2009 Annual Report
1a.Objectives (from AD-416)
Genetically engineer specific fluorescent protein genes into baculoviruses that will be expressed in the envelopes of budded virus and occlusion bodies to serve as barriers against ultraviolet inactivation. Incorporate specific proteins into polyhedra (occlusion bodies) of baculoviruses by a genetic fusion process that would lead to early mortality or inhibit feeding of pest insects. Examine the basis of baculovirus resistance at the cellular level employing cell culture methodology in order to better understand the nature of baculovirus host range.
1b.Approach (from AD-416)
This proposal employs a molecular approach whereby baculoviruses will be stabilized against inactivation by ultraviolet light (UV) as well as to make them faster acting. To achieve these goals, several fluorescent protein genes will be engineered into baculovirus genomes which when expressed in the envelopes of the viral particles and occlusion bodies will provide protection against inactivation by UV light. This methodology is designed to reduce damage to the viral DNA of baculoviruses. To make the bacuoviruses faster acting, specific proteins will be incorporated into occlusion bodies of baculoviruses by a genetic fusion process that will lead to early mortality or inhibit feeding by pest insects since viral replication in the host is not a pre requisite for elaboration of the active proteins. This research project will also examine the nature of baculovirus resistance at the cellular level employing insect cell lines that are refractile to baculoviruses. Recombinants with fluorescent markers under various promoters will elucidate where the block occurs and thus provide knowledge that could be valuable in understanding viral host range.
Baculoviruses are used as biocontrol agents of pest insects. While agriculturally useful, BVs have several drawbacks such as slow actions and narrow host ranges. These issues must be addressed before widespread use can be achieved. In an effort to understand the viral host range we identified proteins that can potentially influence host range. Viral infection led to major shifts in protein expression in insect cells exposed to permissive infection as well as semi-permissive infection. Of the 24 proteins with altered expression in the permissive infection, 21 were either completely absent or present at extremely low expression levels in the infected cells compared to control cells. The other three proteins increased in expression levels by 2- to 4.3-fold relative to controls. In the semi-permissive system, changes in protein expression were similar to results with the permissive system. In contrast, the number and expression levels of identified cell proteins exposed to non-permissive infection after 24 h showed little change from controls. Each of the proteins identified in this study will be examined further to find how their expression impacts infection. Additionally, we are developing several baculoviruses, each with a gene for fluorescence to track the effectiveness of infection both in cell culture and in whole insects. We will determine how well the fluorescence gene protects the virus from UV degradation. Building on the fundamental knowledge of the host range work, the celery looper virus collected from the field was screened to find the most virulent isolates. The virulent isolates were analyzed to study the areas of the genome with increased variability and to relate this information to the host range studies. The most virulent and stable isolates will be tested for potential field use. We also reported that prostaglandins and related biochemical signals mediate insect cellular immune reactions to baculovirus infections.
Determined proteins potentially involved in the block of baculovirus replication in lepidopteran cell lines. Identifying how an insect blocks a baculovirus infection is important in developing tools to undermine the insect immune system for greater control of specific pest insects. By isolating proteins, we discovered proteins that were either present in greater or less abundance when insect cells were infected with baculovirus. We identified these proteins and this information can be used to identify how the insect host range of a baculovirus can be expanded to control a wider range of pest insects. Expanding the host range will allow baculoviruses to be used as biopesticides in a wider array of crops.
Mcintosh, A.H., Grasela, J.J. 2009. Establishment of a Monarch Butterfly (Danaus plexippus, Lepidoptera: Danaidae) Cell Line and its Susceptibility to Insect Viruses. Applied Entomology and Zoology. 44(2):331-336.
Smagghe, G., Goodman, C.L., Stanley, D.W. 2009. Insect Cell Culture and Applications to Research and Pest Management. In Vitro Cellular and Developmental Biology - Animals. 45:93-105.
Mcintosh, A.H., Grasela, J.J. 2009. Early Detection of Baculovirus Expression and Infection in Lepidopteran Larvae Fed Occlusion Bodies of an AcMNPV Recombinant Carrying a Red Fluorescent Protein Gene. In: Halley, G.T., Fridian, Y.T., editors. Environmental Impact Assessments. Hauppauge, NY: Nova Science Publications. p. 457-467.