Submitted to: Journal of Insect Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/1/2005
Publication Date: 8/4/2006
Citation: Shelby, K., Popham, H.J. 2006. Plasma phenoloxidase of the larval tobacco budworm, Heliothis virescens, is virucidal. Journal of Insect Science. 6:16. Interpretive Summary: Baculoviruses are a group of viruses that infect insects and have great potential for the safe control of caterpillars that infest major crops. When a caterpillar feeds on baculovirus particles, it becomes ill to a point that it cannot eat, and dies within a few days. Information on how an insect fights off infection is useful so that additional methods can be developed to ultimately replace chemical pesticides with baculoviruses in the field. Previously, we had found a factor in caterpillar blood that reduced the viral infection by 64 times. In this study, we have identified this factor as phenoloxidase by a series of combinations of caterpillar blood and chemical compounds, and speculate on how this enzyme functions in the blood to block virus infection. This finding will impact scientists working on how an insect resists infection because it begins to explain, along with other studies in the field, why some infections are fatal while others are not and how viruses can be made more useful as a control measure in the field.
Technical Abstract: 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, and Kojic acid), 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. 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.