2006 Annual Report
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:.
Insect cell lines are an essential component of this research because they can be used for quantitation and for the propagation of baculoviruses as well as in 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 (DBM) 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, including the DBM are resistant to 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 (DBM) 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 the most effective of the available baculoviruses against the DBM. This work is very relevant to the agricultural industry relating to both field and vegetable crops.
1. Design and evaluate primers for the engineering of fluorescent protein genes into baculoviruses.
2. Conduct PCR to amplify selected fluorescent genes of interest.
3. Perform gel electrophoresis of PCR products.
4. Isolate and purify PCR products of relevant DNA fragments from gels.
5. Select potential genes for engineering into baculoviruses to improve rapidity of kill or interfere with feeding e.g. proteases, chitinases, Bacillus thuringiensis (Bt) toxin.
6. 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.
7. Select insect cell lines from representative Orders of Lepidoptera, Coleoptera, Diptera and Homoptera.
8. Recover selected insect cell lines from BCIRL’s liquid nitrogen repository cell bank and propagate in culture.
Year 2 (2006)
1. Co-transfection of isolated DNA fragments comprising selected fluorescent genes and wild-type DNA in permissive insect cells.
2. Isolate and purify recombinants expressing fluorescent proteins in the envelopes (calyx) of baculoviruses.
3. Confirm the correct localization of the selected genes by DNA sequencing.
4. Isolate and purify recombinants employing cell culture methodology.
5. Propagate recombinants in cell culture and in larval hosts for production of OB.
6. Purify OB from selected recombinants on sucrose gradients by ultracentrifugation.
7. Identify by PCR techniques routinely used in this lab the authenticity of selected insect cell lines.
8. Propagate selected baculoviruses in insect cell lines and determine their infectivities by TCID50 (tissue culture infective dose fifty).
9. Prepare virus stocks to be used in experiments.
Year 3 (2007)
1. Propagate baculovirus recombinants in cell culture and larval hosts.
2. Isolate and purify by sucrose gradient ultracentrifugation recombinant occlusion bodies (OB).
3. Enumerate OB by fluorescent light microscopy and perform photomicroscopy on samples.
4. Standardize recombinant samples (occlusion body like particles) by protein determinations.
5. Perform bioassays against susceptible larval hosts to determine LT50 and LC50 values.
6. Inoculate selected cell lines from various Orders and determine by TCID50 whether or not replication has occurred.
7. Observe inoculated cell lines using UV fluorescent microscopy at various time intervals post-inoculation and quantitatively record number of cells depicting fluorescence.
8. Determine the nature of the viral block in non-permissive cells following inoculation with a baculovirus.
9. Run RENs on virus samples to establish the identity of viruses recovered from inoculated cell lines.
Year 4 (2008)
1. Perform bioassays of recombinants against several susceptible larval species.
2. Analyze data and calculate LC50 values of various recombinants against susceptible larval species.
3. Analyze data and select the most effective recombinants.
4. 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.
5. 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.
6. Produce additional recombinants with fluorescent protein genes under early and late promoters as needed.
Year 5 (2009)
1. Subject recombinant occlusion bodies to ultraviolet (UV) light under laboratory conditions.
2. Perform bioassays (LC50) on recombinants exposed to UV light to determine their resistance or susceptibility to UV inactivation.
3. Conduct studies of selected recombinants (occlusion bodies) exposed to environmental UV light on leaf surfaces and calculate LC50 values.
4. Propagate selected recombinants in cell culture and larval hosts.
5. Isolate OB or OB-like particles by sucrose gradient utilizing ultracentrifugation.
6. Test the new recombinants for resistance to UV light inactivation.
7. Conduct bioassay studies on susceptible larval hosts to evaluate the effectiveness of the new recombinants.
8. Determine where the block in viral replication took place in non-permissive cell lines by analyzing marker protein or gene transcript expression.
9. 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.
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 the baculovirus Autographia california multiple nuclear polyhedrosis virus (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 and restrictions imposed on baculovirus replication by the cell. User of this research: mainly scientists in this field of research. Problem solved: gene expression occurs in some cell lines that other wise do not permit replication of the virus. Regulatory/policy development/commercialization: None These 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."
Information has been provided to a company interested in commercializing the diamondback moth (DBM) baculovirus, (US Patent 6,042,843) for the control of DBM, a world wide insect pest that attacks crucifers, as well as other lepidopteran pests of agricultural importance. Several other commercial companies have also expressed interest in acquiring the DBM baculovirus for commercial production. 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.
Goodman, C.L., Phipps, S.J., Wagner, R.M., Peters, P., Wright Osment, M.M., Nabli, H., Saathoff, S.G., Vickers, B.V., Grasela, J.J., Mcintosh, A.H. 2005. Growth and development of the knapweed root weevil, Cyphocleonus achates, on a meridic larval diet. Biological Control. 36:238-246.