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LARGE-SCALE PRODUCTION OF VIRAL BIOPESTICIDES IN INSECT CELL CULTURES TO REDUCE WIDE-SPECTRUM INSECTICIDE USE

Objective

Baculoviruses are potentially a valuable, environmentally-safe means of crop pest control. They are capable of infecting, in a highly specific manner, several of the major pest species. Established insect-cell lines and commonly used baculoviruses are poorly suited for biological pest control purposes. Therefore this research project was started in 1993 to:

1. isolate insects and baculoviruses from nature,
2. establish cell lines from the insects,
3. scale-up and optimize insect-cell cultures,
4. produce wild-type baculovirus (PlBs),
5. formulate these PlBs,
6. define dosage and stability of the virus, and
7. use these formulated baculoviruses for field testing in the country of origin of the insects.

Chemical crop protecting agents may impose considerable environmental stress and induce resistance in insects. Therefore, bioinsecticides form a challenging alternative. Especially baculoviruses are of interest, since they are highly specific for the target pest, and no resistance has been reported so far. The aim of this project is the development of an insect-cell-culture process on an industrial scale for the production of a viral biopesticide. Traditionally, baculoviruses are produced in insect larvae. This process is laborious and difficult to scale-up. Moreover, insects are easily contaminated. Modern cell-culture technology facilitates the growth of insect cells in suspension in bioreactors. Successively, those cells may be infected with the extracellular form of the baculovirus, after which the cells will produce the occluded form of the virus (Polyhedra Inclusion Bodies, PlBs) found in nature.
New SeNPV and HaNPV strains were isolated from the Iberian Peninsula, and the NPV isolates were characterized. Insecticidal activity of the isolates were determined but no significant differences were found. Two new cell lines, now established, emerged from Se, with optimized growth in serum-free suspension cultures. The existing Se cell line UCR-Se1 (USe) was adapted to different media including serum-free formulations and to growth in suspension as shake flask- and spinner-cultures. Polyhedral Inclusion Body (PIB) production was satisfactorily. Four new Ha primary cell lines emerged, of which one is proliferating fast with a regular cell morphology. The existing Heliothis zea (Hz) cell line was adapted to serum-free suspension cultures. All cells were characterized and distinguished from each other by DNA-fingerprinting using the RAPD-PCR method, and saved in a cell bank. All cell lines were susceptible to AcMNPV virus. The PlBs produced in Se cells are smaller with a larger number per cell. SeMNPV-lnfected Se cells release a low amount of 17 extracellular viruses (ECV) per cell. Serial passaging of SeMNPV in cell culture resulted in deletions in the viral genome already after 1 passage. Tests of PlBs showed a reduced bioactivity already after 3 passages. Storage condition studies showed SeMNPV hemolymph to be relatively unstable. Hemolymph should be stored at -20 or - 80°C. Large scale production of viral biopesticides (of Se and Ha) in serum-free medium was shown in a 66 liter airlift with Se and Hz cells respectively. Shear effects can be minimized by the effective use of airbubbles in airlifts and/or the use of medium additives. Detailed studies at large scale did not show significant scaleup effects. All the cell lines tested showed the same shear sensitivity.

A mathematical model describing the behaviour of insects, formulations and spray applications in the field was constructed, in order to optimise formulations in a targeted way. Due to the variable and time consuming nature of conventional in vivo baculovirus bioassay methods an in vitro test method was developed. The method was used to quantify baculovirus temperature and simulated sunlight inactivation. Correlation with the in vitro method was good for both stress factors. A gain in efficiency and data quality was clearly demonstrated. Temperature stability studies demonstrated that freeze dried HaSNPV remained infectious when stored at temperatures between -80°C and +35°C for at least 50 days (experiment still ongoing). At 50°C there was a rapid loss of infectivity. UV stability studies confirmed that baculoviruses are rapidly inactivated by UV irradiation and that successful baculovirus formulations need to provide effective UV protection. The UV assay system allowed the testing of potential UV protectants and the protective effect of propyl gallate was demonstrated. The infectivity of in vitro and in vivo produced virus was compared using both bioassays. It was shown that there was no difference in inherent infectivity of in vitro and in vivo produced HaSNPV and AcMNPV. Furthermore the extraction and purification of in vitro produced baculovirus was shown to be much easier and with a much higher efficiency compared to in vivo produced virus. The results of a field trial carried out on alfalfa showed no differences in insecticidal activity between in vitro and in vivo produced SeMNPV.

Field test

In the summer of 1995, the formulated virus was field tested in Spain by the University. together with Zenecca. To support the latter activity, a computer application model for the virus was developed.

- difficulty of production: significant progress has been made in developing better, more productive cell lines and practical production systems
- limited choice of strains: new strains of baculovirus, with activity against key European pests, have been identified
- poor formulation and application methodology: new high-throughput systems for evaluating the stability of virus formulations have been developed, as has a model for simulating application to the crop in order to maximise application efficiency.

Exploitation

The total annual world market for all insecticides is about 6 billion ECU, whereas for biologicals this is 150 million ECU, of which baculoviruses account for less than 10 million ECU of sales. The methods developed, for example the TClDso assay, are available for further optimisation of baculovirus formulations. This assay provides a more rapid, reliable and efficient means of assessing baculovirus efficacy. The computer model can be used to examine the effects of numerous factors influencing the efficacy of baculovirus sprays. This includes plants of different forms, being different crops or different cultivars, and different application methods. The whole development process has also suggested the potential of extending similar methods to a wide range of crop protection situations and therefore assist in the rationalisation of herbicide/fungicide/pesticide and formulation development. An economic analysis indicated that at a medium price of around one ECU per liter, virus can be produced in vitro for 4.5 ECU/ha, which compares favourably with an in vivo production, costing 16.2 ECU/ha. Hence, a large-scale production facility will have a significant commercially potential.
The University of Cordoba isolated and selected new strains of bacteria (denoted as SeMNPV and HaSNPV), developing a practical bioassay technique to test the biological activity of the viruses, and designing and running field trials with bioreactor-produced virus and testing novel spray formulations. An essential requirement for large-scale production of baculovirus in vitro is an optimal cellular substrate. The Technical University of Darmstadt had to develop those. For the replication of SeMNPV a cell line was available, but not optimized for growth in bioreactors. HaSNPV can be propagated in Hz cells, which had to be adapted to conditions for mass production of cells. Therefore, the development of new cell lines from Se and Ha was a major task within the project. Existing and newly generated cell lines were analyzed with regard to growth characteristics and virus replication and adapted to growth in suspension using low-cost, serum-free media. To optimize the in vitro production of viral biopesticides information is needed about large-scale growth of insect cells and their infection kinetics, which was the task for Wageningen Agricultural University. To facilitate large-scale cultivation of insect cells shear stress shas to be minimized in combination with an adequate oxygen supply. Studies in small airlifts or bubble columns showed that damage of cells is associated with cell bubble interactions. The effect of height, gasflow, cell line and medium composition on the death rate of cells was investigated. To show the possibility of producing viral biopesticides, an existing and a newly established cell line of Se and Hz were used for infection kinetics studies and production of baculoviruses. A major factor which has limited the commercial use of baculoviruses to date has been suboptimal formulation technology. The commerical partner, Zeneca, therefore designed formulations and methods of application.

The Lepidoptera (Noctuidae) species Spodoptera exigua (Se) and Heliothis armigera (Ha) are major pests on important crops like tomato, corn and cotton. Baculoviruses, specific for these insect species, are a potential alternative to chemical pesticides.

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Coordinator

WAGENINGEN AGRICULTURAL UNIVERSITY
Address
P.o. Box 8129
6700 EV Wageningen
Netherlands

Participants (3)

TECHNISCHE UNIVERSITÄT DARMSTADT
Germany
Address
Karolinenplatz 5
64287 Darmstadt
UNIVERSIDAD DE CORDOBA
Spain
Address
Etsia Aptdo De Correos 3048
14080 Cordoba
Zeneca Ltd.
United Kingdom
Address
Jealott's Hill Research Station
RG12 6EY Bracknell