Insect Control: Biological and Synthetic Agents - Index of

338 10: Genetically Modified Baculoviruses for Pest Insect Control
recombinant AcMNPV may be able to induce the
RNAi response more efficiently. Of course, a recombinant
virus that is effective at prolonging the
juvenile state would be a disaster as a biological
insecticide. However, these experiments are important
in that they demonstrate the effectiveness of
an RNAi approach in the regulation of insect
neuroendocrinology and the results should be
easily transferred to other significant physiological
effectors.
The use of an authentic, insect-derived protein
to combat the insect is conceptually elegant and
potentially safer because one is only trying to
express an endogenous protein at an inappropriate
time or at higher levels in order to alter the physiology
of the insect. Additionally, there is a perception
that expressing the insect’s own protein at an
inappropriate time in development offers public
relations advantages over the expression of toxins
or proteases as will be discussed below. In practice,
however, overexpression of an insect hormone or
protein involved in hormone metabolism has not
been dramatically effective. For example, the limited
number of JHEs tested to date not enhance the
speed of kill as well as scorpion toxins or proteases.
However, with JHE there is a clear mechanism of
action and an obvious path toward generating improved
viruses by engineering the protein for greater
stability in the insect. The recent homology model of
Thomas et al. (1999) and crystal structure of the
protein will greatly enhance these efforts.
10.2.3. Proteases
In order to better understand the rationale behind
the use of protease encoding genes for improving
speed of kill, a brief review of the midgut barriers
faced by the baculovirus is given. The first barrier
that the ODV encounters in the midgut is the fluid
within the lumen. The high pH (8–11) and endogenous
proteases found in the lumen are initially
required to release ODVs from the polyhedra or
granules. However, digestive enzymes or other factors
within the lumen could also lead to the
inactivation of the released ODV, if the ODVs do
not quickly initiate infection. One such factor is a
lipase, Bmlipase-1, isolated from the digestive juice
of B. mori that shows antiviral activity against
BmNPV (Ponnuvel et al., 2003). The peritrophic
membrane (PM; or peritrophic envelope) is the first
physical barrier that the ODV faces (Richards and
Richards, 1977; Brandt et al., 1978; Tellam, 1996).
The PM is composed of chitin fibrils in a protein–
carbohydrate matrix. The specific composition and
layout of the PM differs in different lepidopteran
species and at different larval instars. The manner
in which the ODVs pass this barrier is still not well
understood. The second physical barrier that the
virus faces is the midgut epithelium. The midgut
epithelium is primarily composed of a single layer
of columnar cells. The columnar cells show directionality
(polarity) in which the luminal border is
formed by microvilli (brush border membrane)
and the hemocoelic border shows characteristic
infolding with large numbers of mitochondria
associated with the infolds. Regenerative and endocrine
cells are also part of the midgut epithelium.
Several pathways have been proposed for how the
nucleocapsids pass through the midgut epithelium
barrier following the fusion of ODVs with the
microvillar membrane (Federici, 1997). One pathway
involves entry of the nucleocapsid into the
columnar cells, translocation through the cytoplasm,
and budding through the hemocoelic border
of the same cell without any viral replication. The
other pathways involve the generation of progeny
nucleocapsids within the columnar cells that can
bud (1) through the hemocoelic border, (2) into the
tracheal matrix via tracheoblasts, or (3) into regenerative
cells. Once in the regenerative cells, the
nucleocapsids may translocate through the cell
without replication and through the hemocoelic
border or virus replication may occur in these cells
as well. Washburn et al. (2003) have proposed a
‘‘hybrid’’ pathway in which some nucleocapsids of
MNPVs uncoat and start replication whereas others
pass directly through the columnar cells. The third
midgut associated barrier to systemic infection is the
basement membrane (or basal lamina). The basement
membrane (BM) is a fibrous matrix composed
primarily of glycoproteins, type IV collagen, and
laminin, which are secreted by the epithelial cells
(Ryerse, 1998). The BM functions in several roles
including structural support of the epithelium or
surrounding tissues, filtration, and differentiation
(Yurchenco and O’Rear, 1993).
10.2.3.1. Enhancins Enhancins are baculovirus
encoded proteins that can enhance the oral infectivity
of a heterologous or homologous baculovirus.
Enhancin, also known as synergistic factor (SF) or
virus enhancing factor (VEF), was first described by
Tanada as a lipoprotein of 93–126 kDa found within
the capsule of the GV of the armyworm Pseudaletia
unipuncta (Yamamoto and Tanada, 1978a,
1978b) that enhances the infection of baculoviruses
in lepidopteran larvae (Tanada, 1959). The enhancin
of the GV of T. ni (TnGV) has been shown to
possess metalloprotease activity (Lepore et al., 1996;
Wang and Granados, 1997). Tanada first suggested