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 and antimicrobial responses. 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. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

The diamondback moth, Plutella xylostella, is reluctant to a baculovirus, Autographa california nucleopolyhedrosis virus (AcNPV) at its oral administration. However, parasitization by an endoparasitoid wasp, Cotesia plutellae, enhances the viral susceptibility. This study analyzed an antiviral activity of P. xylostella in response to the viral infection and determined the parasitic factor inhibiting the antiviral mechanism. For the analysis of antiviral activity of P. xylostella, a recombinant AcNPV expressing enhanced green fluorescence (AcNPV-EGFP) was orally adminstered to lavae of P. xylostella. After 24 h, EGFP expression was observed in the midgut tissue at a confocal-FITC mode. At the same time, a characteristic midgut melanotic response (MMR) was observed in some midgut regions under a phase contrast microscope. Thereafter, the EGFP signal was attenuated, while MMR spread on most midgut region. When the MMR was scored from 0 to 5 by the intensity of melanized cell density, it increased in time- and dose-dependent manners at the viral administration per os. These results suggest that the MMR is an antiviral activity of P. xylostella. This antiviral activity was significantly attenuated by C. plutellae parasitism. The parasitized P. xylostella showed significant decrease in the MMR score compared to nonparasitized larvae when they were orally administered with the same dose of AcNPV. To determine the parasitic factor(s) inhibiting the antiviral activity from the symbiotic polydnavirus of C. plutellae (C. plutellae bracovirus: CpBV), CpBV-IkB, which is a viral homolog of NFkB inhibitor and has been considered as an antiviral factor as in other polydnaviruses, was tested. A recombinant AcNPV expressing CpBV-IkB (AcNPV-IkB) was constructed and administered to P. xylostella larvae. As expected, AcNPV-IkB significantly decreased the antiviral activity measured by the MMR score compared to AcNPV-EGFP treatment. This study suggests that CpBV-IkB plays an antiviral parasitic role in the molecular interactions between P. xylostella and C. plutellae.

Inhibitor <SUB>K</SUB>B (I<SUB>K</SUB>B)-like gene has been found ill the genome of Cotesia plutellae bracovirus (CpBV), which is the obligatory symbiont of an endoparsitoid wasp, C. plutellae. The open reading frame of CpBV-I<SUB>K</SUB>B was 417 bp and encoded 138 amino acids. Four ankyrin repeat domains were found in CpBV-I<SUB>K</SUB>B, which shared high homology with other known polydnavirus I<SUB>K</SUB>Bs. Considering a presumptive cellular I<SUB>K</SUB>B based on Drosophila Cactus, CpBV-I<SUB>K</SUB>B exhibited a truncated structure with deletion of signal-receiving domains, which suggested its irreversible inhibitory role in NF<SUB>K</SUB>B signal transduction pathway of the parasitized host in response to the wasp parasitization. CpBV-I<SUB>K</SUB>B was expressed only in the parasitized diamondback moth, Plutella xylostella. 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-I<SUB>K</SUB>B was conducted and suggested a hypothesis of host antivirus inhibition.