• Korean journal of applied entomology
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• v.56 no.1
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• pp.19-28
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• 2017

Polydnaviruses (PDVs) are a group of double-stranded DNA viruses symbiotic to some endoparasitoid wasps. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to an endoparasitoid wasp, C. plutellae, parasitizing young larvae of Plutella xylostella. An early expressed gene, CpBV-ELP1, plays an important role in the parasitism by suppressing host cellular immunity by its cytotoxic activity against hemocytes. This study aimed to test its oral toxicity against insect pest by expressing it in a recombinant tobacco plant. A recombinant CpBV-ELP1 protein was produced using a baculovirus expression system and secreted to cell culture medium. The cell cultured media were used to purify CpBV-ELP1 by a sequential array of purification steps: ammonium sulfate fractionation, size exclusion chromatography, and ion exchange chromatography. Purified rCpBV-ELP1 exhibited a significant cytotoxicity against Spodoptera exigua hemocytes. CpBV-ELP1 was highly toxic to the fifth instar larvae of S. exigua by injection to hemocoel. It also showed a significant oral toxicity to fifth instar larvae of S. exigua by a leaf-dipping assay. CpBV-ELP1 was cloned into pBI121 vector under CaMV 35S promoter with opaline synthase terminator. Resulting recombinant vector (pBI121-ELP1) was used to transform Agrobacterium tumefaciens LBA4404. The recombinant bacteria were then used to induce callus of a tobacco (Nicotiana tabacum Xanthi) leaves and subsequent generation (T1) plants were selected. T1 generation tobacco plants expressing CpBV-ELP1 gave significant insecticidal activities against S. exigua larvae. These results suggest that CpBV-ELP1 gene can be used to control insect pests by constructing transgenic crops.

• Korean journal of applied entomology
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• v.43 no.3 s.136
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• pp.217-223
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• 2004

Polydnavirus is a symbiotic virus of some endoparasitic wasps and plays crucial roles in inhibiting immune responses and retarding development of the parasitized hosts. Cotesia plutellae bracovirus (CpBV) is a polydnavirus suggesting a major causative to change developmental physiology of the parasitized host. Here, we investigated whether CpBV can interrupt development of non-natural host. Beet armyworm, Spodoptera exigua, is used as a non-permissible host for parasitization of C. plutellae. Extract from the calyx region of C. plutellae contained CpBV, which was confirmed by immunoblotting with a polyclonal antibody raised against CpBV. One female equivalent of CpBV extract was injected into hemocoel of late 4th instar larvae of S. exigua. The injected larvae showed delayed larval period, decrease of body weight gain, and inability of pupal metamorphosis. These inhibitory effect of the CpBV extract was rescued by injection along with CpBV antibody, though the antibody itself did not give any effect on development of the larvae. This result clearly shows that CpBV can interrupt developmental physiology of a non-natural host for its symbiotic wasp.

• BMB Reports
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• v.43 no.4
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• pp.279-283
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• 2010

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 CpBV-SRP1. 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.

• Journal of Microbiology and Biotechnology
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• v.19 no.6
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• pp.610-615
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• 2009

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.

• Korean journal of applied entomology
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• v.53 no.4
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• pp.331-338
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• 2014

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

• Korean journal of applied entomology
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• v.45 no.1 s.142
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• pp.15-24
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• 2006

Inhibitor kB (IkB)-like gene has been found in the genome of Cotesia plutellae bracovirus (CpBV), which is the obligatory symbiont of an endoparsitoid wasp, C. plutellae. The open reading frame of CpBV-IkB was 417 bp and encoded 138 amino acids. Four ankyrin repeat domains were found in CpBV-IkB, which shared high homology with other known polydnavirus IkBs. Considering a presumptive cellular IkB based on Drosophila Cactus, CpBV-IkB exhibited a truncated structure with deletion of signal-receiving domains, which suggested its irreversible inhibitory role in NFkB signal transduction pathway of the parasitized host in response to the wasp parasitization. CpBV-IkB was expressed only in the parasitized diamondback moth, Plutella flostella. Its expression was estimated by quantitative RT-PCR during parasitization period, showing a constitutive expression pattern from the first day of parasitization. An indirect functional analysis of CpBV-IkB was conducted and suggested a hypothesis of host antivirus inhibition.

• The Korean Journal of Pesticide Science
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• v.19 no.3
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• pp.279-286
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• 2015

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. Several CpBV genes play important roles in suppressing immune responses of the parasitized larvae. This study tested a hypothesis that the CpBV genes inducing host immunosuppression could be applied to develop a potent recombinant baculovirus. Based on a previous study, a recombinant baculovirus expressing CpBV-ELP1 (AcMNPV-ELP1) was selected and multiplied using larvae of the beet armyworm, Spodoptera exigua. The recombinant viruses were produced in a yield of $5{\times}10^{10}$ polyhedral inclusion body (PIB)/larva. The cultured AcMNPV-ELP1 exhibited a much higher pathogenicity against S. exigua larvae. However, its insecticidal activity was varied among larval instars of S. exigua, in which first and late instars were high susceptible. Spray of the recombinant baculovirus ($5{\times}10^6PIB/mL$) exhibited higher control efficacy (${\approx}$ 88%) against S. exigua larvae infesting cabbage than a chemical insecticide, tebufenozide, at 7 days after treatment. These results indicate that AcMNPV-ELP1 mass-cultured using host insect system is highly pathogenic and can be applied to develop a novel microbial control agent.

• The Korean Journal of Pesticide Science
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• v.12 no.3
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• pp.283-290
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• 2008

Baculoviruses have been used to control some serious lepidopteran pests. However, their narrow target insect spectrum and slow efficacy are main limitations to be used in various applications. This study introduces a technique to overcome these limitations by inhibiting insect immune defence to enhance the viral pathogenicity. Polydnaviruses are an insect DNA virus group and symbiotic to some ichneumonid and braconid endoparasitoids. Cotesia plutellae bracovirus (CpBV) is a braconid polydnavirus and encodes several immunosuppressive genes. We selected seven CpBV genes and recombined them to wild type Autographa California multiple nucleopolyhedrovirus (AcNPV). A bioassay of these seven recombinants indicated that most recombinants had similar or superior efficacy to wild type AcNPV against beet armyworm, Spodoptera exigua, and diamondback moth, Plutella xylostella. Recombinant AcNPV with CpBV-ELP was the most potent in terms of lethal time by shortening more than 2 days compared to wild type AcNPV. This recombinant was further proved in its dose-dependent pathogenicity and its efficacy by spray application on S. exigua infesting cabbage cultivated in pots. We discussed the efficacy of CpBV-ELP recombinant AcNPV in terms of suppressing antiviral activity of target insects.

• BMB Reports
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• v.44 no.6
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• pp.393-398
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• 2011

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.

• YAKHAK HOEJI
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• v.48 no.2
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• pp.111-116
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• 2004

The ethanol extracts of mixed vegetables (Bioactive Vegetables, BV), mixed fruits (Bioactive Fruits, BF) and their liquid formulation (Chungpae Plus , CP) were evaluated for their antioxidative and antigenotoxic activities. They were shown to possess the significant free radical scavenging effect against 1,1-diphenyl-2-picryl hydrazine (DPPH) radical generation and were revealed to show the inhibitory effect of lipid peroxidation as measured by malondialdehyde (MDA) formation although the potencies were not higher than those of vitamin C. They did not possess any pro-oxidant effect on bleomycin-Fe(III) dependent DNA degradation, whereas vitamin C showed strong pro-oxidant effect. Furthermore, oral administration of BV and BF inhibited micronucleated reticulocyte (MNRET) formation of mouse peripheral blood induced by KBrO3 treatment in vivo. CP also showed significant inhibition under same experimental condition. Therefore, the liquid formulation (CP) containing BV and BF may be a useful natural antioxidative and antigenotoxic agent by scavenging free radicals, inhibition of lipid peroxidation and protecting chromosomal damage.