Use of viral proteins for controlling the cotton boll weevil and other insect pests

A protein extract from Chilo iridescent virus or the whole virus controls insects, particularly the cotton boll weevil and the cotton aphid, respectively. These compositions/agents may be used to directly control insects or genes for active proteins may be cloned into vectors for transformation of plants or plant colonizing microorganism, thereby providing a method for controlling insect infestation.

FIELD OF THE INVENTION
 This invention relates to a method of controlling insects, including
 particularly weevils, cotton boll weevils etc. by use of a viral protein
 extract which may be applied directly to the boll weevil or larva or
 cotton plants. In another aspect, the invention relates to an isolated
 gene from the viral toxin which can be transferred to plant crops such as
 cotton so that toxin will be produced in such engineered plants.
 BACKGROUND OF THE INVENTION
 The use of natural products, including proteins, is a well-known method of
 controlling many insect pests. Endotoxins of Bacillus thruringiensis
 (B.t.) are used to control both lepidopteran and coleopteran insect pests.
 Genes producing the B.t. toxin have been introduced and expressed in
 several plants, including cotton, tomato, and tobacco, and have also been
 expressed by various microorganisms. However, there are several
 economically important insect pests that are not susceptible to
 B.t.endotoxins, and this group includes the cotton boll weevil.
 Researchers at the Monsanto Co. have identified a bacterial enzyme
 (cholesterol oxidase) that induces mortality and stunting in boll weevil
 larvae and in several lepidopteran species. These workers have isolated
 the gene for this enzyme and expressed it in plant-colonizing bacteria and
 in cotton tissue culture. Experience with B.t. toxins suggests that
 development of resistance will be a problem with use of protein toxins for
 insect control, and a number of approaches have been recommended to
 minimize this. These include the use of refugia, dosage control, and use
 of multiple toxins, etc. An important strategy against the development of
 resistance will be the identification of alternate toxins that have a
 different mode of action. This approach will allow use of lower doses for
 all toxins and will minimize the probability of mutations that result in
 resistance to two or more toxins.
 There are, however, several economically important insect pests that are
 not susceptible to B.t. endotoxins. One such important pest is the cotton
 boll weevil. There is also a need for additional proteins which control
 insects for which B.t. or other toxins provides control in order to manage
 any development of resistance in the population.
 Interest in the biological control of insect pests has arisen as a result
 of disadvantages of conventional chemical pesticides. Chemical pesticides
 generally affect beneficial as well as nonbeneficial species. Insect pests
 tend to acquire resistance to such chemicals so that new insect pest
 populations can rapidly develop that are resistant to these pesticides.
 Furthermore, chemical residues pose environmental hazards and possible
 health concerns. Biological control presents an alternative means of pest
 control which can reduce dependence on chemical pesticides.
 The primary strategies for biological control include the deployment of
 naturally-occurring organisms which are pathogenic to insects
 (entomopathogens) and the development of crops that are more resistant to
 insect pests. Approaches include the identification and characterization
 of insect genes or gene products which may serve as suitable targets for
 insect control agents, the identification and exploitation of previously
 unused microorganisms (including the modification of naturally-occurring
 nonpathogenic microorganisms to render them pathogenic to insects), the
 modification and refinement of currently used entomopathogens, and the
 development of genetically engineered crops which display greater
 resistance to insect pests.
 In 1972 McLaughlin et al. published their work on the effect of CIV on the
 cotton boll weevil. They showed that infection with whole virus arrested
 metamorphosis and death. Researchers in France showed that soluble
 extracts from CIV inhibited host protein synthesis and gene expression in
 cell cultures from mosquitoes and some caterpillar species (Cerutti and
 Devauchelle, 1980). However, no group has previously shown that a protein
 fraction from CIV kills boll weevil larvae, nor has any group shown whole
 virus or viral protein preparations causing inhibition of protein
 synthesis in boll weevil cell lines or induction of programmed cell death
 (apoptosis) in any cell line.
 The cotton boll weevil will have an economic impact exceeding $500 million
 per year in Texas alone. More than 9,200 jobs will be lost and at least 60
 cotton gins will close if no new technology is developed. Chemical control
 of the weevil is not working well because of resistance problems and
 adverse effects on beneficial insects. In addition, there are difficulties
 in discovering new chemistry and problems with insecticide contamination
 of ground reserves. Therefore, the development of alternative, biological
 (especially microbial) control systems is critical. Because larvae develop
 inside the cotton boll and cannot be sprayed externally, the best control
 strategy will be to engineer transgenic cotton that produces insecticidal
 proteins.
 Our laboratory has shown that Chilo iridescent virus (CIV) induces
 metamorphic deformity in boll weevil larvae, and kills them. We have shown
 that CIV replicates efficiently in this host. A protein extract from the
 virus induces mortality in neonate larvae. The Extract also inhibits host
 protein synthesis and minduces programmed cell death or apoptosis as
 evidenced by cell blebbing and DNA fragmentation. Heating at 60 degrees C.
 for 30 minute or treatment with protease destroys these activities.
 Prior art has shown that Chilo iridescent virus (CIV) induces mortality and
 metamorphic deformity in the cotton boll weevil. Prior art also shows that
 CIV protein extracts inhibited host gene expression in lepidopteran and
 dipteran cells. However, the use of CIV protein extracts in the control of
 in sect infestation have not been demonstrated nor has it been
 demonstrated that CIV protein extract induces programmed cell death or
 apoptosis in any cell line or organism.
 What is needed is a biological pesticide which reduces the adverse effects
 of chemical pesticide. A biological pesticide is preferred because it
 creates less of an environmental hazard than a chemical pesticide. A
 pesticide that causes insect death more rapidly is additionally needed.
 What is also needed to the identification and isolation of a gene that
 codes for a protein which will control insect development. Such a gene or
 its protein product could then be incorporated into various organisms for
 the improved biological control of insect pests.
 SUMMARY OF THE INVENTION
 It has been discovered that a protein extract from purified Chilo
 iridescent virus (CIV) particles will control infestations by boll
 weevils, and whole virus particles will control aphid populations.
 It has been discovered that proteins or extracts of proteins that are
 insecticidal proteins provide composition and methods for using certain
 viral inhibitors to protect plants otherwise susceptible to insect
 infestation by one or more of Mexican bean beetle, red flower beetle,
 confused flower beetle, boll weevil, Colorado potato beetle, 3-line potato
 beetle, rice weevil, maze weevil, granularly weevil, Egyptian alfalfa
 weevil, bean weevil, yellow mill worm weevil, asparagus beetle and a
 variety of other insects including other beetles and weevils.
 We have shown that a protein fraction from purified Chilo iridescent virus
 particles causes mortality in freshly hatched larvae of the cotton boll
 weevil. This toxin preparation also inhibits wholesale protein synthesis,
 and induces programmed cell death (apoptosis) in cell cultures of the
 cotton boll weevil, Anthonomus grandis. Knowledge of the viral toxin will
 allow isolation of the gene responsible for the toxin. The gene would then
 be transferred to crop plants (such as cotton) so that toxin will be
 produced in such engineered plants. Pest insects will be arrested in their
 development or die upon contact with toxin producing plant tissues. The
 toxin is the only way viral protein component is known to kill boll weevil
 larvae. It is also the only viral toxin that inhibits host protein
 synthesis and induces programmed cell death in boll weevil cells. The
 toxin will be used to engineer pest resistance for all plants. Its unique
 mechanism of action will also reduce development of resistance to other
 toxins.
 The protein extract is lethal to boll weevil larvae and will interrupt
 protein synthesis in boll weevil cells and induce programmed cell death or
 apoptosis in them. This mechanism of action is distinct from that of
 cholesterol oxidase, which alters the insect gut environment by inducing
 changes in lipids surrounding essential enzymes, such as alkaline
 phosphatase. CIV particles will induce mortality in aphid populations. The
 protein extract or virus may be applied directly to the plants or
 introduced in other ways, such as expression in plant-colonizing
 microorganisms or in crop plants, after isolation of the toxin gene. Tests
 on the effect of toxin on aphid populations are in progress.
 As used herein, the term "controlling insect infestation" means reducing
 the number of insects which cause reduced yield, through either mortality,
 retardation of larval development (stunting), or reduced reproductive
 efficiency.
 Present technology utilizes chemical insecticides to control the boll
 weevil and other insect pests. Microbial insecticides are being developed
 to address problems of resistance and environmental damage. A number of
 protein toxins against caterpillars have been identified, and at least one
 (the B.t. toxin) has been used to engineer caterpillar-resistant plants
 (Meeusen, R. L. and Warran, G. 1989. Ann. Rev.Entomol. 34:373-381). Thus
 far only one other class of toxin, cholesterol oxidase (Purcell et al.,
 Biochem. Biophys.Res.Comm. 196: 1406-1413. 1993; Corbin et al., U.S. Pat.
 Appl. Nos. 475,964; 083,948, 1995), has been identified against the boll
 weevil. It should be emphasized that our toxin works by a mechanism that
 is different from that of cholesterol oxidase and is in a different class
 altogether. For any pest, it is critical to develop several different
 toxins or genes, each working through a different mechanism, in order to
 avoid the problem of resistance in the target population. Thus, the toxin
 we have developed will play a novel and useful role in pest control.
 Accordingly, it is an object of the present invention to provide a gene and
 its gene product that are useful in the control of insect pest.
 It is another object of the present invention to provide a recombinant
 virus that is a more effective pesticide than wild type virus. It is yet
 another object of the present invention to provide a genetically
 engineered virus that is an effective pesticide and is also
 environmentally acceptable. It is yet another object of the present
 invention to provide a modified biological pesticide that express es a
 genetically inserted gene.
 It is another object of the present invention to provide a novel use of
 viral proteins and protein extracts for controlling cotton boll weevil and
 other insects. In yet another object of the invention is the demonstration
 that a protein fraction from purified Chilo iridescent virus particles
 causes mortality in freshly hatched larvae of the cotton boll weevil.
 It is another object of the present invention to provide a modified
 biological pesticide that inhibits with viral extract which induces
 cellular suicide (apoptosis) in boll weevil, bud worm cells and aphids.
 Also contemplated is insecticidal compositions, those comprising an
 agriculturally suitable carrier and genetically modified insect parasite.
 An insect parasite is an organism which lives or replicates in close
 association with an insect larva, and has adverse affects on that larvae
 An insect parasite can be a bacterium, a fungus, a virus or another
 insect. Such a genetically modified insect parasite comprising toxin gene
 will be improved as an insect control agent by the insertion and
 expression of a toxin gene.
 Any of the above-noted insecticidal compositions may further comprise
 ingredients to stimulate insect feeding. The insecticidal compositions of
 the present invention can be ingested by insect pests after plant
 application, and those insect pests susceptible to the insect control
 agent in the insecticidal composition will exhibit reduced feeding and
 will die.
 It is therefore an object of the present invention to provide proteins
 capable of controlling insects, such as boll weevils and lepidopterans,
 and genes useful in producing such proteins. It is a further object of the
 present invention to provide genetic constructs for and methods of
 inserting such genetic material into microorganisms. It is another object
 of the present invention to provide transformed microorganisms containing
 such genetic material.

DETAILED DESCRIPTION OF THE INVENTION
 The use of CIV protein extracts for controlling insects is within the scope
 of this invention. Additionally, it is contemplated herein that the
 compositions of the inventions will include isolation and expression of
 the toxin gene in plant-colonizing microbes and in crop plants. Virus
 purification and extraction Virus production: The procedures for
 purification of CIV are well known in the art and used for other
 iridescent viruses. Chilo iridescent virus was reared in the greater wax
 worm, Galleria mellonella. Waxworm larvae were nicked with sharpened
 forceps that had been dipped in a virus suspension (0.5 .mu.g/ml). Larvae
 were checked every three days in order to remove dead and pupated insects.
 All other larvae were frozen at -20 degrees C. two weeks after
 infestation. Virus was purified from the waxworm larvae by maceration in
 Tris-NaCl buffer (50 mM Tris-HCl, 150 .mu.M NaCl, ph 7.4) using a Waring
 blender. The slurry was filtered through cheesecloth into Sorvall GSA
 centrifuge tubes to remove large particulate material. The supernatant was
 then transferred into SS-34 centrifuge tubes and centrifuged at 17,000 rpm
 for 30 minutes at 4 degrees C. After overnight resuspension of the virus
 pellet in Tris-NaCl buffer, the suspension underwent another round of
 differential centrifugation in SS-34 tubes. After overnight resuspension
 of the second pellet, the virus pellet in Tris-NaCl buffer, the suspension
 underwent another round of differential centrifugation in SS-34 tubes.
 After overnight resuspension of the second pellet, the virus was layered
 on top of 10-60 percent sucrose (w/v) gradients and centrifuged for 2
 hours at 36,000 rpm at 4 degrees C. Viral layers were harvested, pelleted,
 resuspended, and run on a second set of sucrose gradients. The resulting
 virus layers were pelleted, resuspended, and filtered through a series of
 0.45 and 0. 22 .mu.m pore-size filters. The concentration of the virus was
 determined by spectrophotometric analysis. One unit of absorbance at 260
 nm (A260) equals 55 .mu./ml of virus. Production of viral protein
 extracts: The preparation of viral protein extracts are well known in the
 art and used for several viruses, including iridescent viruses. CHAPS
 extractions: Five milligrams of sucrose gradient-purified virus is
 pelleted and resuspended in CHAPS extraction buffer (10 .mu.M Tris-HCL, 10
 mM CHAPS, and 1M KCl, pH 7.4) in a final volume of 10 .mu.g/ml. The
 suspension is then incubated at 30 degrees C. for 15 minutes and 5 .mu.l
 of the suspension is layered on top of 6 ml of 20 percent sucrose in SW-41
 centrifuge tubes. The suspension is centrifuged for 2 hours at 36,000 rpm
 at 4 degrees C. The supernatant above the sucrose is then collected and
 subjected to four rounds of ultrafiltration using a YM-10 membrane with
 storage buffer (50 mM Tris-HCl, 150 mM NaCl, ph 7.4) to a final volume of
 approximately 1 ml. The extract is then filtered through a 0.22 .mu.m
 filter and stored at -80 degrees C. Membrane Filtration: Virus is
 resuspended in Borate buffer (0.01 M Borate, pH 7.5) and stirred overnight
 at 4 degrees C. to facilitate protein release. The suspension is then
 filtered through a YM-100 membrane, followed by concentration to
 approximately 1 ml using a YM-10 membrane. The extract is then filtered
 through a 0.22 im filter and stored at -80 degrees C.
 Bioefficacy Essays
 Effect of viral protein extract on neonate boll weevil larvae: Boll weevil
 growth medium containing eggs of the cotton boll weevil, Anthonomus
 grandis (obtained from the GAST laboratory, Starkville, Miss.) were
 divided into square sections just large enough to fit standard Petri
 dishes. Each dish contained approximately 100 eggs. Upon hatching of the
 eggs, the medium surface was sprayed with approximately 500 .mu.l of
 either a soluble protein extract (5 .mu.g/ml) and the antibiotic
 gentamicin (50 .mu.g/ml) or identical buffer without viral protein. Eight
 days after initial treatment, insects were removed from the dishes,
 observed for mortality, and again sprayed with approximately 500 .mu.of
 the appropriate treatment. Larvae were observed daily for the next eight
 days.
 FIG. 1 shows that treatment with viral protein extract killed 37 percent of
 neonate larvae populations, whereas only seven percent of buffer-treated
 and five percent of untreated larvae were killed. The data show that viral
 protein extract for Chilo iridescent virus has insecticidal properties.
 Active extracts contained approximately seven major and eleven minor
 polypeptides of varying relative abundance.
 Effect of Chilo iridescent virus on the cotton aphid, Aphis gossypii:
 Cotton leaves were brushed with 10 .mu.g/ml purified (twice through
 sucrose gradients) Chilo iridescent virus in Tris NaCl buffer (50 mM
 Tirs-HCl, 150 mM NaCl, pH 7.4) containing 15 .mu.g/ml casein as a carrier
 protein and the protease inhibitors Leupeptin (2 .mu.g/ml) and Pepstatin A
 (1 .mu.g/ml). Mock treatments (consisting of buffer preparation) and
 untreated leaves served as negative controls. After brushing, 15 aphids
 were placed in a small area on the underside of cotton leaves. In order to
 contain the aphids, the bottom portion of a 30-.mu.m plastic Petri dish
 (with a covered hole for ventilation) was placed over the leaf surface
 containing aphids, while the top portion was inverted and placed on the
 opposite side of the leaf. The apparatus was held together with clamps and
 the leaves were supported on a shelf. After three days incubation, treated
 and control leaves were removed from the cotton plants and aphid numbers
 and mortality were determined using a dissecting microscope. Results are
 shown as percent decrease in live aphid numbers with respect to untreated
 samples. The assay was performed in triplicate.
 FIG. 2 shows that compared to untreated control groups, aphid population
 growth is reduced by 65 percent when treated with Chilo iridescent virus.
 Mock treatments with casein reduced population growth by only 30 percent.
 The results suggest a significant viral effect and indicate that CIV is an
 effective viral insecticide against aphids.
 Inhibition of Protein Synthesis by Viral Protein Extract
 Boll weevil (BRL-Ag-3A: AG) and spruce budworm (CF124T; CF) and cells
 (6.25.times.10.sup.5 cells/ml) were seeded into 24-well tissue culture
 trays. After overnight attachment at 28 degrees C., culture medium was
 removed and cells were washed with unsupplemented medium. Subsequently,
 the cells were incubated for 3 hours at 28 degrees C. and starved of
 methionine for two hours in 100 .mu.l ExCell 401 methionilne-eficient
 medium (JRH Biosciences). Following starvation, the cells were labeled for
 one hour with 150 .mu.l ExCell 401 methionine-deficient medium containing
 80 .mu.Ci/ml .sup.35 S-methionine. Cells were then removed from well
 matrices using a rubber policeman; samples were centrifuged for 30 seconds
 and resuspended in SDS-PAGE sample buffer. Samples were then boiled for 5
 minutes and analyzed on a 10 percent SDS-polyacrylamide gels. Gels were
 immersed in protein fixing solution (30 min), submerged in En.sup.3 Hance
 (Dupont NEN) for 15 minutes, and precipitated in ice-cold water for 15
 minutes. Gels were then dried onto filter paper and exposed to Hyperfilm
 MP X-ray film (Amersham) at -80 degrees C. before developing.
 FIG. 3 shows that host protein synthesis in both boll weevil and spruce
 budworm lines is drastically reduced with viral protein concentrations of
 10 .mu.g/ml. Untreated cells or cells treated with heated viral protein
 did not inhibit host synthesis and neither did treatment with Proteinase K
 (Sigma: 50 .mu.g/ml, 37 degrees C., 2 hr). These data indicate that the
 inhibiting viral factor is a protein. Thus, a polypeptide (or
 polypeptides) in the viral extract has an inhibitory effect on protein
 synthesis in boll weevil and spruce budworm cells.
 Induction of Programmed Cell Death by Viral Protein Extract
 Two major manifestations of virus-induced programmed cell death or
 apoptosis are the formation of blebs and fragmentation of cellular DNA. We
 show that protein extracts from purified CIV induce both of these effects
 in boll weevil and spruce budworm cells.
 Bleb formation: Serial ten-fold dilutions of the toxin preparation (in the
 range 150 .mu.g/ml to 150 pg/ml) were prepared and used for treating cell
 cultures. An equal volume of cell suspension (BRL-AG-3A cells at
 5.times.10.sup.5 cells/ml or CF 124T cells at 7.5.times.10.sup.5 cells/ml)
 and the appropriate dilution of toxin solutions were mixed, and 15 ml of
 this preparation was added to each well of a 60-well Terasaki plate. The
 plates were then placed in a sandwich bag along with a moistened paper
 towel and incubated at 28 degrees C. The assay was done in duplicate using
 mock-treated cells (buffer only) as controls. Cells were examined at 24
 hours post treatment for cytopathology.
 FIG. 4 shows that both boll weevil and spruced budworm cells manifest
 blebbing. This is characteristic cells undergoing apoptosis. The formation
 of coronas and blebs are more numerous in the spruce budworm cells, but
 significant levels of this effect are evident in boll weevils cells also.
 Table 1 shows the dose-response endpoints for apoptotic cytopathology in
 boll weevil and spruce budworm cells. The minimum dose eliciting a 50
 percent response is 6 nanograms per ml in spruce budworm cells and 30
 nanograms per ml in boll weevil cells.
 DNA fragmentation: Spruce budworm, CF124T, cells (3.times.10.sup.6
 cells/well) were added to 6-well total volume of 3 ml. The trays were
 incubated at 28 degrees C. overnight to allow for attachment of cells. The
 cell monolayers were washed once with unsupplmented medium (TNMFH). One
 milliliter each of larval derived virus (10 .mu.g/ml), toxin (CHAPS; 10
 .mu.g/ml), and actinomycin D (4 .mu.g/ml; positive control) were then
 added into the respective wells. Boll weevil, BRL-AG-3A, cells were
 treated as above except that the actinomycin concentration was 1 .mu.g/ml
 and viral protein extract was used at 4 .mu.g/ml and 7.5 .mu.g/ml.
 Mock-enfected (sans virus), mock-treated (sans viral extract), and
 untreated cells were used as negative controls. The treatments were
 adsorbed for 1 hour at 21 degrees C. on a Bellco rocker platform set at
 2.5 rpm. After adsorption, the volume in each well was made up to 3 ml
 with complete TNMFH (medium containing 10 percent fetal bovine serum and
 0.5 .mu.g/ml gentamicin). Virus-infected cells were incubated for 24 hours
 at RT and toxin-treated samples were incubated at 28 degrees C. DNA was
 harvested 24 hours after infection or treatment using 0.4M Tris-HCl pH7.5,
 0.1M EDTA, 0.1 percent SDS, and 200 .mu.g/ml proteinase K solution. After
 incubation at room temperature for 12-16 hours, samples were phenol
 extracted, ethanol precipitated, and digested with 20 .mu.g/ml of RNase A
 (Sigma) for 30 min. at 37 degrees C. 20 .mu.g/ml of each sample were then
 analyzed by agarose gel electrophoresis.
 FIG. 5A shows that virus protein extract induces significant host DNA
 fragmentation at 2.5 .mu.g/ml in budworm cells. DNA fragmentation is this
 cell line results in the formation of a ladder effect due to cleavage of
 cellular DNA into precise lengths. The extent of fragmentation is
 comparable to that induced by actinomycin D (positive control).
 Virus-treated cells did not induce any fragmentation with whole virus
 suggest that establishment of an infection cycle and subsequent expression
 of a viral apoptosis inhibitor gene might be responsible for the
 suppression of programmed cell death and ensuing effects. Such inhibitor
 genes have been detected in viruses.
 FIG. 5B shows that DNA fragmentation is induced in boll weevil cells, but
 to a lesser extent compared to the positive (actinomycin D) control. The
 mode of DNA fragmentation is cell line dependent; fragmentation in boll
 weevil cells has always resulted in a smear rather than a ladder effect,
 due to incremental cleavage of cellular DNA.
 The analysis of future expected losses attributable to the boll weevil are
 based on experiences and trends already evident on the Texas High Plains
 and are projected 5-10 years into the future. In the furthest north areas
 where cotton is grown, the damage caused by the boll weevil historically
 has been light. However, in the High Plains, serious economic damages may
 be experienced as far north as Floyd, Hale, Lamb, Briscoe and Bailey
 counties. From these counties and south, annual yield losses of about 15%
 are projected, along with an increase in insect control cost of $35 to $45
 per acre on irrigated cotton and about $20 per acre on dryland cotton.
 These losses, combined with acreage shifts to alternative crops are
 reflected in reduced farmer net income from $189 million to $47 million (a
 loss of $142 million).
 Because yield and cost effects of the boll weevil, about 500,000 acres of
 cotton production is expected to shift to alternative crops, which offers
 greater profit than cotton under boll weevil pressure. This suggest s a
 reduction in cotton production of 800,000 bales. The loss of this
 production would result in the closure of approximately 60 of the current
 190 cotton gins in the region.
 Compared to other crops, cotton generates relatively more jobs per dollar
 of production, and has a significantly greater impact on the regional
 economy. The gross income from acres traditionally planted to cotton is
 expected to decline from $862 million to $668 million (a loss of $194
 million). This will impact the economy of the region by reducing business
 activity $500 million with a loss of more than 9,000 jobs. The majority of
 the losses will occur in Floyd, Hale, Lamb, Bailey and Briscoe and
 counties to the south.
 The results based on the most likely estimate of future boll weevil impact
 indicates that the boll weevil is indeed a serious threat to cotton
 production and to the economy of the Texas High Plains. Due to uncertainty
 on exactly how the boll weevil will spread and survive on the Texas High
 Plains, an upper bound or "worst case" and a lower or "best case" scenario
 was evaluated to put bounds on the estimates.
 Previously we talked about McLaughlin 1972 showing that certain viruses
 kill boll weevil larvae. We also presented that Cerutti and Devauchelle
 (1980) taught that viral extract inhibits gene expression/protein
 synthesis in mosquitos and bud worms. The present invention shows that a
 protein fraction or extract from purified Chilo iridescent virus partially
 causes mortality in freshly hatched larvae. A viral extract induces
 cellular suicide (apoptosis) in boll weevil and bud worm cells. The viral
 extract inhibits protein synthesis in boll weevil cells. Purified, active
 protein can be isolated from the viral protein extract and its amino acid
 sequence can be determined. This information can then be used to isolate
 and identify the gene for the active polypeptide.
 All publications and patents mentioned in this specification are herein
 incorporated by reference as if each individual publication or patent was
 specifically and individually stated to be incorporated by reference.
 From the foregoing, it will be seen that this invention is one well adapted
 to attain all the ends and objects hereinabove set forth together with
 advantages which are obvious and which are inherent to the invention.
 It will be understood that certain features and subcombinations are of
 utility and may be employed without reference to other features and
 subcombinations. This is contemplated by and is within the scope of the
 claims.
 Since many possible embodiments may be made of the invention without
 departing from the scope thereof, it is to be understood that all matter
 herein set forth or shown in the accompanying drawings is to be
 interpreted as illustrative and not in a limiting sense.