Cotesia Plutellae Bracovirus Research Articles

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28–50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.

Transient expression of a viral histone H4, CpBV-H4, suppresses immune-associated genes of Plutella xylostella and Spodoptera exigua

A viral histone H4, CpBV-H4, is encoded in the Cotesia plutellae bracovirus (CpBV) genome. This polydnavirus is symbiotic with C. plutellae, an endoparasitoid wasp . When the wasp parasitizes its host, Plutella xylostella, the symbiotic CpBV is delivered to host hemocoel and infects different internal tissues. CpBV-H4 encoded in the virus exhibits high sequence similarity to host histone H4, except for an extended N-terminal tail (38 amino acids long). When the CpBV-H4 cloned in a eukaryotic expression vector was transiently expressed in P. xylostella and a nonhost, Spodoptera exigua, it clearly inhibited several immune-associated genes, including cecropin, gloverin, serpin, apolipophorin III, and transferrin. However, its truncated construct, prepared by deleting 38 amino acids at the N-terminal tail, lost its inhibitory activity against immune-associated genes of the both species. This study has verified an inhibitory activity of CpBV-H4 against host immune-associated genes and has provided a possibility to expand its activity spectrum to the genes of other insect species.

A SERI technique reveals an immunosuppressive activity of a serine-rich protein encoded in Cotesia plutellae bracovirus

Polydnavirus genome is segmented and dispersed on host wasp chromosome. After replication, the segments form double- stranded circular DNAs and embedded in viral coat proteins. These viral particles are delivered into a parasitized host along with parasitoid eggs. A serine-rich protein (SRP) is predicted in a polydnavirus, Cotesia plutellae bracovirus (CpBV), genome in its segment no. 33 (CpBV-S33), creating CpBVSRP1. This study explored its expression and physiological function in the diamondback moth, Plutella xylostella, larvae parasitized by C. plutellae. CpBV-SRP1 encodes 122 amino acids with 26 serines and several predicted phosphorylation sites. It is persistently expressed in all tested tissues of parasitized P. xylostella including hemocyte, fat body, and gut. Its physiological function was analyzed by injecting CpBV-S33 and inducing its expression in nonparasitized P. xylostella by a technique called SERI (segment expression and RNA interference). The expression of CpBV-SRP1 significantly impaired the spreading behavior and total cell count of hemocytes of treated larvae. Subsequent RNA interference of CpBV-SRP1 rescued the immunosuppressive response. This study reports the persistent expression of CpBV-SRP1 in a parasitized host and its parasitic role in suppressing the host immune response by altering hemocyte behavior and survival.

Translation inhibitory factors encoded in Cotesia plutellae bracovirus require the 5′-UTR of a host mRNA target

Physiological processes of the diamondback moth, Plutella xylostella, larvae parasitized by Cotesia plutellae are altered by several parasitic factors including a polydnavirus, C. plutellae bracovirus (CpBV). Two homologous genes, CpBV15alpha and CpBV15beta, have been proposed as host translation inhibitory factors (HTIFs). This study analyzed their effects on host gene expression at a post-transcriptional level. A proteomic approach using two dimensional electrophoresis revealed that the parasitization resulted in 24.0% (60/250 spots) reduction of gene expression compared to nonparasitized control. It also indicated that the transient expression of CpBV15alpha or CpBV15beta in nonparasitized larvae resulted in 26.0% (65/240 spots) or 28.0% (70/240 spots) reduction, respectively. Seven spots that were not detected in the transiently expressed samples were further analyzed by a tandem mass spectrometry. These proteins were predicted to be associated with host cell signaling and metabolism. To investigate translation inhibitory effects of CpBV15alpha and CpBV15beta, capped mRNA of a storage protein 1 (SP1) of P. xylostella, a common inhibitory target of both HTIFs, was prepared by in vitro transcription and translated in vitro in the presence or absence of recombinant HTIFs prepared from Sf9 cells by recombinant baculoviruses. Translation of SP1 mRNA containing 5'-untranslated region (5'-UTR) was inhibited by both HTIFs. However, translation of SP1 mRNA without 5'-UTR was insensitive to the exposure of both HTIFs. Both HTIFs inhibited the host gene translation in a dose-dependent manner. In addition, these two factors showed cooperative inhibition. This study suggests that CpBV15alpha and CpBV15beta inhibit host mRNAs directly by acting on translation machinery, in which 5'-UTR of target mRNAs would be required for the inhibitory action.

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.

Sequence and gene organization of 24 circles from the Cotesia plutellae bracovirus genome

Twenty-four genomic segments of Cotesia plutellae bracovirus (CpBV) were completely sequenced, and their genomic structures were analyzed. The aggregated genome size is 351,299 bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3%, and 125 putative open reading frames (ORFs) are predicted. More than half (52.5%) of predicted genes are annotated as hypothetical, but they share sequence similarities with those of other bracoviral genomes. The annotated ORFs can be classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest, including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode ankyrin-like and EP1-like genes, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Phylogenetic analyses based on PTP, ankyrin and EP1-like gene groups revealed no correlation between bracoviruses.

Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus.

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp , Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.

Analysis of promoter activity of selected Cotesia plutellae bracovirus genes

In a previous study, we cloned 27 discrete genome segments of Cotesia plutellae bracovirus (CpBV) and provided the complete nucleotide sequences and annotation. Seven putative coding regions were predicted from one of the largest segments, CpBV-S30. The activity of promoters associated with six predicted ORFs from this segment were investigated using both transient and baculovirus expression assays with enhanced green fluorescent protein as a reporter gene. CpBV promoters showed activity earlier than the polyhedrin promoter and the activity of some of these promoters was superior to that of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter in the baculovirus expression assays. The promoter of ORF3004 showed the highest level of activity in insect cells, exhibiting 24 % of the activity obtained with the AcMNPV polyhedrin promoter in Sf9 cells. In Spodoptera exigua larvae, the ORF3006 promoter showed the highest activity, with about 35 % of the activity measured with the polyhedrin promoter. In addition, analysis of the ORF3006 promoter revealed that the region between -382 and -422 from the translation start point was critical for activity of this promoter. These results suggest that the CpBV-S30 promoters characterized here could be useful tools in a variety of biotechnological applications, such as gene expression analyses and insecticide development.

A Technique of Segment Expression and RNA Interference (SERI) Reveals a Specific Physiological Function of a Cysteine-rich Protein Gene Encoded in Cotesia plutellae Bracovirus

As a provirus, polydnavirus has a segmented DNA genome on chromosome(s) of host wasp. It contains several genes in each segment that presumably play critical roles in regulating physiological processes of target insect parasitized by the wasp. A cysteine-rich protein 1 (CRP1) is present in the polydnavirus Cotesia plutellae bracovirus (CpBV) genome, but its expression and physiological function in Plutella xylostella parasitized by the viral host C. plutellae is not known. This CpBV-CRP1 encoding 189 amino acids with a putative signal peptide (20 residues) was persistently expressed in parasitized P. xylostella with gradual decrease at the late parasitization period. Expression of CpBV-CRP1 was tissue-specific in the fat body/epidermis and hemocyte, but not in the gut. Its physiological function was analyzed by inducing transient expression of a CpBV segment containing CpBV-CRP1 and its promoter, which caused significant reduction in hemocyte -spreading and delayed larval development. When the treated larvae were co-injected with double-stranded RNA of CpBV-CRP1, the expression of CpBV-CRP1 disappeared,whereas other genes encoded in the CpBV segment was expressed. These co-injected larvae significantly recovered the hemocyte-spreading capacity and larval development rate. This study reports that CpBV-CRP1 is expressed in P.xylostella parasitized by C.plutellae and its physiological function is to alter the host immune and developmental processes.

Insecticidal activities of recombinant Autographa californica nucleopolyhedrovirus containing a scorpion neurotoxin gene using promoters from Cotesia plutellae bracovirus

To investigate the usefulness of early-expressed promoters from Cotesia plutellae bracovirus (CpBV) in improvement of insecticidal activities of baculovirus, recombinant AcNPVs, Ac3003ProAaIT, Ac3004ProAaIT, Ac3005ProAaIT and Ac3006ProAaIT expressing scorpion neurotoxin, AaIT, under the control of ORF3003, ORF3004, ORF3005 and ORF3006 promoter, respectively were constructed. Among these recombinant viruses, Ac3006ProAaIT showed highest insecticidal activity against 3rd instar larvae of Spodoptera exigua. These results suggested that early promoters from CpBV could be successfully applied to improve pathogenicity of baculoviruses.

A putative actin destabilizer, CpBV-RTX, encoded in Cotesia plutellae bracovirus and its expression in the parasitized Plutella xylostella

A novel gene was identified in Cotesia plutellae bracovirus (CpBV), which acts as a symbiotic virus of the endoparasitoid wasp C. plutellae. The gene was encoded in the genome of CpBV, but not in the genome of its natural host, Plutella xylostella. The gene has an open reading frame comprised of 94 amino acids. A bioinformatic analysis indicated that the gene encodes a signal peptide comprised of 18 residues at its amino terminus and 4 glycosylation sites at threonine and serine residues, as well as two asparagine residues. The expression of the gene by C. plutellae was specific in parasitized P. xylostella, but not in unparasitized larvae. In parasitized larvae, the expression of the gene occurred on the first day after parasitization, and its expression resumed on the fourth day. The gene showed tissue specific expression in the fat body and epidermis, but not in hemocytes and gut tissue. Its sequence showed some similarity with that of a bacterial toxin, RTX, of Vibrio spp., especially in its actin cross-linking domain. The viral gene has been named CpBV-RTX, and its putative physiological function is discussed in terms of host-parasite molecular interactions.

A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behaviour of the diamondback moth, Plutella xylostella

Histone H4 is highly conserved and forms a central-core nucleosome with H3 in eukaryotic chromatin. Its covalent modification at the protruding N-terminal region from the nucleosomal core can change the chromatin conformation in order to regulate gene expression. A viral H4 was found in the genome of Cotesia plutellae bracovirus (CpBV). The obligate host of the virus is an endoparasitoid wasp, C. plutellae, which parasitizes the diamondback moth, Plutella xylostella, and interrupts host development and immune reactions. CpBV has been regarded as a major source for interrupting the physiological processes during parasitization. CpBV H4 shows high sequence identity with the amino acid sequence of P. xylostella H4 except for an extended N-terminal region (38 aa). This extended N-terminal CpBV H4 contains nine lysine residues. CpBV H4 was expressed in P. xylostella parasitized by C. plutellae. Western blot analysis using a wide-spectrum H4 antibody showed two H4s in parasitized P. xylostella. In parasitized haemocytes, CpBV H4 was detected predominantly in the nucleus and was highly acetylated. The effect of CpBV H4 on haemocytes was analysed by transient expression using a eukaryotic expression vector, which was injected into non-parasitized P. xylostella. Expression of CpBV H4 was confirmed in the transfected P. xylostella by RT-PCR and immunofluorescence assays. Haemocytes of the transfected larvae lost their spreading ability on an extracellular matrix. Inhibition of the cellular immune response by transient expression was reversed by RNA interference using dsRNA of CpBV H4. These results suggest that CpBV H4 plays a critical role in suppressing host immune responses during parasitization.

Differential expression profile of genes encoded in a genome segment of Cotesia plutellae bracovirus in a parasitized host, Plutella xylostella

AbstractThe Polydnaviruses are an insect DNA virus group. The segmented genome of a polydnavirus is located on the host wasp chromosome as a provirus. After replication, virions are delivered into the parasitized host, where their expression products play significant roles in specific processes of parasitism. However, little is known about how viral gene expression is controlled. In the present study, we tested whether different genes in a genome segment are expressed concomitantly. Cotesia plutellae bracovirus (CpBV) is a polydnavirus mutualistic to an endoparasitoid, Cotesia plutellae (Braconidae: Hymenoptera). Its circular genome segments in viral particles were captured by a transposon containing pUC origin sequence and replicated in Escherichia coli. CpBV‐S30 (23.5 kb), one of the large CpBV genome segments, was fully sequenced, and seven open reading frames (ORF) were predicted. We analyzed expression patterns of the seven ORF in the diamondback moth, Plutella xylostella, parasitized by C. plutellae. Transcription analyses indicated that all ORF of CpBV‐S30 in the parasitized P. xylostella were expressed from the first day of parasitization and then expression levels decreased over the period of parasitization, except for CpBV‐H4, which showed an additional expression peak. In terms of tissue specificity in their expression patterns, all ORF were found to be expressed in the fat body, hemocytes and gut of the parasitized host. Promoter sequence analysis showed that all seven ORF had typical promoter elements, including TATA binding sites, but promoter component numbers and kinds varied. These results suggest that genes in a genome segment of CpBV can differentially express in the parasitized host, presumably by their different promoter components.

Transient expression of an EP1-like gene encoded in Cotesia plutellae bracovirus suppresses the hemocyte population in the diamondback moth, Plutella xylostella

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). This on-going research has identified seven EP1-like (ELP) genes in the CpBV genome. A group of ELP genes has been speculated as an immunosuppressant encoded in Cotesia-associated bracoviruses. This study analyzed gene expression of these seven CpBV-ELPs in the parasitized diamondback moth, Plutella xylostella. Of these, six CpBV-ELPs were expressed in P. xylostella parasitized by C. plutellae. However, their expression levels varied in different tissues and parasitization stages. Especially, CpBV-ELP1 showed a persistent and ubiquitous expression pattern in both reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence assays. When nonparasitized P. xylostella was transfected with a recombinant CpBV-ELP1 in a eukaryotic expression vector, CpBV-ELP1 was expressed for at least 3 days and the proteins were detectable in the cytoplasm of hemocytes. The transfected larvae showed significant reduction in total hemocyte numbers, compared with larvae injected with the cloning vector alone. Co-transfection with double-stranded RNA could knock down the expression of CpBV-ELP1 and prevented the reduction of the hemocyte population. This study demonstrates that CpBV-ELP1 plays a physiological role in suppressing host immune response presumably by its hemolytic activity during C. plutellae parasitization.

Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a final multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.

Two Pseudogenes of Cotesia plutellae Bracovirus in Parasitized Diamondback Moth, Plutella xylostella

It has been proposed that two novel genes have been found in Cotesia plutellae bracovirus (CpBV) based on the results of a full genome sequencing project. The putative open reading frame (ORF) of CpBV-DBL1 encodes 644 amino acids with one intron, while that of CpBV-DUF1 does 646 amino acids with four introns. Analyses of Southern hybridization and genomic polymerase chain reaction (PCR) confirmed that both genes were located on CpBV genome in the C. plutellae chromosome. However, Northern hybridization and reverse transcriptase-PCR revealed no specific expression in Plutella xylostella that had been parasitized by C. plutellae. Moreover, no putative promoter/enhancer region was found at the 5′ upstream end of CpBV-DBL1. Additionally, although CpBV-DUF1 contained a putative promoter/enhancer, its ORF was interrupted by another putative ORF in a reverse reading direction. These results indicate that both CpBV-DBL1 and CpBV-DUF1 are pseudogenes in parasitized P. xylostella.

Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: Sequence analysis, expression profile, and a possible biological role in host immunosuppression

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). On this process, several putative open reading frames have been proposed, among which there was a large gene family coding for protein tyrosine phosphatases (PTPs). This study analyzed the deduced amino acid sequences of 14 CpBV-PTPs in terms of conserved domains with other known polydnaviral PTPs and determined their expression patterns in diamondback moth, Plutella xylostella, parasitized by C. plutellae. The analyzed CpBV-PTPs share the common 10 motifs with classical type of PTPs. However, there are variations among CpBV-PTPs in active site sequence and phosphorylation sites. Quantitative real-time polymerase chain reaction (PCR) indicated that most PTPs in the parasitized P. xylostella were expressed from the first day of parasitization and increased the expression levels during parasitization. All 14 PTPs were expressed in both immune-associated tissues of fat body and hemocytes in the parasitized host. During last instar, the PTP enzyme activity of the parasitized P. xylostella was significantly lower than that of the nonparasitized. The reduction of the PTP activity was observed in cytosolic fraction, but not in membrane fraction. The hemocytes of parasitized P. xylostella markedly lost their spreading ability in response to a cytokine (PSP1: plasmatocyte-spreading peptide 1). The functional link between the reduced PTP activity and the suppressed hemocytic behavior was evidenced by the inhibitory effect of sodium orthovanadate (a specific PTP inhibitor) on hemocyte-spreading behavior of nonparasitized P. xylostella. These results suggest that CpBV-PTPs are expressed in the parasitized P. xylostella and affect cellular PTP activity, which may be associated with host immunosuppression.

Structure and Expression Profiles of Two Putative Cotesia plutellae Bracovirus Genes (CpBV-H4 and CpBV-E94a) in Parasitized Plutella xylostella

Endoparasitoid wasp, Cotesia plutellae, parasitizes diamondback moth, Plutella xylostella, and interrupts host development and immune processes. Its obligate symbiotic virus, C. plutellae bracovirus (CpBV), has been regarded as a major source of interrupting the physiological alteration during the parasitization. On the process of genome sequencing of CpBV, two putative genes were detected and homologous to E94 of baculovirus and histone H4 subunit of a nucleosome, respectively. The open reading frame of CpBV-E94a was 642 bp encoding 214 amino acid residues, while that of CpBV-H4 was 423 bp encoding 141 amino acid residues. Both genes were on the same segment of CpBV genome. They were expressed only in the P. xylostella parasitized by C. plutellae in which all three tissues of hemo-cytes, fat body, and gut expressed both genes. Quantitative RT-PCR analyses indicated that their expression patterns in the parasitized host were similar and exhibited bimodal peaks at early and late parasitization periods. We discussed their physiological functions based on the gene structures.

Gene Expression of Cotesiaplutellae Bracovlrus EP1-like Protein (CpBV-ELP1) in Parasitized Diamondback Moth, Plutellae xylostella

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). This on-going research has identified several open reading frames (ORFs) including an EPl-like protein (ELP1). This study was intended to analyze gene expression of CpBV-ELPl in the parasitized diamondback moth, Plutella xylostella. CpBV-ELP genomic DNA contains one intron (778 bp long). Its ORF consists of 726 bp encoding 241 amino acid residues. The hypothetical CpBV-ELPl protein is predicted as 27, 787.83 Da and possesses N-terminal signal peptide plus three potential N-glycosylation sites. Its amino acid sequence exhibits high homology with EP1 genes from C. congregata or C. karyai bracovirus. A reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that CpBV-ELPl was expressed only in P. xylostella parasitized by C. plutellae. The expression levels were measured by real time quantitative RT-PCRs during entire parasitization period at 25 °C culturing temperature. The expression began at the first day of parasitization and increased with the parasitization period. The ORF PCR product was cloned, over-expressed, and molecular weight of the purified protein was about 30 kDa.