2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? A goal of sustainable agricultural practices and integrated pest management (IPM) strategies is the minimization of the use of classical chemical insecticides as a means of crop protection because of their lack of specificity to pest insects and their negative impacts on human health, non-target insects and the environment. In addition, pest insects have often developed resistance to these chemicals. As a result, appropriate alternatives are needed. Research conducted under this project is focused on finding and developing alternative pest management tools, specifically to develop naturally-occurring baculoviruses of pest insects into safe, environmentally stable and effective products. The goal of this research is the improvement of these pesticidal baculoviruses so that they may be more efficacious in the control of agronomically important insects. Baculoviruses are effective biological control agents for the control of pest insects, but are not as fast-acting as conventional chemical insecticides, usually possess a limited host range, and do not persist well in the environment (mainly because of inactivation by ultraviolet light). The present research project addresses some of these limitations of baculoviruses by focusing on:. 1)the development of baculoviruses that will be more resistant to ultraviolet light in the environment, . 2)the development of faster acting baculoviruses and. 3)the determination of the resistance to baculoviruses at the cellular level for the expansion of the host range of baculoviruses. Insect cell lines are an essential component of this research because they can be used for quantitation and for the propagation of baculoviruses, in their quantitation as well as for molecular biological procedures. In addition, such cell lines can be readily preserved in liquid nitrogen for future use. The targets of our baculoviral research are several lepidopteran pests including the cotton bollworm/budworm complex and the diamondback moth which attack numerous types of crops and vegetables on a world-wide scale. Therefore, management of these and other pest insects via biological control instead of chemical control can aid in the reduction of insect damage to crops while minimizing the health and/or environmental risks associated with conventional chemical insecticides. The world-wide bollworm/budworm pest-complex attacks more than 30 food and fiber crops. Many geographical populations of these pests are resistant or are becoming resistant to currently used synthetic chemical insecticides. In addition, the chemical insecticides now in use are being phased out by regulations, and their replacement by less toxic insecticides is limited by the increased cost of development. In the USA these insect pests cause more than a billion dollars damage each year, with another 1/4 billion dollars spent each year for control. Concern over environmental pollution, quality of ground water, and increased resistance of pest insects to insecticides has further intensified the need for research directed toward the use of natural, safe, effective biological control products. The development and availability of novel baculoviral products to manage this insect pest complex may eliminate many of the problems and concerns associated with the use of chemical insecticides. Baculoviral products for the control of the cotton bollworm/budworm complex are attractive viable alternatives to chemical insecticides. They are highly specific, effective, non-polluting and have not induced stable resistance development in field insect pests. It is essential to develop alternative methods to replace or reduce the use of chemical insecticides for the control of insect pests to prevent increased crop losses and increases in the cost of production. Baculovirus isolates with faster killing properties and improved persistence in the environment, which can be readily produced in cell culture, are urgently needed. The diamondback moth is also a worldwide pest of many vegetable crops costing billion of dollars attributed to economic losses and control measures. A new baculovirus isolated at this laboratory several years ago has proven to be most effective of the available baculoviruses against the diamondback moth. This work is very relevant to the agricultural industry relating to both field and vegetable crops. 2.List the milestones (indicators of progress) from your Project Plan. Objective 1. Development of baculoviruses that will be more resistant to ultraviolet light in the environment. Year 1 (2005) Design and evaluate primers for the engineering of fluorescent protein genes into baculoviruses. Conduct PCR to amplify selected fluorescent genes of interest. Perform gel electrophoresis of PCR products. Isolate and purify PCR products of relevant DNA fragments from gels. Year 2 (2006) Co-transfection of isolated DNA fragments comprising selected fluorescent genes and wild-type DNA in permissive insect cells. Isolate and purify recombinants expressing fluorescent proteins in the envelopes (calyx) of baculoviruses. Confirm the correct localization of the selected genes by DNA sequencing. Year 3 (2007) Propagate baculovirus recombinants in cell culture and larval hosts. Isolate and purify by sucrose gradient ultracentrifugation recombinant occlusion bodies (OB). Enumerate OB by fluorescent light microscopy and perform photomicroscopy on samples. Year 4 (2008) Perform bioassays of recombinants against several susceptible larval species. Analyze data and calculate LC50 values of various recombinants against susceptible larval species. Year 5 (2009) Subject recombinant occlusion bodies to ultraviolet (UV) light under laboratory conditions. Perform bioassays (LC50) on recombinants exposed to UV light to determine their resistance or susceptibility to UV inactivation. Conduct studies of selected recombinants (occlusion bodies) exposed to environmental UV light on leaf surfaces and calculate LC50 values. Objective 2. Development of faster acting baculoviruses. Year 1 (2005) Select potential genes for engineering into baculoviruses to improve rapidity of kill or interfere with feeding e.g. proteases, chitinases. Bacillus thuringiensis (Bt) toxin. Employ a fusion method, developed in this lab, to locate the gene of choice linked to the polyhedrin gene so that when expressed it will be localized in OB protein. Since the candidate protein will be localized in OB it will not depend on viral replication to be effective. Year 2 (2006) Isolate and purify recombinants employing cell culture methodology. Propagate recombinants in cell culture and in larval hosts for production of OB. Purify OB from selected recombinants on sucrose gradients by ultracentrifugation. Year 3 (2007) Standardize recombinant samples (occlusion body like particles) by protein determinations. Perform bioassays against susceptible larval hosts to determine LT50 and LC50 values. Year 4 (2008) Analyze data and select the most effective recombinants. Employing the methodology in Objective 1 produce recombinants that will have the properties of UV light resistance and fast acting activity against target insect pests. Year 5 (2009) Propagate selected recombinants in cell culture and larval hosts. Isolate OB or OB-like particles by sucrose gradient utilizing ultracentrifugation. Test the new recombinants for resistance to UV light inactivation. Conduct bioassay studies on susceptible larval hosts to evaluate the effectiveness of the new recombinants. Objective #3. Determination of the resistance to baculoviruses at the cellular level for the expansion of host range. Year #1 (2005) Select insect cell lines from representative Orders of Lepidoptera, Coleoptera, Diptera and Homoptera. Recover selected insect cell lines from BCIRL's liquid nitrogen repository cell bank and propagate in culture. Year #2 (2006) Identify by PCR techniques routinely used in this lab the authenticity of selected insect cell lines. Propagate selected baculoviruses in insect cell lines and determine their infectivities by TCID50 (tissue culture infective dose fifty). Prepare virus stocks to be used in experiments. Year #3 (2007) Inoculate selected cell lines from various Orders and determine by TCID50 whether or not replication has occurred. Observe inoculated cell lines using UV fluorescent microscopy at various time intervals post-inoculation and quantitatively record number of cells depicting fluorescence. Analyze infected cell lysates by SDS-PAGE to determine the protein profile following infection. Identify useful marker proteins to follow to study viral progression. Run RENs on virus samples to establish the identity of viruses recovered from inoculated cell lines. Year #4 (2008) Analyze marker proteins to study progression of infection in select permissive, non-permissive and semi-permissive cell lines either by PCR, western blot, or SDS-PAGE. Produce additional recombinants with fluorescent protein genes under early and late promoters as needed. Year #5 (2009) Determine where the block in viral replication took place in non-permissive cell lines by analyzing marker protein or gene transcript expression. Design experiments to overcome block in viral replication. For example if the block is due to lack of viral penetration into the cells, transfection procedures could be used whereby the baculovirus DNA is introduced directly into cells and then observed for expression of the marker gene as well as for viral replication. 4a.What was the single most significant accomplishment this past year? A New Baculovirus Isolate Associated with an Insect Parasite The discovery and isolation of a new baculovirus associated with an insect parasite. This virus has been shown to be highly infectious for several lepidopteran pests and is being tested against several other pest species. Restriction endonuclease (REN) gel profiles has shown it to be different from other baculovirus isolates currently used in this laboratory. In collaboration with CRIS 3622-22000-027 this newly discovered virus has the potential for usage as a biological control agent against other lepidopteran pests of agricultural importance. These studies are a continuation of and complement previous studies under CRIS 3622-22000-022 which ended in April 2004 and was replaced by an interim CRIS 3622-22000-028 to produce more efficacious baculoviruses with the properties of UV light resistance, faster acting and determination of resistance to baculoviruses on a cellular basis. 4b.List other significant accomplishments, if any. Susceptibility of Insect Cell Lines from Various Orders to AcMNPV Insect cell lines from Arthropoda represented by Lepidoptera (e.g. loopers), Coleoptera ( e.g.beetles), Diptera (e.g. mosquitoes) and Homoptera (e.g. leafhoppers) were evaluated for their ability to support replication of AcMNPV. Of the 10 lepdopteran cell lines tested, only 3 cell lines failed to support replication of the virus whereas none of the cell lines from the 3 other orders replicated AcMNPV. However when employing AcMNPV carrying a red fluorescent protein (RFP) gene as a marker, 2 of the Dipteran cell lines as well as 2 of the nonpermissive cell lines from Lepidoptera expressed the RFP. No expression was observed in any of the beetle or leafhopper cell lines. These systems provide a means for studying virus resistance at the cellular level. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This research was initiated to test the resistance of two baculovirus recombinants carrying fluorescent protein genes to inactivation by ultraviolet light. Occlusion bodies of the two recombinants containing viral particles were exposed to ultraviolet light for a prolonged period. One recombinant contained the green fluorescent protein (GFP) in the viral envelope and the other recombinant contained a red fluorescent protein (RFP) in the occlusion body. The research was conducted at the Biological Control of Insects Research Laboratory (BCIRL) in Columbia, MO, and collaborators were L. Lua, University of Queensland, Brisbane, Australia and S. Braunagel, Texas A & M University, College Station, Texas. It was demonstrated that the recombinants were 25 to 100 fold more resistant to ultraviolet light inactivation compared with the parental wild-type virus (non-engineered) and results of this study are in press (Journal of Insect Science). This finding has a far reaching impact to provide a means whereby biological control agents can be expected to persist in the environment for longer periods of time thereby resulting in better control of insect pest populations. These findings that have been demonstrated in the above accomplishments 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." 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Insect cell lines, baculoviruses and cell culture and virological methods have been provided to other scientists in the field. Information has been provided to a commercial company interested in using the diamondback moth baculovirus, patented by ARS, for the control of this world wide insect pest that attacks crucifers. The science and technology becomes available to the end-user upon completion of the proposed project via publications as well as contacts with industry. There should be no constraints other than potential information and products that might require patents which usually are involved and take a long time to process. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None.