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Wednesday, 17 November 2004 - 1:00 PM
1048
The role of plasma phenoloxidase in resistance of Heliothis virescens larvae to baculovirus infection
Kent S. Shelby, shelbyk@missouri.edu and Holly JR. Popham, pophamh@missouri.edu. USDA Agricultural Research Service, Biological Control of Insects Research Laboratory, 1503 S. Providence Rd, Research Park, Columbia, MO
Heliothis virescens larval plasma contains high levels of an antiviral activity against Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) budded virus in vitro. Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect. However it is known that other enzymes which generate antimicrobial reactive oxygen intermediates and reactive nitrogen intermediates are present in hemolymph which could contribute to the observed virucidal activity. To elucidate the contributions of phenoloxidase and other candidate activities to plasma innate immune response against baculovirus infection we have used specific metabolic inhibitors. In vitro the general inhibitors of melanization (N-acetyl cysteine, ascorbate and glutathione), and specific inhibitors of phenoloxidase (phenylthiourea, Kojic acid and Sodium benzoate), completely blocked virucidal activity up to the level seen in controls. Addition of the enzyme catalase to plasma did not affect virucidal activity; however addition of superoxide dismutase exhibited a modest inhibitory effect. Pull-down assays in which plasma phenoloxidase was depleted from samples using increasing concentrations of rabbit polyclonal anti-PO antisera reduced virucidal activity in a concentration dependent manner. Immunoblots confirm that phenoloxidase was removed from plasma by this treatment. Inhibitors of Nitric oxide synthase activity did not affect virucidal activity. Our results confirm that phenoloxidase is the predominate activity in larval plasma accounting for inactivation of HzSNPV in vitro, and that phenoloxidase-dependent superoxide production may contribute to this virucidal activity.
Species 1: Lepidoptera Noctuidae Heliothis virescens (tobacco budworm)
Keywords: immunity, reactive oxygen
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