of genetically modified baculoviruses for insect control
Baculoviruses are insect pathogens that are used as biological control
agents of insect pests as alternatives to chemical insecticides. Safety
testing confirms that baculoviruses are insect specific and cause no hazards
to beneficial insects, other animals or to plants. The major drawback
for the commercial use of baculoviruses is their slow action, and this
is especially relevant for crops which have low damage thresholds. They
also have a limited host range and are costly to produce. These problems
called for the improvement of the insecticidal ability of baculovirus
strains by genetic modification.
This study aimed to test the biosafety of genetically modified baculovirus
so that their behaviour in the environment can be predicted.
Approach and methodology
The behaviour of genetically-modified baculoviruses was assessed by the
construction of baculoviruses with increased virulence, but reduced persistence
and survival in the environment. The baculovirus Autographa californica
nuclear polyhedrosis virus (AcNPV) provided a suitable model. AcNPV was
marked, and either a suicide mechanism incorporated or deletions introduced
to reduce its persistence and survival in the field. The biosafety of
the recombinant AcNPV baculovirus was compared to that of the wild type.
we designed and tested a microcosm system and carried out limited field
release of the genetically modified AcNPV.
Main findings and outcome
The deletion of six AcNPV genes, believed to affect the persistence, survival
and spread of AcNPV in the environment, did not affect the replication
of AcNPV or its efficiency as an insecticide. Thus, deletion mutants in
these genes may have a reduced ability to spread in the environment. Single,
double and triple mutants were constructed, with and without the lacZ
reporter gene. The promoters of the deleted genes were left intact to
avoid disturbing the overall transcription during virus replication.
Attempts were made to construct a suicide recombinant by placing the Escherichia
coli lac operator (lacO) up- or downstream of the polyhedrin
(ph) gene transcriptional start site and the lac repressor (lacI)
under the control of the Drosophila heat shock promoter hsp70.
Thus, in theory, the expression of ph and the production of polyhedra
is blocked in the field and unblocked by the addition of IPTG. In practice,
the introduction of lacO was insufficient to block the expression
To study UV-persistence, dry virus deposits were inactivated by direct
exposure to artificial UV-sunlight. There was no detectable difference
in half-life between the wild-type AcNPV and an AcNPVpe- mutant
(resulting in envelope-less polyhedra). The loss of the polyhedron envelope
does not appear to increase UV sensitivity.
Comparison of the six AcNPV deletion mutants in a sensitive host showed
that the infectivity of single deletion mutants does not differ significantly
from the wild type. However, double deletions may be less infective.
The survival and spread of genetically modified viruses was compared with
that of non-modified viruses after their release into the environment.
Mixed infections of wild type and recombinant AcNPV were passaged in cohorts
of insects. After each passage the proportion of each virus type in the
mixture was estimated and the remainder re-passaged. After several passages
the recombinant was lost from the system, suggesting reduced biological
fitness. Similar experiments were run in which one virus was introduced
a set time after the other. This showed that the first virus does not
prevent infection by a second virus, but does interfere with its replication,
significantly reducing its titre. Consequently, the opportunities for
recombination between genetically modified and non-modified baculoviruses
are considerably reduced when infection with another virus is asynchronous.
Due to concerns that baculoviruses may interact with non-target Lepidoptera
we examined the extent of infection in permissive, semi-permissive and
poorly permissive hosts. In the semi-permissive host the level of infection
was determined by the dose/host ratio, mortality only occurring at a certain
dose threshold. In the poorly permissive host no mortality occurred regardless
of the dose.
Genetically modified AcNPV were tested in a contained microcosm prior
to field release to obtain data about their yield and dispersal. Two mutants
both carrying a LacZ reporter gene were extensively tested. The
microcosm was separated into two compartments by an insect screen that
prevented larval migration but allowed air circulation. Each compartment
contained soil, sugar beet plants and second instar larvae of S. exigua.
Wild-type or recombinant AcNPVs were applied to one compartment. After
one larval generation the quantity of virus in the soil, plants and water
was determined. There was no difference between the infectivity of the
wild type and that of the recombinant AcNPV. The spread of wild type AcNPV
was however five times greater than the recombinant. Equal quantities
of non-modified and genetically modified viruses resulted in only small
differences in larval mortality (39 and 48% respectively). The recombinants
were better retained in the upper layers of the soil whereas the wild
types penetrated the throughout the soil and contaminated infiltration
We successfully generated stable genetically modified baculoviruses with
multiple deletions whilst maintaining their specificity and biological
activity. We tested the behaviour of single deletion mutants in a microcosm
and demonstrated that they have a reduced ability to spread. Genetically
modified baculoviruses may be less competitive than the wild type during
asynchronous mixed infections.
The trachea was identified as the main conduit for dissemination of infection
through the insect hosts body.
October 1991 September 1993
Wageningen Agricultural University (NL)
Institute of Virology and Environmental Microbiology
Biologische Bundesanstalt für Land- und Forstwirtschaft