Entomopoxvirus Research Articles

Genomic analysis of hyperparasitic viruses associated with entomopoxviruses.

Polinton-like viruses (PLVs) are a diverse group of small integrative dsDNA viruses that infect diverse eukaryotic hosts. Many PLVs are hypothesized to parasitize viruses in the phylum Nucleocytoviricota for their own propagation and spread. Here, we analyze the genomes of novel PLVs associated with the occlusion bodies of entomopoxvirus (EPV) infections of two separate lepidopteran hosts. The presence of these elements within EPV occlusion bodies suggests that they are the first known hyperparasites of poxviruses. We find that these PLVs belong to two distinct lineages that are highly diverged from known PLVs. These PLVs possess mosaic genomes, and some essential genes share homology with mobile genes within EPVs. Based on this homology and observed PLV mosaicism, we propose a mechanism to explain the turnover of PLV replication and integration genes.

Search for genes gained by horizontal gene transfer in an entomopoxvirus, with special reference to the analysis of the transfer of an ABC transporter gene

Although it is generally believed that large DNA viruses capture genes by horizontal gene transfer (HGT), the detailed manner of such transfer has not been fully elucidated. Here, we searched for genes in the coleopteran entomopoxvirus (EV) Anomala cuprea entomopoxvirus (ACEV) that might have been gained by ACEV by HGT. We classified the potential source organisms for HGT into three categories: the host A. cuprea; other organisms, including viruses unrelated to EVs; and organisms with uncertain host attribution. Of the open reading frames (ORFs) of the ACEV genome, 2.1 % were suggested to have been gained from the host by ACEV or its recent ancestor via HGT; 8.7 % were possibly from organisms other than the host, and 3.7 % were possibly from the third category of organisms via HGT. The analysis showed that ACEV contains some interesting ORFs obtained by HGT, including a large ATP-binding cassette protein (ABC transporter) ORF and a tenascin ORF (IDs ACV025 and ACV123, respectively). We then performed a detailed analysis of the HGT of the ACEV large ABC transporter ORF—the largest of the ACEV ORFs. mRNA sequences obtained by RNA-seq from fat bodies—sites of ACEV replication—and midgut tissues—sites of initial infection—of the virus's host A. cuprea larvae were subjected to BLAST analysis. One type of ABC transporter ORF from the fat bodies and two types from the midgut tissues, one of which was identical to that in the fat bodies, had the greatest identity to the ABC transporter ORF of ACEV. The two types from the host had high levels of identity to each other (approximately 95 % nucleotide sequence identity), strongly suggesting that the host ABC transporter group consisting of the two types was the origin of ACV025. We then determined the sequence (12,381 bp) containing a full-length gene of the A. cuprea ABC transporter. It turned out to be a transcription template for the abovementioned mRNA found in both tissues. In addition, we determined a large part (ca. 6.9 kb) of the template sequence for the mRNA found only in the midgut tissues. The results showed that the ACEV ABC transporter ORF is missing parts corresponding to introns of the host ABC transporter genes, indicating that the ORF was likely acquired by HGT in the form of mRNA. The presence of definite duplicated sequences adjacent to the ACEV ABC transporter genes—a sign of LINE-1 retrotransposon-mediated HGT—was not observed. An approximately 2-month ACV025 transcription experiment suggested that the transporter sequence is presumed to be continuously functional. The amino acid sequence of ACV025 suggests that its product might function in the regulation of phosphatide in the host-cell membranes.

Persistence of perorally inoculated entomopoxvirus spindles in the midgut of Bombyx mori larvae

Abstract Peroral inoculation of entomopoxvirus (EV) spindles, microstructures composed of the protein fusolin, enhances the infectivity of some insect viruses by disrupting the physical barrier against microbe infection, the peritrophic matrix, in the insect midgut. Here, we examined the temporal persistence of spindles of Anomala cuprea EV (ACEV) that infect Coleopteran species in Bombyx mori larva midgut because spindle solubility over time in the midgut is closely associated with the degree of the enhancement of microbe infectivity by fusolin. A number of ACEV spindles fed to B. mori larvae were retained in the digestive systems even 20 h after the completion of feeding spindles, and a number of spindles were found to be excreted still almost intact in feces under a light microscope. In an in vitro experiment, most ACEV spindles remained intact in B. mori midgut juice 1 h after the start of incubation and some of spindles appeared even overnight in contrast to Bombyx mori nucleoplyhedrovirus polyhedra, which were immediately dissolved in midgut juice. These results suggest spindles are not generally dissolved readily in the midgut of many insects. The difficulty in solubility of ACEV spindles is considered to be mainly due to that fusolin contains many cysteine residues that form a 3D network of disulfide bonds between fusolin dimers. To use spindles at a low cost as additives in microbial insecticides, increasing the solubility of spindles by protein engineering is important to utilize full spindles inoculated.

Entomopoxvirus infection induces changes in both juvenile hormone and ecdysteroid levels in larval Mythimna separata.

Insect viruses are among the most important pathogens of lepidopteran insects. Virus-infected larvae often show developmental defects including a prolonged larval period and a failure to pupate, but the mechanisms by which insect viruses regulate host development need further investigation. In this study, the regulation of host endocrinology by a lepidopteran entomopoxvirus (EPV), Mythimna separata EPV (MySEV), was examined. When fourth instar M. separata were inoculated with MySEV occlusion bodies, pupation was prevented and the insects died during the final (sixth) larval instar. Liquid chromatography-MS analysis revealed that juvenile hormone (JH) titres in the haemolymph of MySEV-infected sixth instars were higher than those in mock-infected larvae. JH esterase (JHE) activity was also examined by kinetic assay using a colorimetric substrate. The level of JHE activity in the haemolymph of MySEV-infected larvae was generally lower than that found in mock-infected larvae. In contrast, ecdysteroid titre in the haemolymph of final-instar MySEV-infected larvae was lower than that found in mock-infected larvae when measured by radioimmunoassay. A statistically significant difference in the release of ecdysteroids from prothoracic glands (PGs) that were dissected from MySEV- or mock-infected sixth instar Day 3 larvae was not found following prothoracicotropic hormone (PTTH) exposure. Our results indicate that the release of ecdysteroids was reduced not by infection of the PGs by MySEV, but by reduced PTTH production from the brain. Taken together our study suggests that EPVs retard host development by both reducing ecdysone titre and maintaining status quo levels of JH by preventing its metabolism.

Structure of viral spindles, in vivo crystals boosting insecticidal activity

Entomopoxviruses (EV) produce two types of microcrystals in infected cells: virus-containing spheroids representing their main infectious form; and spindles, bipyramidal crystals of the viral fusolin protein, that contribute to the oral virulence of these viruses. In co-feeding experiments, spindles also enhance the insecticidal activity of unrelated insect pathogens, which suggested their use as bioinsecticide additives. To understand how fusolin contributes to virulence and assembles in vivo, we determined the structures of EV spindles by X-ray microcrystallography using crystals isolated from EV-infected common cockchafers. This structure reveals that fusolin is composed of a fibronectin III domain followed by an extended C-terminal molecular arm (CT). The globular domain is structurally homologous to CBP21, a protein that is secreted by Gram-negative bacteria to degrade chitin as a source of energy. Like CBP21, fusolin has all the hallmarks of a lytic polysaccharide monooxygenase enzyme (LPMOs) with two conserved histidine residues forming a copper binding site and a prominent di-tryptophan motif positioned to bind the planar surface of crystalline chitin. The LPMO domain assembles in vivo into ultra-stable crystals crosslinked by CT. This molecular arm mediates the formation of domain-swapped dimers and their assembly into a crystalline lattice stabilized by a 3-D network of inter-dimer disulfide bonds. Overall, the molecular organization of spindles indicates a mode of action where controlled released of the LPMO domain of fusolin by proteolytic removal of the CT extension leads to disruption of the chitin-rich peritrophic matrix of larvae to facilitate the initial steps of viral invasion of the host.

New Insights into the Evolution of Entomopoxvirinae from the Complete Genome Sequences of Four Entomopoxviruses Infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.

Further research on the biological function of inclusion bodies ofAnomala cupreaentomopoxvirus, with special reference to the effect on the insecticidal activity of aBacillus thuringiensisformulation

Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation.

Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan

Three entomopoxviruses (EPVs) isolated from diseased Adoxophyes honmai larvae at different localities (Tsukuba, Itsukaichi, and Miyazaki) in Japan were compared for biochemical identity and key parameters of virus fitness, fatal infection, speed of kill, and virus yield. When the structural peptides of occlusion bodies (OBs) and occlusion-derived viral particles were compared using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, no difference in banding patterns was observed. However, DNA restriction endonuclease analysis showed that the three isolates were genotypically different, but many commonly sized DNA fragments were observed. Five tortricid species, A. honmai, Adoxophyes orana, Adoxophyes dubia, Homona magnanima, and Archips insulanus were susceptible to all isolates. No significant differences in the key viral fitness parameters were detected among the isolates in A. orana. However, the Miyazaki isolate had a different effect on H. magnanima; it allowed infected insects to survive longer and develop to a larger size, but had a lower yield of OBs per larva at any given time to death. OB yields per unit cadaver weight for the Miyazaki isolate, which indicate the conversion rate of the insect to virus, were lower over time compared to the other two isolates. The implications for selecting a candidate isolate to control tortricid pests are discussed.

Biological control of tortricidae in tea fields in Japan using insect viruses and parasitoids

Tea is a perennial and evergreen plant. Cultivated tea trees provide a habitat for insect pests and their natural enemies. In Japan, granuloviruses (GVs) have successfully controlled two of the most important pests of tea, Adoxophyes honmai and Homona magnanima (Tortricidae: Lepidoptera). The GVs are produced in vivo and a single application sustains pesticidal efficacy throughout a year, which encompasses 4 to 5 discrete generations of both species. A. honmai and H. magnanima also have various natural enemies, especially hymenopteran parasitoids. Such resident natural enemies also play a role in reducing the pest density in virus-controlled fields, but the effect of virus infection on parasitoids sharing the same host larva has not been well studied. Survival of one of the major parasitoids of A. honmai, Ascogaster reticulata (Braconidae: Hymenoptera), is reduced by virus infection of the host. Viruses, including GV and entomopoxvirus (EPV), and certain koinobiont endoparasitoids, including A. reticulata, are both known to regulate host endocrinology. However, the GV and EPV have distinct host regulation mechanisms, and consequently have different impacts on the survival of A. retuculata, when A. reticulata parasitizes a host that is infected with either GV or EPV. These additional effects on host regulation displayed by both viruses and parasitoids affect the outcome of virus-parasitoid interactions.

Stability of the viral-enhancing ability of entomopoxvirus spindles exposed to various abiotic factors

The proteinaceous paracrystaline structures called spindles that are produced by some entomopoxviruses (EPVs) are known to strongly enhance the infectivity of various nucleopolyhedroviruses (NPVs). These proteinaceous bodies thus may be potential synergists of bio-control agents (such as NPVs) for insect pests. Although the infectivity-enhancing ability of spindles needs to be highly stable when they are used as synergists in the field, little is known regarding the effects of various abiotic factors, e.g. high temperature and ultraviolet (UV) irradiation, on this stability. In the present study, we exposed Anomala cuprea EPV (AcEPV) spindles to various abiotic factors and examined their subsequent ability to enhance the infectivity of Bombyx mori NPV in B. mori larvae. The enhancing activity of AcEPV spindles was stable against high temperature, UV-B irradiation, ethyl alcohol, or the bactericide benzalkonium chloride and persisted after long exposure to formaldehyde. These results suggest that spindles are stable materials, even though they are composed of proteins, and thus are promising potential synergists of various viral insecticides.

NAD+‐Dependent DNA Ligase: A novel target waiting for the right inhibitor

DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single stranded or double stranded breaks between adjacent 5' phosphoryl and 3' hydroxyl groups of DNA. These enzymes are important for survival because they are involved in major cellular processes like DNA replication/repair and recombination. DNA ligases can be classified into two groups on the basis of their cofactor specificities. NAD(+)-dependent DNA ligases are present in bacteria, some entomopox viruses and mimi virus while ATP-dependent DNA ligases are ubiquitous. The former have recently been drawing a lot of attention as novel targets for antibiotics to overcome current drug resistance issues. Currently a diverse range of inhibitors have been identified. There are several issues to be addressed in the quest for optimized inhibitors of the enzyme. In the first part of the review we summarize current structural work on these enzymes. Subsequently we describe the currently available classes of inhibitors. We also address modalities to improve the specificity and potencies of new inhibitors identified using protein structure based rational approaches. In conclusion, NAD(+)-dependent ligases show great promise and represent a novel drug target whose time has come.

Spindles of an Entomopoxvirus Facilitate Its Infection of the Host Insect by Disrupting the Peritrophic Membrane

The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.

Poxvirus genomes: a phylogenetic analysis.

The evolutionary relationships of 26 sequenced members of the poxvirus family have been investigated by comparing their genome organization and gene content and by using DNA and protein sequences for phylogenetic analyses. The central region of the genome of chordopoxviruses (ChPVs) is highly conserved in gene content and arrangement, except for some gene inversions in Fowlpox virus (FPV) and species-specific gene insertions in FPV and Molluscum contagiosum virus (MCV). In the central region 90 genes are conserved in all ChPVs, but no gene from near the termini is conserved throughout the subfamily. Inclusion of two entomopoxvirus (EnPV) sequences reduces the number of conserved genes to 49. The EnPVs are divergent from ChPVs and between themselves. Relationships between ChPV genera were evaluated by comparing the genome size, number of unique genes, gene arrangement and phylogenetic analyses of protein sequences. Overall, genus Avipoxvirus is the most divergent. The next most divergent ChPV genus is Molluscipoxvirus, whose sole member, MCV, infects only man. The Suipoxvirus, Capripoxvirus, Leporipoxvirus and Yatapoxvirus genera cluster together, with Suipoxvirus and Capripoxvirus sharing a common ancestor, and are distinct from the genus Orthopoxvirus (OPV). Within the OPV genus, Monkeypox virus, Ectromelia virus and Cowpox virus strain Brighton Red (BR) do not group closely with any other OPV, Variola virus and Camelpox virus form a subgroup, and Vaccinia virus is most closely related to CPV-GRI-90. This suggests that CPV-BR and GRI-90 should be separate species.

A putative DNA helicase and novel oligoribonuclease in the Diachasmimorpha longicaudata entomopoxvirus (DlEPV).

Diachasmimorpha longicaudata entomopoxvirus (DlEPV) is a symbiotic entomopoxvirus (EPV) of the parasitic wasp Diachasmimorpha longicaudata. It has a double-stranded DNA genome of 250-300 kb and is >60% A-T rich. We describe ten ORFs (RI-35-1 to -10) contained within a 5.64 kb clone, RI-35, from a DlEPV EcoRI genomic library. Our goal was to identify unique motifs and compare them with others in the database, particularly those of poxviruses. Two ORFs (RI-35-1 and RI-35-7, respectively) encode putative proteins (113 aa and 219 aa) that are probably involved in regulating gene expression based on their predicted nuclear localization and the presence of SPxx motifs, leucine-zipper like sequences (113 aa), and a basic domain (219 aa). The largest gene (RI-35-3) is under the control of an intermediate/late promoter and is presumed to encode a cytoplasmic 480 aa DNA-dependent DNA helicase with conserved motifs that are characteristic of DExH helicases. Amino acid analysis of the DNA helicase sequence showed that DlEPV is close to but distinct from the Genus B EPVs. The DlEPV helicase is also distinct from that of the Diadromus pulchellus ascovirus 1a from the D. pulchellus parasitic wasp, with less than 10% amino acid identity. DlEPV encodes a 207 aa oligoribonuclease (RI-35-8) of the DEDDh family of exoribonucleases. The second largest ORF (RI-35-9) is under the control of a poxvirus early promoter and encodes a protein of 329 aa that is likely DlEPV-specific. Three ORFs (RI-35-4, -5, and -6) overlap (in the anti-sense strand) with ORFs encoding putatively important virus replication proteins (which were also under the control of intermediate promoters) and are presumably not expressed in DlEPV. These results support earlier reports that DlEPV is a member of the sub-family Entomopoxvirinae, most likely in Group C, and is the first symbiotic EPV described to date from a parasitic wasp.

Disintegration of the peritrophic membrane of silkworm larvae due to spindles of an entomopoxvirus

Mode of action by which entomopoxvirus (EPV) spindles enhance nucleopolyhedrovirus (NPV) infection remains unclear. Spindles of Anomala cuprea entomopoxvirus (AcEPV), a coleoptran EPV, are known to enhance Bombyx mori NPV (BmNPV) infection in silkworm larvae. AcEPV spindles were orally administered to silkworm larvae with or without BmNPV polyhedra, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles with or without the polyhedra had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles also was observed by the histological sectioning of the midgut. However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly. In contrast, the administration of AcEPV spheroids caused neither the disintegration of PMs nor the enhancement of BmNPV infection in silkworm larvae. These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reaching the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.

DNA Ligases: Structure, Function and Mechanism

DNA ligases play an essential role in DNA replication, repair, and recombination and are classified as ATP-dependent or NAD-dependent based on their adenylation cofactor requirement. ATP-dependent ligases have been found in bacteriophages, viruses, archaea, eukaryotes and bacteria. NAD-dependent ligases exist in eubacteria and entomopoxviruses. Both types of ligases share a catalytic core which consists of conserved motifs found in nucleotidyl transferase superfamily. These motifs are essential for adenylation cofactor binding, metal ion coordination, and ligation chemistry, as validated by X-ray crystal structure determination and mutational analyses. Viral ATP ligases are generally small in size and have been used as models for elucidating the catalytic mechanism. More complex genomes, such as yeast and humans, encode multiple ATP ligases with additional non-catalytically essential domains at N-terminal and / or C-teminal regions of the catalytic core. Some of these additional domains are involved in protein-protein interactions, which target different ATP ligases to specific loci to fulfill specific cellular replication and repair functions. As a consequence of multiple protein interactions, ligase activity may be enhanced to complete a specific strand joining reaction. In humans, three ligase genes encoding four ligases (ligase I, ligase IIIα, ligase IIIβ, and ligase IV) are required for replication, recombination, repair, and nonhomologous end-joining (NHEJ). In addition to ligation activities, other enzymatic activities such as AP lyase activity have been detected in ATP ligases, implicating an expanded role of ligases in DNA repair. The discovery of ATP ligases in eubacteria indicates their ubiquity in living organisms. NAD ligases are composed of a nucleotidyl transferase catalytic core and additional C-terminal domains. The discovery of NAD ligases in entomopox virus genomes changes the notion that NAD ligases are only associated with eubacteria. While NAD and ATP ligases complement each other in rescuing growth defects, specific cellular functions such as DNA repair appear to require proteinprotein interactions specified by domains associated with NAD or ATP ligases. A ligase-DNA complex structure is needed to better understand the recognition and catalytic mechanism during DNA strand joining. The complex protein-protein interactions have to be elucidated to define the cellular functions. Newly identified putative ATP ligases in bacteria may open a new dimension in DNA ligation. Keywords: dna ligase, viral atp ligase

Purification and Partial Characterization of an Entomopoxvirus (DlEPV) from a Parasitic Wasp of Tephritid Fruit Flies.

An insect poxvirus [entomopoxvirus (EPV)] occurs in the poison gland apparatus of female Diachasmimorpha longicaudata, a parasitic wasp of the Caribbean fruit fly, Anastrepha suspensa and other tephritid fruit flies. The DlEPV virion is 250–300 nm in diameter, has a “bumpy” appearance and a unipartite double stranded DNA genome of 290–300 kb. DlEPV DNA restriction fragment profiles differed from those reported for Amsacta moorei EPV (AmEPV) and Melanoplus sanguinipes EPV (MsEPV), the only two EPVs whose genomes have been sequenced, and from those reported for vaccinia (Vac), a vertebrate poxvirus (chordopoxvirus, ChPV). Blast search and ClustalW alignment of the amino acids deduced from the 2316 nucleotides of a DlEPV DNA fragment cloned from an EcoR1 genomic library revealed 75–78% homology with the putative DNA-directed RNA polymerases of AmEPV, MsEPV, and two ChPV homologs of the Vac J6R gene. Of the deduced 772 amino acids in the DlEPV sequence, 28.4% are conserved/substituted among the four poxviruses aligned, 12.9% occur in at least one EPV, 6.5% in at least one ChPV, 3.1% in at least one EPV and one ChPV, and 49.1% occur only in DlEPV. Although the RI-36-1 fragment represents a portion of the gene, it contains nucleotides that encode the NADFDGDE consensus sequence of known DNA-directed RNA polymerases. Western blots using a mouse polyclonal anti-DlEPV serum recognized six major protein bands in combined fractions of sucrose-purified DlEPV, at least one band in homogenates of male and female wasps, and at least two bands in host hemolymph that contained DlEPV virions. A digoxigenin-labeled DlEPV genomic DNA probe recognized DNA in dot-blots of male and female wasps. These results confirm that DlEPV is a true EPV and probably a member of the Group C EPVs. Unlike other EPVs, DlEPV does not express the spheroidin protein. Since it also replicates in both the wasp and fly, members of two different insect Orders, DlEPV may represent a new EPV Group, or a subgroup of the Group C viruses.

Involvement of spindles of an entomopoxvirus (EPV) in infectivity of the EPVs to their host insect.

The biological function of entomopoxvirus (EPV) spindles (inclusion bodies that lack virions), has not been elucidated. We characterized the function of EPV spindles in the cupreous chafer, Anomala cuprea (Coleoptera: Scarabaeidae). Spheroids or spheroids mixed with spindles of Anomala cuprea EPV were administered per os to the A. cuprea larvae. A significant increase in infectivity was induced by the addition of spindles to the spheroids, strongly suggesting that the spindles play an important role in the infection of the host insect.

The spheroidin of an Entomopoxvirus isolated from the grasshopper Anacridiumaegyptium (AaEPV) shares low homology with spheroidins from lepidopteran or coleopteran EPVs

Based on virion morphology, the current virus taxonomy groups entomopoxviruses (EPVs) (Poxvirus: Entomopoxvirinae) from coleopteran and dipteran hosts in separated genera, wilts it keeps viruses infecting either lepidopteran or orthopteran hosts in the same genus. In contrast to the morphological criteria, the few data available from recent studies at the genetic level have suggested that EPVs infecting different insect orders are phylogenetically distant. In order to elucidate EPVs phylogeny we have cloned and sequence the highly conserved/highly expressed spheroidin gene of Anacridium aegyptium entomopoxvirus (AaEPV). This gene and its promoter is of interest for the development of genetic engineering on EPVs. The spheroidin gene was located in the AaEPV genome by Southern blot and hybridisation with specific degenerated oligonucleotides probes synthesised after partial sequencing of the purified spheroidin protein. A total of 3489 bp were sequenced. This sequence included the coding and promoter region of 969 residues 108.8 kDa protein identified as spheroidin. AaEPV spheroidin contains 21 cysteine residues (2.2%) and 14 N-glycosylation putative sites distributed along the sequence. The cysteine residues are particularly abundant at the C-terminal end of the protein, with 11 residues in the last 118 aa. Our results confirm that the spheroidin is highly conserved only between EPVs isolated from the same insect order. Polyclonal antibodies raised against AaEPV spherules specifically revealed spheroidin in Western Blots failing to cross-react with MmEPV or AmEPV spheroidins or MmEPV fusolin. Comparison of spheroidins at the aa level demonstrate that AaEPV spheroidin shares only 22.2 and 21.9% identity with the lepidopteran AmEPV and the coleopteran MmEPV spheroidins, respectively, but 82.8% identity with the orthopteran MsEPV spheroidin. Only two highly conserved domains containing the sequence (V/Y)NADTG(C/L) and LFAR(I/A) have been identified in all known spheroidins. The phylogenetic tree constructed according to the clustalx analysis program revealed that EPVs are clearly separated in three groups — lepidopteran, coleopteran and orthopteran — according to the insect order of the virus hosts. In base to our results, the split of the genus Entomopoxvirus B dissociating lepidopteran and orthopteran EPVs into two different genera is suggested.

Characterization of an Overexpressed Spindle Protein during a Baculovirus Infection

The nucleopolyhedrovirus CfDEFNPV contains a gene encoding a viral protein, which accumulates as bipyramidal inclusion bodies (spindles) in the cytoplasm of infected cells. The spindles appear as early as 24 h postinfection, approximately 1 day earlier than viral occlusion bodies (OBs). Purification and characterization of the spindle protein was complicated by the fact that the OBs copurified with the spindles. We therefore modified CfDEFNPV by replacing the polyhedrin gene (plh) with a cassette containing the green fluorescent protein (GFP) gene. The recombinant virus did not produce OBs; however, the synthesis and morphogenesis of the spindles were not altered. When analyzed by SDS–PAGE, the spindles produced a 50-kDa protein, which was termed spindlin. Tunicamycin inhibition and endoglycosidase studies showed that spindlin was glycosylated. The N-terminus of spindlin was sequenced and its gene (gp50) was located on the viral genome. The gene was cloned and sequenced. Homologs of gp50 were found in several baculoviruses as well as in entomopoxviruses (EPV). In the latter virus, the homologous gene is that of fusolin, which also encodes a protein that forms spindle-shaped inclusion bodies in the cytoplasm of infected cells. Immunoblot analysis indicated that spindlin and fusolin were not serologically related, even though they share conserved polypeptide domains. Sequence analysis showed that gp50 of CfDEFNPV contains two late promoter motifs (TTAAG) in its 5′ flanking region. Both were used, but the proximal motif (−14 to −18 nt relative to the ATG) was the primary sequence from which most of the mRNA was initiated. When gp50 was cloned in a heterologous baculovirus expression system, spindlin was synthesized, although the spindles were irregular in shape. This suggested that the spindle structure may be species-specific or it may require more than one gene product for its morphogenesis.