggests that it facilitates infection of arthropods.

Isolation and genomic characterization of a cypovirus from Clanis bilineata.

Clanis bilineata Walker, soybean hawkmoth, belongs to the subfamily Ambulicinae (Sphingidae, Lepidoptera) and is an edible insect that usually grows on soybean leaves. In this study, we isolated a new cypovirus from naturally diseased Clanis bilineata larvae (named CbCPV), scanned its structure, sequenced its genome, and studied its phylogenetic relationship to other cypoviruses. Microscopy showed that CbCPV polyhedral occlusion bodies were about 1.878 µm on average and contained many virions in the ultrathin sections. The complete genome sequence of CbCPV is 22,812 bp comprising 10 segmented double-stranded RNAs. Apart from segment 1 containing one open reading frame (ORF) and one sub-ORF, the other nine segments all contain one open reading frame and encoded one putative protein. The non-coding regions contained conserved sequences at 5' termini (AGUCAAA) and 3' termini (AGC), except segment 4 containing a different 5' termini (AUGUUUA). The whole sequence of the polyhedrin gene in CbCPV contained 892 nucleotides, encoding a protein of 246 amino acids. Based on amino acid sequences of polyhedrin or RNA dependent RNA polymerase (RdRp), the phylogenetic analysis indicated that CbCPV was closely related to DnCPV-23. The putative function of all segments differed from each other, but the most closely related species of segments were DnCPV-23 with 98.2-99.8% nucleotide identity. Overall, the evidence of morphology, protein analysis and nucleic acids (genomic pattern) showed that CbCPV is a new isolate in the cypovirus-23 type and can be termed Clanis bilineata cypovirus type 23 (CbCPV-23).

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Functional analysis of a putative Bombyx mori cypovirus miRNA BmCPV-miR-10 and its effect on virus replication.

Bombyx mori cypovirus (BmCPV) is an important pathogen of silkworm (B. mori), the economically beneficial insect. The mechanism of its interaction with host immune defence system in the process of infection is still not yet completely clear. Researches have demonstrated that virus-encoded microRNAs (miRNA) play a crucial role in regulating host-pathogen interaction, but few reports are available so far on miRNAs encoded by insect viruses, especially the RNA viruses. In this study, a putative miRNA encoded by the 10th segment of BmCPV genomic RNA, BmCPV-miR-10, was identified and functionally analysed. The expression of the putative BmCPV-miR-10 could be detected via stem-loop RT-PCR (reverse transcription-Polymerase Chain Reaction) in the midgut of silkworm larvae infected with BmCPV. BmCSDE1 (B. mori cold shock domain E1 protein) gene was predicted to be a candidate target gene for BmCPV-miR-10 with the miRNA binding site located in 3' untranslated region of its mRNA. The regulation effect of the putative BmCPV-miR-10 on BmCSDE1 was verified in HEK293 cells by lentiviral expression system, in BmN cells by transfecting BmCPV-miR-10 mimics. The qRT-PCR (quantitative real-time PCR) results showed that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1. By injection of BmCPV-miR-10 mimics into the silkworm larvae infected with BmCPV, it was further proved that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1 in vivo, then inhibit the expression of BmApaf-1 (B. mori apoptotic protease activating factor 1), while enhance the replication of BmCPV genomic RNAs to a certain extent. These results implied that the putative BmCPV-miR-10 could down-regulate the expression of BmCSDE1, then suppress the expression of BmApaf-1, thereby created a favourable intracellular environment for virus replication and proliferation.

Transcriptional responses of Daphnis nerii larval midgut to oral infection by Daphnis nerii cypovirus-23.

BACKGROUND: Daphnis nerii cypovirus-23 (DnCPV-23) is a new type of cypovirus and has a lethal effect on the oleander hawk moth, Daphnis nerii which feeds on leave of Oleander and Catharanthus et al. After DnCPV-23 infection, the change of Daphnis nerii responses has not been reported. METHODS: To better understand the pathogenic mechanism of DnCPV-23 infection, 3rd-instar Daphnis nerii larvae were orally infected with DnCPV-23 occlusion bodies and the transcriptional responses of the Daphnis nerii midgut were analyzed 72 h post-infection using RNA-seq. RESULTS: The results showed that 1979 differentially expressed Daphnis nerii transcripts in the infected midgut had been identified. KEGG analysis showed that protein digestion and absorption, Toll and Imd signaling pathway were down-regulated. Based on the result, we speculated that food digestion and absorption in insect midgut might be impaired after virus infection. In addition, the down-regulation of the immune response may make D. nerii more susceptible to bacterial infections. Glycerophospholipid metabolism and xenobiotics metabolism were up-regulated. These two types of pathways may affect the viral replication and xenobiotic detoxification of insect, respectively. CONCLUSION: These results may facilitate a better understanding of the changes in Daphnis nerii metabolism during cypovirus infection and serve as a basis for future research on the molecular mechanism of DnCPV-23 invasion.

Identification of long noncoding RNAs in silkworm larvae infected with Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the most important pathogens causing severe disease to silkworm. Emerging evidence indicates that long noncoding RNAs (lncRNAs) play importantly regulatory roles in virus infection and host immune response. To better understand the interaction between silkworm, Bombyx mori and BmCPV, we performed a comparative transcriptome analysis on lncRNAs and mRNAs between the virus-infected and noninfected silkworm larvae midgut at two time points postinoculation. A total of 16,753 genes and 1845 candidate lncRNAs were identified, among which 356 messenger RNA (mRNAs) and 41 lncRNAs were differentially expressed (DE). Target gene prediction revealed that most of DEmRNAs (123) were coexpressed with 28 DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans- regulation by BmCPV-induced lncRNAs, and a regulatory network of DElncRNAs and DEmRNAs was then constructed. According to the network, many genes involved in apoptosis, autophagy, and antiviral response, such as ATG3, PDCD6, IBP2, and MFB1, could be targeted by different DElncRNAs, implying the essential roles of these genes and lncRNAs in BmCPV infection. In all, our studies revealed for the first time the alteration of lncRNA expression in BmCPV-infected larvae and its potential influence on BmCPV replication, providing a new perspective for host-cypovirus interaction studies.

Histologic lesions of experimental infection with Lymantria dispar multicapsid nucleopolyhedrovirus and Lymantria dispar cytoplasmic polyhedrosis virus in European gypsy moth caterpillars (Lymantria dispar dispar).

European gypsy moths (Lymantria dispar dispar) are an invasive species in North America, and are listed by the International Union for the Conservation of Nature as one of the 100 most destructive invasive species worldwide. They have several known viruses, some of which are used as biological control agents. However, there are no detailed descriptions of many entomopathogenic viral infections, including in European gypsy moths, using bright-field microscopy. In this study, 11 European gypsy moth caterpillars were evaluated histologically: 4 were experimentally infected with Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV; Baculoviridae); 4 were experimentally infected with Lymantria dispar cytoplasmic polyhedrosis virus (LdCPV; Reoviridae); 3 control animals were uninfected. A complete tissue set was evaluated in all animals from all groups using bright-field microscopy, including epidermis, cuticle, striated muscle, tracheae, foregut, midgut, hindgut, Malpighian tubules, hemocytes, fat body, and nervous system. LdMNPV-infected caterpillars had marked karyomegaly and intranuclear viral inclusions in cells of the epidermis, tracheae, fat body, and hemocytes. LdMNPV-infected caterpillars also had hyperplasia and hypertrophy of epidermal and tracheal epithelial cells. LdCPV-infected caterpillars had numerous granular eosinophilic intracytoplasmic viral inclusions in midgut epithelial cells. Both LdMNPV-infected and LdCPV-infected caterpillars had atrophy of fat body adipocytes; this change was more pronounced in LdCPV-infected caterpillars. This work provides the first detailed descriptions of these viral infections in European gypsy moth caterpillars using bright-field light microscopy and provides images of normal histology from control caterpillars.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2',3'-cGAMP-degrading enzyme family.

Analysis of reassortant and intragenic recombination in Cypovirus.

Cypoviruses (CPVs) are RNA viruses with segmented double-stranded genome and major pathogens of various insects, including economic insects like silkworms and pest insects for agricultural crops and forests. Genome reassortment and recombination are common phenomenon for viruses as a mechanism to expand host range and increase virulence. In the present study, we analyzed the reassortant and recombination events for CPVs. The results showed that two genome segments (S1 and S4) of BmCPV1-YN shared higher nucleotide identity with the corresponding segment of BmCPV1-I while others were all more closely to BmCPV1-SZ, suggesting BmCPV1-YN was originated from reassortant events between BmCPV1-I and BmCPV1-SZ. Recombination analyses revealed that S6 of BmCPV1-YN was a recombinant segment derived from BmCPV1-I and BmCPV1-SZ, and S10 of DpCPV1 was a recombinant segment emerged from BmCPV1-I and LdCPV1. Our findings provide the evidence for the fact that CPVs could undergo reassortant and recombinant events and enrich the knowledge about etiology and molecular epidemiology of CPVs.

Antiviral defense against Cypovirus 1 (Reoviridae) infection in the silkworm, Bombyx mori.

Recent years have shown a large increase in studies of infection of the silkworm (Bombyx mori) with Cypovirus 1 (previously designated as B. mori cytoplasmic polyhedrosis virus), that causes serious damage in sericulture. Cypovirus 1 has a single-layered capsid that encapsulates a segmented double-strand RNA (dsRNA) genome which are attractive features for the establishment of a biotechnological platform for the production of specialized gene silencing agents, either as recombinant viruses or as viral-like particles with nonreplicative dsRNA cargo. For both combatting viral disease and application of Cypovirus-based pest control, however, a better understanding is needed of the innate immune response caused by Cypovirus infection of the midgut of lepidopteran larvae. Studies of deep sequencing of viral small RNAs have indicated the importance of the RNA interference pathway in the control of Cypovirus infection although many functional aspects still need to be elucidated and conclusive evidence is lacking. A considerable number of transcriptome studies were carried out that revealed a complex response that hitherto remains uncharacterized because of a dearth in functional studies. Also, the uptake mechanism of Cypovirus by the midgut cells remains unclarified because of contrasting mechanisms revealed by electron microscopy and functional studies. The field will benefit from an increase in functional studies that will depend on transgenic silkworm technology and reverse genetics systems for Cypovirus 1.

A new cypovirus from the Japanese peppered moth, Biston robustus.

A cypovirus was isolated from larvae of the Japanese peppered moth, Biston robustus. The viral genome is 23,954 bp comprising 10 segmented double-stranded RNAs with a new electropherotype among cypoviruses. Each segment encodes one putative protein and has non-coding regions that contain conserved sequences at their 5' and 3' termini, 5'-AGAA(U/A)U-3' and 5'-UGC-3', respectively. Seven proteins encoded in the genome are homologous to those of other cypoviruses at a cut-off E-value of 1 × 10-5. The maximal sequence identities of these proteins with cypovirus homologs are 24.30%-39.40%. These results indicate that the virus isolated is a novel cypovirus; herein designated as Biston robustus cypovirus 24 (BrCPV-24). This newly isolated BrCPV-24 infects the larvae of the silkworm Bombyx mori.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

Exogenous gene can be expressed by a recombinant Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitro-transcribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.

Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

Receptor-mediated endocytosis using a ß1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

Polyhedra structures and the evolution of the insect viruses.

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.

Peptidoglycan Recognition Protein S5 of Bombyx mori Facilitates the Proliferation of Bombyx mori Cypovirus 1.

Bombyx mori cypovirus 1 (BmCPV1), a primary pathogen of the silkworm, is a typical dsRNA virus belonging to the Reoviridae family. In this study, a total of 2520 differentially expressed genes (DEGs) were identified by RNA-seq analysis of the silkworm midgut after BmCPV1 infection and Gene Ontology (GO) functional annotation showed that the DEGs predominantly functioned in binding (molecular function), cell (cellular component), and cellular processes (biological process). Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation revealed that the DEGs were mainly distributed in global and overview metabolism maps, translation, and signal transduction. Among the identified DEGs, BmPGRP-S5 belongs to the peptidoglycan recognition protein (PGRP) family. Previous studies have revealed that PGRPs were involved in the interactions between silkworm and BmCPV1. Here, we explored the effect of BmPGRP-S5 on BmCPV1 replication and demonstrated that BmPGRP-S5 promotes the proliferation of BmCPV1 in BmN cells through overexpression or knockdown experiments. Knocking down of BmPGRP-S5 in silkworm larvae similarly promoted the proliferation of BmCPV1. Through experimental validation, we therefore determined that BmPGRP-S5 acts as a proviral host factor for BmCPV1 infection. This study clarifies the proliferation mechanism of BmCPV1 and provides new insights into the functional role of BmPGRP-S5.

The Impact of a Cypovirus on Parental and Filial Generations of Lymantria dispar L.

Recently, we found that the spongy moth Lymantria dispar L. is susceptible to infection by a Dendrolimus sibiricus cytoplasmic polyhedrosis virus (DsCPV-1). In the present study, we evaluated the pathogenicity of DsCPV-1 against L. dispar larvae and its impact on surviving insects after the infection. Offspring of virally challenged insects were tested for susceptibility to a stress factor (starvation). In addition, we used light microscopy and quantitative polymerase chain reaction (qPCR) to test the ability of DsCPV-1 to be transmitted vertically. We found insect mortality of the L. dispar parents following the infection was positively associated with DsCPV-1 dose. DsCPV-1 was lethal to second-instar L. dispar larvae with a 50% lethal dose (LD50) of 1687 occlusion bodies per larva. No vertical transmission of DsCPV-1 to offspring larvae was detected, while the majority of insect deaths among offspring larvae were caused by microsporidia (Vairimorpha lymantriae), which was harbored by the parents. The offspring of virally challenged parents exhibited a higher number of detected microsporidia compared to the control. Our findings suggest that the application of DsCPV-1 is effective in controlling pests in terms of transgenerational impact following virus exposure.

A New Cypovirus-1 Strain as a Promising Agent for Lepidopteran Pest Control.

Now more than ever researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus, possessing attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe the morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (the half-lethal dose is 25 occlusion bodies per second-instar larva) and to have a relatively wide host range (infecting representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for the possible use of this strain in pest control, we call on virologists, pest control specialists, and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides. IMPORTANCE In this article, we describe a newly discovered cypovirus strain that displays features ideally suited for the development of a modern biological insecticide: high potency, relatively broad host range, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, and ecologically friendly. Based on an alignment of CPV genomes, we suggest that the enhanced host range of this new strain is the sequence of evolutionary events that occurred after coinfections involving different CPV species within the same host. These findings suggest that we need to positively reconsider CPVs as prospective agents as biocontrol products.

Les polyèdres viraux : armures cristallines des virus d'insectes.

Viral polyhedroses are very common diseases of insects. They were first identified as leading causes of losses in the silk industry. This heterogeneous group of diseases is characterized by the formation of crystals in infected cells that are called viral polyhedra or occlusion bodies and represent the infectious form of the viruses. Polyhedra have similar role in the infectious cycle of the two groups of viruses responsible for polyhedroses, the cypoviruses - members of the Reoviridae family - and the Baculoviridae. Polyhedra embed virus particles within infected cells in a robust crystalline matrix that protects viral infectivity after release in the environment. Upon ingestion by a new host, crystals dissolve readily thereby releasing the infectious particles to initiate a new viral cycle. Owing to their unique molecular organization, these atypical infectious forms have long intrigued virologists and biochemists alike. They attracted particular interest because of the in vivo crystallization process and the contrast between rapid release upon ingestion and extreme stability. It is only recently that novel approaches and technologies allowed the structure determination of such tiny crystals by X-ray crystallography. Cypovirus and baculovirus polyhedra share the same role in the virus cycle, the same crystalline lattice with a cubic centered symmetry, and matrix proteins called polyhedrins of similar sizes. However, their building blocks differ by their folds and packing in polyhedra. The two classes of polyhedra therefore harbour distinct molecular architectures and appear to have emerged independently in the virosphere. The role of tyrosine clusters in polyhedra dissolution and the use of molecular arms to achieve in vivo crystallization may thus represent striking cases of convergent evolution. This review summarizes our understanding of viral polyhedra with an emphasis on the recent structural studies. We also provide examples of biotechnological applications entailing structure-based engineering of polyhedra as novel types of crystalline microparticules.

Development of a cypovirus protein microcrystal-encapsulated Bacillus thuringiensis UV-tolerant and mosquitocidal δ-endotoxin.

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.