2008 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.
The objective of this study was to determine whether or not recombinant baculoviruses carrying fluorescent protein genes were more resistant to UVB as compared with their parental wild-type viruses. Natural sunlight which includes UVB (280-320nm) is very detrimental to microbials in the environment including baculoviruses which are used as biological control agents for major insect pests of agricultural importance. Baculovirus recombinants were produced by modification of their genomes to accept green, blue and red fluorescent protein genes (FPG) so that when these genes are expressed the fluorescent proteins become localized in the envelopes surrounding the viral particles or the occlusion bodies (OB) which contain viral particles. OB at a concentration of 1 x 10**7/ml were exposed to UVB light from an Atlas Suntest instrument for 4h to give a total delivery of 7100 kj/mm**2 which is several times the intensity compared with that of natural sunlight. After exposure the OB were fed to 24h T. ni larvae and lethal concentration fifty (LC50) values calculated. The results show that some recombinants gave up to 7X better protection against UVB than did the wild-type parent. We examined the nature of virus resistance at the cellular level by employing cell lines that were both resistant to the virus as well as susceptible. Susceptible cell lines were inoculated with 2 different baculoviruses and at certain intervals following inoculation the samples were processed and run on 2D-gel electrophoresis to determine protein expression. Results showed that a large number of proteins that were expressed in uninoculated (controls) cells were dramatically suppressed in the virus challenged cultures signifying that the virus utilized the cell machinery to produce viral components necessary for virus replication and production. Similar experiments with a non-permissive cell line are under study. The nature of virus resistance is an important entity in biological control as it could explain why some viruses are capable of infecting certain species of insects and not others.
The findings of the investigations are in concert with and supportive of goals set forth in the National Program Action Plan 304 and as such is part of the program component for Development of new and Improved Pest Control Technologies.
Establishment of a monarch butterfly cell line. Although there are many insect cell lines established from insect pests there are very few from nontarget species such as the monarch butterfly. This study describes only the second cell line established from the monarch. A cell line was successfully established from adult monarch butterflies and was passaged continuously in culture and preserved in liquid nitrogen. It was shown to be susceptible to some insect viruses but not to all. Its identity was confirmed by DNA amplification fingerprinting and the polymerase chain reaction (DAF-PCR).
This cell line will enable the research community to investigate a host of factors such as toxins and viruses that might affect this much valued nontarget insect that is highly revered not only in our society but worldwide. Specialized tests employing this cell line will be highly beneficial and thus avoid the sacrifice of the living host.
The findings demonstrated in the above accomplishment are consistent with and supportive of goals set out in the National Program Action Plan 304 and as such is part of the program component for Development of New and Improved Pest Control Technologies, "particularly biologically-based methods."
5.Significant Activities that Support Special Target Populations
|Number of the New MTAs (providing only)||3|
Mcintosh, A.H., Grasela, J.J., Long, S.H., Shelby, K. 2008. Isolation and characterization of a baculovirus associated with the insect parasite Cotesia marginiventris. Journal of Insect Science. 8:1-19.