| Abstract |
Baculoviruses have a significant potential as biological pesticides.
Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) could
thus be applied to protect plants against the African Cotton Leafworm. For
the in vitro production of SpliMNPV a cellular system has to be
established. For this purpose three new continuous cell lines were
established from embryonic tissues of S. littoralis. These three cell
lines were designated as Spli-C, Spli-S and Spli-B. They consist mostly of
spherical cells in addition to spindle and giant cells. The population
doubling time for Spli-C, Spli-S and Spli-B were 30.5, 31 and 44.5 h,
respectively, at passage 19, while at passage 120 it was 26, 27 and 32 h,
respectively. The DNA fingerprinting techniques RAPD and DAF confirmed
that the cell lines originated from S. littoralis tissues. Lactate
dehydrogenase (LDH) isozyme analysis showed distinguishable differences
between the three new cell lines and the other insect cell lines used in
our laboratory. Susceptibility tests of the three cell lines showed that
all the cell lines were non-permissive to Autographa californica
multicapsid nucleopolyhedrovirus (AcMNPV), with the cells dying a few
hours post-infection and forming apoptosis-like bodies. Nuclear DNA
fragmentation was observed in all AcMNPV-infected cell lines by DNA gel
electrophoresis analysis. At same time, the susceptibility of the three
cell lines to SpliMNPV showed that all the cell lines were susceptible,
whereby many viral occlusion bodies (OBs) were observed inside the
infected cells. The SpliMNPV OB productivity of the three cell lines
showed significant differences. The cell line Spli-C had the highest
susceptibility to SpliMNPV compared to the other two cell lines. The
average of OB production was about 1x10^7 OBs/ml in the Spli-C cell line,
while in Spli-S and Spli-B it was 3.3x10^6 and 2x10^6 OBs/ml, respectively.
In addition, the SpliMNPV polyhedrin gene expression and DNA replication
in the three cell lines were also investigated over time using
quantitative PCR (qPCR). The Spli-C cell line that showed higher OB
production than the other two cell lines was immobilized using sodium
cellulose sulfate (NaCS) and poly-diallyldimethylammonium chloride
(PDADMAC) capsules to protect cells from shear stress caused by agitation
and gas sparging during supply of sufficient oxygen. Cell culture in
capsules leads to high cell densities resulting in increased viral OB
production per volume. The Spli-C cell densities were increased from
4-5x10^6 cells/ml in suspension culture to 1.3x10^7 cells/ml in capsules.
Summarizing, our results suggest that large-scale production of SpliMNPV
as a biopesticide is possible with the new S. littoralis cell lines Spli-C,
Spli-S and Spli-B.
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