Insect Control: Biological and Synthetic Agents - Index of

358 10: Genetically Modified Baculoviruses for Pest Insect Control
vEGT - PTTHM infected larvae were in the conjugated
form in comparison to vEGTDEL infected
larvae.
An elegant approach to further improve the egt
gene deletion construct has been to insert an insect
selective neurotoxin gene cassette into the egt gene
locus, thus generating a toxin expressing, egt
gene inactivated construct. For example, Chen et al.
(2000) inserted an AaIT-GFP gene cassette (aait and
green fluorescent protein (gfp) genes driven by the
polh gene promoters of HaSNPV and AcMNPV,
respectively) into the egt gene locus of HaSNPV
(also known as HearNPV) in order to generate
HaCXW2. In time-mortality bioassays, they found
that the ST50 of second instar H. armigera infected
with HaCXW2 was reduced by 32% (63.9 versus
94.4 h) and 8% (63.9 versus 69.5 h) in comparison
to control larvae infected with wild-type HaSNPV
or HaCXW1 (a recombinant HaSNPV carrying gfp
at its egt gene locus). Sun et al. (2002, 2004) also
showed that HaCXW1 and HaCXW2 were effective
pest control agents under field conditions. In
their field experiments, second instar H. armigera
were allowed to feed on HaCXW1 or HaCXW2
treated plots (1.8 10 10 polyhedra per 0.015 hectare
plot containing 800 cotton plants) for 12 h, then
the larvae were transferred to artificial diet and
reared at ambient temperature. The ST50 values of
the larvae infected with HaCXW1 or HaCXW2 in
the field were reduced by 15% (101 versus 119 h)
and 26% (88 versus 119 h), respectively, in comparison
to control larvae that were field infected with
HaSNPV. Treatment of cotton plants in the field
with HaCXW1 or HaCXW2 reduced leaf consumption
by approximately 50% and 63%, respectively.
The results of additional field trials using HaSNPV-
AaIT (a recombinant HaSNPV expressing AaIT
under a chimeric ph-p69p promoter at the egt gene
locus) are discussed below.
From these results it appears that deletion of the
egt gene from the baculovirus genome is a useful
strategy to improve its speed of kill and consequently
its insecticidal activity, but by itself the reduction
in speed of kill is too small to be of commercial
value. Deletion of the egt gene, however, reduces
the number of progeny viruses that are produced in
comparison to the wild-type virus. Thus, the fitness
of the egt gene deleted virus appears to be reduced in
comparison to the wild-type virus. Conceptually,
removal of an endogenous gene from its genome
is safer than the insertion of a heterologous gene
because a ‘‘new’’ gene will not be introduced into
the environment. In practice, however, it is believed
that there will be no significant difference in the risk
of a recombinant baculovirus pesticide in which
an endogenous gene is deleted from the genome
or in which a heterologous gene is inserted into the
genome. Safety issues and fitness of the virus are
further discussed below.
10.4.2. Removal of Other Nonessential Genes
In a comparative study of seven completely sequenced
lepidopteran baculovirus genomes, Hayakawa et al.
(2000a) identified 67 putative genes that were common
to all seven. When Herniou et al. (2001, 2003)
added four additional lepidopteran NPVs and a GV
(i.e., a total of 12 completely sequenced lepidopteran
baculoviruses) 62 common genes were identified.
Considering that the genomes of most baculoviruses
encode well over 100 putative genes, these findings
suggest that a large number of genes in the genome
of lepidopteran baculoviruses may serve auxiliary or
host specific functions or help to improve the fitness
of the virus. This hypothesis is consistent with the
finding that 61 out of the 136 putative genes of
BmNPV appear not to be essential for viral replication
in BmN cells, i.e., some level of viral replication
was detected even when a gene was deleted by replacement
with a lacZ gene cassette (Kamita et al.,
2003b; Gomi, Kamita, and Maeda, unpublished
data). These findings suggest that there are a
number of target genes that can be removed from
the baculovirus genome in order to decrease fitness,
alter host specificity, or simply to obtain a virus that
may replicate faster or more efficiently.
Dai et al. (2000) have identified a mutant of
S. exigua MNPV (SeXD1) that is lacking 10.6 kb
of sequence (encoding 14 complete or partial genes
including an egt gene homolog) that corresponds to
the region between 13.7 and 21.6 map units in the
wild-type SeMNPV genome. Dose-mortality bioassays
in third instar larvae of S. exigua showed that
the LD50s of SeXD1 and SeMNPV (403 and 124
polyhedra per larva, respectively) were not significantly
different. Time-mortality bioassays indicated
that the ST50 of third instar S. exigua infected
with SeXD1 was reduced by 25% (70.2 versus
93.1 h) in comparison to control larvae infected
with SeMNPV. Dai et al. (2000) did not further
examine which gene or genes in this 10.6 kb region
are involved in decreasing the virulence of SeXD1
in comparison to SeMNPV. Considering that deletion
of the egt gene from other baculoviruses has
been shown to reduce the survival times of larvae
infected with these viruses, the egt gene homolog of
SeMNPV appears to be a prime candidate as the
gene involved in reducing the virulence. However,
several of the other genes that were deleted in
this region are commonly found in other baculoviruses
and may play roles in enhancing infection