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FAIR-CT96-3222
Field testing & safety assessment of viral biopesticides for use in integrated pest management systems |
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Contract No: | FAIR-CT96-3222 |
| Date Prepared: | March 2001 | |
| Source: | Final Report Abstract |
Objectives
The objectives of this project are:
Activities
These were arranged on a task basis. The aim task 1 of the project were to develop a baculovirus formulation with added protection from UV protective adjuvants, to develop a new formulation which will allow protective adjuvants to be in close proximity to the PIBs to allow greater efficiency of the protective effect, to evaluate the activity and UV stability of an in vitro produced, formulated viral biopesticide in laboratory based assays and glasshouse trials and finally, to evaluate the measurable parameters of a mathematical model developed for the AIR1-CT92-0386 project
A suspension concentrate (SC) type formulation containing PIBs but no protective adjuvants was produced for an in vitro produced HaSNPV baculovirus as a 'working' formulation. Development of a suspension concentrate (SC) type formulation containing both PIBs and UV protectent followed. Also developed was a liposome type formulation, which encapsulated the PIBs in the liposome and allowed protective adjuvants to be incorporated into the formulation and surround the PIBs.
A series of bioassays were carried out to evaluate and optimise the intrinsic activity and UV stability of the HaSNPV virus, using SC formulations containing UV protectants and feeding stimulants, and liposome formulations encapsulating PIBs. Results from laboratory based studies indicated that the addition of UV protectant to the formulation significantly improved the UV stability of the virus.
The parameters of a mathematical model (developed in 1995 at Zeneca, Jealott's Hill) that could be measured and compared to a 'real-life' field situation were identified and measurements of some of the parameters took place in France in 1999. It was found that the mathematical model was suitable for use under controlled conditions e.g. for predicting behaviour and plant growth in a glasshouse, but not suitable for use under field conditions.
Several field and glasshouse trials were carried out in Spain, France and the Netherlands to evaluate the intrinsic efficacy of the SC and liposome formulations. Results from the trials were inconclusive due to poor natural populations of larvae, incremental weather conditions and In the case of a glasshouse trial in the Netherlands, artificially infested larvae escaping from plants. However, the trials did indicate that there was no significant difference in intrinsic activity between the SC and liposome formulations and that the formulations could be used equally well for both HaSNPV and SeMNPV baculoviruses.
Another objective of the project was the optimisation of the production process. Part of this objective was the selection of susceptible insect cells and stable virus strains for optimal infectivity, productivity and stability. For this experiments were carried out for the Selection of the optimal cell/virus combination with regards to bloactivity rather than growth and/or production using Holicoverpa armigers and Spodoptera exigua baculovirus (HaSNPV, SeMNPV).
From three H. armigera cell lines, the HzAM-1 cell line was selected. This cell line was susceptible for the used HaSNPV isolates, produced most PIBs per cell and grow very well on a tissue culture medium (CCM3 complemented with 2.5% FBS), which is used In a large-scale bioreactor. HaSNPV- SP1 isolate appeared to be the best isolate to infect insect cells and therefore optimisation of the virus- cell system was carried out. DNA analysis showed similarity with an isolate from China, HaSNPV- WIW. H. armigera larvae were infected with HaSNPV-SP1 isolated for the production of haemolymph and PIBs. HzAM-1 cells with a high passage number appeared to be less susceptible for the HaSNPV- SP1 isolate. Unfortunately there was not enough time left to work on the instability of the HaSNPV- SP1 virus during serial passage in cell culture. However previous experiments have indicated that HzAM-1 cells and HaSNPV-SP1 form a stable virus-cell system for the production of bioactive HaSNPV-SP1 in vitro.
From three S. exigua cell lines, at first the Se-IZD2109 cell line showed to be the most susceptible and productive cell line for the SeMNPV-US virus. However at a later stage it appeared that the Se30l cell line showed the best results. Both cell lines grow very well on the tissue culture medium, which is used in a large-scale bloreactor. Once Se30l cells were adapted to suspension in the bioreactor, however, they had become 250 fold less susceptible for the virus. S. exigua larvae were infected with SeMNPV-US and infectious haemolymph and PIBs were obtained. Analysis of the stability of SeMNPV-US (DNA) upon serial passage in cell culture of both Se30l (most susceptible) and SeUCR call line has been carried out. It appeared that the SeMNPV-US virus isolate is not stable during serial passage in cell culture. Deletions in the viral DNA could be observed not only in the most susceptible, but also in the other S. exigua cell line. The cell line used influences the rate and extent of deletion of the viral DNA. SeMNPV-US virus seems to be more stable during serial passage an Se3Ol than on SeUCR cell line.
The results obtained suggest that the generation/selection procedure of SeMNPV-US with genomic deletions is an intrinsic problem in the SeMNPV insect-cell system. In a pendent project it was found that a selection of deleted forms of SeMNPV occurred. There are more genotypes with dfferent biological activities within the field isolate. To enhance the stability of SeMNPV a novel strategy was employed involving the alternate cloning of SeMNPV in cell culture (using plaque assays) and in vivo production. The clones that were finally obtained in this way appeared to be mutants of SeMNPV, lacking about 12 kb of sequence information in their genome. They retained their biological activity and were easily produced in cell culture, which shows that the alternate cloning strategy is a successful and promising method to enhance viral stability.
The second part of the optimisation of the process involved the growth of cells and subsequent production of virus. The minimum inoculation density is an important parameter in the scale-up. Lower inoculation densities mean the number of scale-up steps can be reduced and low MOI's can be used, which in turn postpones the passage effect. Thus the effect of the inoculation density on the growth of dfferent insect cell lines growing in dfferent media was studied. Lower inoculation densitiesresulted in some cell lines having lower maximum growth rates in batch culture. The relation depends on the cell line and medium used. Hz-AM1, which had been maintained in the CCM3 medium in suspension culture for over a year, did not show a significant decline in growth rate even at the lowest measurable inoculation density of 1x104 cells/ml. In general this study showed that inoculation densities in suspension culture of 5x104 cells/ml, which is substantially lower than the standard 2-3x105 cells/ml, are achievable, also in STRS. The duration of the lag-phase showed no significant change.
Another important parameter is the maximum cell density reached that can be reached. More cells in general also means more PIB production. Using genetic algorithms, fed-batch fermentations of insect cells could be quickly optimised. An increase in density from 3.7x106 calls/ml to 14x
106 cells/ml was obtained after only 4 generations of optimisation. Through the use of low-volume additions of powdered components, a further increase is anticipated.
Further optimisation may be achieved through use of models to predict cell growth and subsequent PIB production as a function of process variables. For this purpose, a model, VCIMGP (virus cell interaction, metabolic growth and production) was developed to simulate the total cel growth, the total production of virus particles or heterologous protein and the total production of virions. The VCIMGP model consists of two parts, a virus-cell interaction part and a metabolic part. The virus-cell interaction part of the model is based on a previous developed model (VIPRO). The metabolic part predicts the production of new virions, new heterologous protein and growth of now cells. The consumption of the dfferent substrates is linked to these production processes and the maintenance of the living cells. The model could successfully simulate different reactor-systems with a wide range of parameter values. The model is stable at any realistic parameter value.
To obtain the parameter values for the model and obtain insight in the physiology of the insect cells a continuous culture with a slowly decreasing dilution rate (A-stat) was conducted. It was shown that the A-stat yields more information in the same amount of time and is comparable in results to chemostats at multiple steady states. Insect calls appeared to use a very high proportion of the glucose for maintenance, and only a small fraction for biomass synthesis.
In order to be able to perform metabolic flux analysis and thus gain more insight in insect-cell metabolism a number of analytical procedures were adapted from the literature and optimised for insect cells. Thus a method for determining the dry weight, ash content and C, H and N ratio was developed. The AAA-direct method of Dionex was acquired to be used for the analysis of amino acids and some sugars. The glucose and lactate concentration can be measured accurately with the Analox methods. The Lowry method turned out to be the best method for measuring proteins in insect cells and their culture medium, while the Anthrone method turned out to be the best method to measure the amount of carbohydrates in insect cells and call culture medium. The lipids could be measured with chloroform extraction.
Production on a pilot scale level (66 litre) was accomplished in this study using a very low multiplicity of infection (MOI) and early time of infection (TOI). However, for this strategy to work in an optimal way process variables, such as the virus titre, infection kinetics and cell growth must be precisely known. Therefore, further research to determining the titre of the virus and modelling of virus production is required.
For routine production a master and working cell bank should be developed. For this purpose a freezing procedure could be optimised to give a high success rate upon thawing. Furthermore the routine production requires that all procedures be conducted in the same way so as to assure reproducible runs. For this purpose a total of 38 tested SOPs were created.
The objectives of task 3 were to evaluate the virus half-life and the persistence of the virus in soil and on the leaf surface, study the effect on non-target species, evaluate the biosafety of the product and the process and finally to produce virus in vivo for product comparison. The effect of environmental factors (plant characteristics, UVB-irradiation from sunligtt) on the efficacy of the baculovirus HaSNPV was thoroughly studied. It was shown that the performance of HaSNPV is affected by the plant species on which the virus is transferred to the host Insect. Activity of HaSNPV was significantly reduced on foliage of cotton and alfalfa in comparison to cucumber and tomato.
In contrast, no significant effect of induced resistance in the food plant, caused by infection with a pathogen, by treatment with a botanical inducer or by herbivore damage on beculovirus activity was found. An improvement of the UV-persistence of HaSNPV formulated as liposomes was confirmed in experiments where polyhedra-layers on glass cover slips were exposed to simulated sunlight. Application of PIBs on plant foliage and subsequent irradiation did not allow any conclusions, as the variation of observed mortality was too high.
To assess biosafety of in vitro produced HaSNPV in field applications performed in Cordoba/Spain (1998) and Toulouse/France (1999), occurrence and persistence of the virus in soil samples of the field sites was evaluated by diet-incorporation bioassays with Helicoverpa armigera in the laboratory. Experiments showed that the viability of a virus deposit in the soil is limited to a short time period after field application.
The host range of in vitro and in vivo produced HaSNPV was investigated in bioassays including non-target species from several insect families. The beneficial lacewing Chrysoperla carnea, an important egg predator of the target pest H. armigera in the field, was not affected by virus treatment. Only few closely related Lepidopteren specieses (Heliothis virescens, Spodoptera littoralis, Spodoptera exigua) were susceptible to high virus inocula. Biological activity and pathogenesis of HaSNPV for Spodoptera exigua was thoroughly studied, supported by the use of a recombinant isolate of HaSNPV. Mortality was dose-dependent but signftantly delayed in S. exigua. Virus replication took place only in particular tissues of this heterologous host.
Storage stability assays were conducted using both formulated (suspension concentrate) and unformulated HaSNPV. The virus retained activity when stored at 4°C over a period of twelve months. Formulated virus appeared to be more stable at 25°C than unformulated virus, but both samples lost activity when stored at 40"C for one month. Studies were carried out over a twelve- month period. Concerning shelf life of highly diluted working solutions of both in vivo and in vitro produced HaSNPV, storage at 4°C in the dark was found to be a suitable way to preserve virus activity over longer time periods. It was found that the activity of freshly diluted virus suspensions was variable and dropped to a lower but more stable level after one month of storage. Virus stability of highly diluted suspensions was better preserved in Polyamid vials than in glass vials. To obtain reliable parameters of biological activity (LD50), bioessays should be repeated several times to reduce variation of results.
For product quality comparison and host range studies, continuous mass rearing cultures of 3 Lepidopteran species were established and batches of eggs or pupae of several other species were reared until hatching of larvae for the use in bioassays. Several isolates of HaSNPV were propagated in vivo, purified and enumerated.
The biosafety of the in vitro produced baculovirus was evaluated on non-host vertebrate organisms using in vitro techniques. The work carried out in this project consisted of a preliminary phase of obtaining and banking the cell lines intended for use, establishing and optimising the methods for the biosafety assessment. This was followed by a series of cytotoxicity studies, where different virus preparations, namely the polyhedrin included bodies (PIB), alkaline liberated occluded virus (ALOV) and non-occluded virus (NOV), were used to assess their cytotoxic potential on various mammalian cell lines, an amphibian (A6) and a fish (RTG-2) cell line as well as rat hopatocytes and human and mouse lymphocytes. The cytotoxicity tests used were the XTT test, where the vitality gf the cells after virus treatment was assessed, along with growth curve analysis and lymphoproliferation. The data showed that none of the virus preparations exerted any biologically relevant adverse effects on the tested cells. The cytotoxicity tests were followed by genotoxicity studies. The used test system was the in vitro micronucieus assay, which can analyse the mutagenic and aneugenic potential of the test items.
The different virus preparations were applied to all mentioned cell lines and human lyrtiphocytes. The data obtained indicated no increased rates of micronucleated cells after treatment with the virus. In order to analyse long term effects of exposure to the virus the cell lines were repeatedly treated with NOVs for over 4 weeks and then analysed for growth characteristics increased micromuclous rates or susceptibility to aneugens. The results showed that the long-term exposure to the virus did not affect the cells in any of the analysed parameters.
Finally, a series of studies were done to analyse whether any virus-host DNA interactions occur, which would lead to primary DNA breakage. The single cell gel electrophoresis (comet assay) method was used to analyse primary DNA damage. It could be shown that any supposed interactions of virus DNA with host DNA does not lead to DNA breakage, which is an indication of a lack of such DNA interaction. It can be concluded that the novel methods used to analyse the potential biohazard of the in vitro produced virus could not find any biologically relevant effects, thus indicating the biosefety of the test item.
Conclusions
Overall it can be concluded that the production of a baculovirus biopesticide is possible. Production at pilot-scale is possible and the product is biosafe with respect to the performed tests. The virus can be formulated in such a way that UV degradation is minimised. Furthermore, the virus can be stored for periods of at least one year.
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