Production and efficacy of baculviruses

Several baculvirusus of nuclear polyhedrosis virus (NPV) have been produced and tested for microbial control of various Lepidoptera spp. To date, there are three registered preparations of NPV that are exempt from the requirement of tolerance by the Environmental Protection Agency (EPA) in the United States (US). The first and only commercially available viral preparation used in agriculture was developed by Sandoz, Inc. under the name of Elcar® for control of Heliothis spp. on cotton. The other two baculovirus preparations were developed and registered by the US Department of Agriculture (USDA) for control of Douglas-fir tussock moth and gypsy moth on forests. Several methods are being used for production of NPV viruses: (1) field collection of diseased larvae, (2) laboratory rearing of insects followed by infection with viral inoculum, (3) tissue culture. and (4) tissue culture and mass rearing larvae. Recent progress in mass production of insect virus points toward the adoption of tissue culture with the whole organism technology for production of a standardized viral product. The practical usefulness of various baculovirus preparations has been demonstrated for protection of forests from defoliation by various lepidopterous species. In agriculture, Elcar® has been successfully marketed and has been very well received for use in integrated pest management on cotton. Recent development also demonstrated that use of adjuvants further increase the efficacy of Elcar® against Heliothis spp. on cotton.     

Replication and serial passage of infectious Heliothis nucleopolyhedrosis virus in an established line of Heliothis zea cells

An established cell line derived from the ovary of adults of the cotton bollworm, Heliothis zea, supported growth of the Heliothis nucleopolyhedrosis virus (NPV). Typical NPV symptoms were obtained when infected cells were fed to neonatal bollworms; however, the cell line never produced free virions or inclusion bodies containing virions. Infectious virus was passed through the cell line 7 consecutive times, using only infected cells from the previous pass. Infectivity at the 7th serial-pass represented a dilution of >10⁻⁸ of the original inoculum.     

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm,Helicoverpa armigera (Hubner)

American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha￣?) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶ and 1 × 10⁵ POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.     

Effect of dew from cotton and soybean foliage on activity of Heliothis nuclear polyhedrosis virus

The ionic composition of dew collected from foliage of cotton and soybean plants as well as its potential for inactivation of Heliothis NPV were compared. Cotton dew had a mean pH of 8.8 and was more alkaline than soybean dew (pH 7.8). Cotton dew had a higher concentration of ions that did soybean dew but there was no qualitative difference in the cation content of dew from the two hosts. In bioassay tests, no loss of activity occurred when polyhedra were held in dew of either plant species. If the dew in which polyhedra were suspended was air-dried and resuspended daily in deionized water, polyhedra in soybean dew remained active but in cotton dew retained little activity after 7 days. Also, electron microscopical examination of polyhedra pelleted from these cotton dew preparations showed much dissolution after 7 days. Although there was dissolution of polyhedra in cotton dew when dried, an examination of polyhedra on the upper surface of either plant species in the field showed little degradation after 7 days.     

Cross-infectivity and activation studies with four baculoviruses

The NPVs of 3Spodoptera species and 1Heliothis species were bioassay tested for cross-infectivity. The progeny virus from the test insects was purified and examined by specific identification criteria. This demonstrated that activation of virus was much more common than cross-infection. Stress experiments using chemicals and varying environmental conditions failed to activate any virus in the stock insect cultures. Experiments were designed to test for possible mixed inoculum virus as an explanation of the activation effect but this theory was disproved. The mechanism of the activation is unexplained but it seems clear that when independent identification techniques are used it can be demonstrated that infecting an insect larva with a NPV from another host can result in death due to infection with the NPV normally associated with that host rather than that used as inoculum. This can occur even though no latent virus can be detected in the insect population by conventional methods.     

An in vitro method for increasing UV-tolerance in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) nucleopolyhedrovirus

A method for increasing tolerance to ultraviolet (UV) radiation in a strain of nucleopolyhedrovirus of cotton bollworm, Helicoverpa armigera (Hübner) (HearNPV) using a solar simulator is described. The Coimbatore isolate (CBE I) of HearNPV was subjected to a five-step sequence of selection to increase its UV tolerance. Each step consisted of irradiation of wet deposits of the virus to near UV (at energy level of 300W/m²), bioassay against second instar H. armigera larvae and propagation in early fifth instar larvae. Selection steps carried out at 15, 30, 60 and 90 minutes of exposure revealed that the continuous exposure of HearNPV-CBE I at low doses of UV irradiation (270–540 KJ/m²) did not significantly affect the virus activity as measured by its biological activity against second instar larvae. Selection at higher doses (1620 KJ/m²) led to loss of viral activity in the first two exposure cycles; however, there was retention of virulence coupled with increased tolerance to UV doses from third cycle onwards. Further, studies on the persistence of UV tolerant strain of HearNPV-CBE I in comparison with original strain showed that the tolerant strain had more persistence even after 7 days of weathering both under exposed (18% original activity remaining) and shaded (26% original activity remaining) condition on potted cotton plant.     

Evaluation of seven viral isolates as potential biocontrol agents against Pseudoplusia includens (Lepidoptera: Noctuidae) caterpillars

The caterpillar Pseudoplusia includens (Walker, 1857) (Lepidoptera, Noctuidae), known as soybean looper, is a pest that has recently assumed greater importance in soybean in Brazil. Isolates of nucleopolyhedroviruses (NPVs) of this pest have been identified from cotton in Guatemala and soybean farms in Brazil, providing an interesting perspective of potential use of viral insecticide against the insect in lieu to chemical insecticides. With the objective to contribute to the characterization studies of this virus, morphological and molecular analyses and biological activity were carried out with seven P. includens viral isolates (I-A to I-G). Electron microscopy of viral samples, purified from macerated infected larvae, showed particles with typical morphology of the Baculoviridae family, genus Alphabaculovirus (Nucleopolyhedrovirus - NPV) presenting virions with only a single nucleocapsid per envelope (SNPV) occluded in a protein matrix, forming occlusion bodies (OB). This virus was then classified as P. includens single nucleopolyhedrovirus (PsinSNPV). OB particles analyzed in SDS-polyacrylamide gel showed an intense band corresponding in size to NPV polyhedrin protein. DNA restriction profiles of the PsinSNPV isolates showed differences in the fragment size and number suggesting the existence of genotypic variants, except between I-E and I-F profiles that were similar. Among the isolates tested for infectivity against P. includens, I-A, I-E and I-F were the most virulent. Survival times (ST₅₀) varied according to viral concentration, with significant differences among isolates for the three higher concentrations.     

Susceptibility of the parasitoidMicroplitis croceipes [Hymenoptera: Braconidae] to field rates of selected cotton insecticides

Microplitis croceipes (Cresson), a parasitoid ofHeliothis spp., was treated topically with field rates of 5 formulated insecticides commonly used in cotton to controlHeliothis spp. Treatment with the pyrethroid fenvalerate, a mixture of the formamidine chlordimeform plus fenvalerate, and the carbamate thiodicarb resulted in nearly 100% survival at both the lowest and highest field rates recommended for these insecticides. The organophosphate acephate and the carbamate methomyl were extremely toxic to adultM. croceipes, causing 100% mortality at the lowest recommended field rates. Comparisons are made to previously reported laboratory toxicity data for some of these insecticides for adultM. croceipes. Choice of insecticides to control pyrethroid-resistantHeliothis spp., and the effects of these insecticides on adultM. croceipes survival, are discussed. This article reports the results of research only. Mention of a proprietary product does not constitute endorsement or a recommendation for its use by the U. S. Department of Agriculture.     

in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence,structure analysis of crystal proteins for stability

Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125^th amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.     

Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus

The susceptibility of Heliothis armiger larvae of different ages to a commercial nuclear polyhedrosis virus (NPV), Elcar, was determined by bioassay. The median lethal dosage (LD₅₀) increased 150-fold during the first week of larval life at 25°C, i.e., during development to early fourth instar, but daily feeding rate and thus potential virus acquisition also increased. A linear relationship was determined between log LD₅₀ and larval length, indicating that larval length constitutes a useful index for estimating the susceptibility of larval populations. Median lethal times (LT₅₀s) were similar for larvae tested at ages of 0 to 7 days and ranged from 3.6 to 8.0 days at 30°C. The amount of virus produced in a single, infected neonate was equivalent to 1.4 × 10⁶ LD₅₀s for neonates, a 900,000-fold increase on the dose supplied. The data support the practice of directing the NPV against neonates, but, on the basis of larval susceptibility alone, the age of larvae at treatment may not always be critical.     

Measurement of Surface Charge of Baculovirus Polyhedra

The isoelectric points of three baculoviruses, Trichoplusia ni nuclear polyhedrosis virus (NPV), T. ni granulosis virus, and Spodoptera littoralis NPV were identified by cell electrophoresis. At neutral pH polyhedra were negatively charged. T. ni NPV polyhedra were reacted with a number of reagents which could potentially attach to or degrade their surface structure. This gave information on the components that contribute to the charge profile of T. ni NPV. This is discussed in relation to the use of polyhedra as biological control agents against insect pests.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

Nucleopolyhedrovirus introduction in Australia

Nucleopolyhedrovirus (NPV) has become an integral part of integrated pest management (IPM) in many Australian agricultural and horticultural crops. This is the culmination of years of work conducted by researchers at the Queensland Department of Primary Industries and Fisheries (QDPI&F) and Ag Biotech Australia Pty Ltd. In the early 1970's researchers at QDPI&F identified and isolated a virus in Helicoverpa armigera populations in the field. This NPV was extensively studied and shown to be highly specific to Helicoverpa and Heliothis species. Further work showed that when used appropriately the virus could be used effectively to manage these insects in crops such as sorghum, cotton, chickpea and sweet corn. A similar virus was first commercially produced in the USA in the 1970's. This product, Elcar(R), was introduced into Australia in the late 1970's by Shell Chemicals with limited success. A major factor contributing to the poor adoption of Elcar was the concurrent enormous success of the synthetic pyrethroids. The importance of integrated pest management was probably also not widely accepted at that time. Gradual development of insect resistance to synthetic pyrethroids and other synthetic insecticides in Australia and the increased awareness of the importance of IPM meant that researchers once again turned their attentions to environmentally friendly pest management tools such NPV and beneficial insects. In the 1990's a company called Rhone-Poulenc registered an NPV for use in Australian sorghum, chickpea and cotton. This product, Gemstar(R), was imported from the USA. In 2000 Ag Biotech Australia established an in-vivo production facility in Australia to produce commercial volumes of a product similar to the imported product. This product was branded, ViVUS(R), and was first registered and sold commercially in Australia in 2003. The initial production of ViVUS used a virus identical to the American product but replicating it in an Australian Helicoverpa species, H. armigera. Subsequent research collaboration between QDPI&F and Ag Biotech reinvigorated interest in the local virus strain. This was purified and the production system adapted to produce it on a commercial scale. This new version of ViVUS, which was branded ViVUS Gold(R), was first registered and sold commercially in 2004. Widespread insect resistance to insecticides and a greater understanding of integrated pest management is leading to increased adoption of technologies such NPV in Australian agriculture.     

Acylamines enhance the infection of a baculovirus of the armyworm, Pseudaletia unipuncta (Noctuidae,Lepidoptera)

The acylamines (cationic detergents) enhance the infection of a nuclear polyhedrosis virus (NPV) in larvae of the armyworm, Pseudaletia unipuncta. Those with 6 carbons (hexylamine) or 12 carbons (dodecylamine) in the acyl group are more effective in enhancing the NPV than those with 4, 8, or 10 carbons. Hexylamine and dodecylamine increase the rate of infection beyond the 50% level (ID₅₀) by 10 to 100 times when based on the concentration of the virus polyhedra (inclusion bodies).     

The effect of magnesium ions on the solubility of polyhedral inclusion bodies and its possible role in the inactivation of the nuclear polyhedrosis virus of Spodoptera littoralis by the cotton leaf gland exudate

Polyhedral inclusion bodies of Spodoptera littoralis nuclear polyhedrosis virus (NPV) treated with solutions of cotton leaf gland exudate (a process which has been shown to inactivate the NPV), exhibited a marked decrease in solubility in dilute alkali. This process is reversible with EDTA and can be simulated by MgCI₂. The effect of magnesium ions on solubility did not involve the enzyme, alkaline protease. It is suggested that this decrease in solubility may be involved in the mechanism by which the cotton leaf inactivates the NPV. However, since MgCI₂ alone could not be shown to inactivate NPV in vitro it must be assumed that other constituents of the leaf gland exudate also play a part.     

Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

Effect of larval maturation on mortality induced by nuclear polyhedrosis and granulosis virus infections of Heliothis armigera

All the instars of Heliothis armigera larvae were found to be susceptible to both nuclear polyhedrosis virus (NPV) and granulosis virus (GV). An inverse relationship between mortality and larval age was found in the case of the NPV, while the GV displayed a rather erratic mortality pattern. A degree of maturation immunity against the NPV was found to exist, but the same is not true for the GV. The important role that pupation plays on the effect of a lethal infection is also discussed.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

A high incidence of parasitism ofHeliothis SPP. [Lep.: Noctuidae] larvae in cotton in Southeastern Arkansas

During 1981 and 1982, bollworm,Heliothis zea (Boddie), and tobacco budworm,H. virescens (F.), larvae (n=3,666) were collected from 41 cotton fields near Portland, Arkansas (USA) to assess the occurrence of parasitism. Three strategies were employed to controlHeliothis spp. in these fields: (1) release ofTrichogramma pretiosum Riley; (2) insecticidal control; or (3) inaction (check). Insecticide use in nonchemical control fields was reduced, but not eliminated.Heliothis spp. larvae collected in cotton had higher parasitism rates in 1981 (30.9%) and 1982 (50.1%) than had been reported for cotton since the advent of organochlorine insecticide usage. Four species of larval parasites and 1 species of larval-pupal parasite were recorded. The larval parasiteMicroplitis croceipes (Cresson) comprised 90.6% and 94.5% of all parasitic insects reared from field collectedHeliothis spp. in 1981 and 1982, respectively. No difference (P>0.05) in level of parasitism existed betweenH. zea andH. virescens. Differences between treatments occurred only in 1982 whenH. zea larvae were parasitized at a greater (P<0.05) rate in check fields (68.3%) than in insecticidal control fields (44.3%). Higher levels of larval parasitism in cotton fields may be a consequence of reduced insecticide usage and changes in materials applied, particularly the pyrethroids. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

The nuclear polyhedrosis virus (NPV) of Porthetria dispar was isolated and purified through a two-step zonal centrifugation procedure. The LD₅₀ of the purified NPV was determined by a dose-response assay. Quantitative analyses were made of whole polyhedra and of separated fractions of polyhedral protein, virus rods, and denatured material, i.e., the pellet obtained from low speed centrifugation of dissolved polyhedra, to determine the protein, DNA, and “RNA” (orcinol-positive material) present in this NPV. Approximately one-half the “RNA” was present in the denatured material. Trace elements were also determined, and four, Fe, Mg, Cu, and Zn, of the ten assayed were present in the polyhedral protein fraction, while only Mg and Zn were in the virus rod fraction.