Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-08-31
2001-01-30
Mosher, Mary E. (Department: 1641)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S069100, C435S235100, C435S320100
Reexamination Certificate
active
06180098
ABSTRACT:
This invention relates to the production of recombinant
Helicoverpa armigera
nuclear polyhedrosis viruses (HaSNPV) capable of expressing heterologous DNA sequences. In one particular application of the invention, the recombinant HaSNPVs are used as biological insecticides for the control of Heliothis and Helicoverpa pest species.
Nuclear polyhedrosis viruses (NPVs) are large double-stranded DNA viruses of insects and crustaceans in which the rod-shaped nucleocapsids are enclosed in a lipoprotein membrane. NPVs are a genus in the family Baculoviridae, and are characterised by multiple virus particles being occluded in a large, virally encoded, pseudo-crystalline protein matrix known as the polyhedron. Also included in the Baculoviridae are the granulosis viruses (in which only a single virus particle is occluded into the polyhedron) and the non-occluded baculoviruses (that produce no inclusion body at all).
The polyhedra of NPVs are characteristically polyhedral or cuboidal in shape and between 1-15 &mgr;m in diameter. Within the polyhedron the nucleocapsids are arranged in two distinct ways. In the case of the multiple enveloped NPVs (MNPVs) one or more nucleocapsids share the same lipoprotein membrane, while in the case of the single enveloped NPVs (SNPVs) each nucleocapsid has its own membrane. In both SNPVs and MNPVs the polyhedron itself is comprised almost entirely of a single protein, polyhedrin, encoded by a single viral gene. It is around this gene sequence, and in particular the DNA sequences (promoter) that control its expression, that technologies have been developed for the insertion of heterologous DNA sequences into the baculovirus genome. Indeed, three species of NPV namely Alfalfa looper
Autographa californica
(AcMNPV), the SNPV of the silkworm
Bombyx mori
(BmSNPV) and the SNPV of the corn earworm
Helicoverpa zea
(HzSNPV), have been engineered in this manner to express a wide range of viral, bacterial, plant and animal genes and have been expressed in permissive hosts in both cell culture and insects.
The promoters of genes other than polyhedrin have also been utilised for expression of heterologous DNA sequences in AcMPNV. For instance, the promoter of the gene, p10, has been used to express a wide range of genes and the promoters of basic core protein and ORF 603, has also been used to drive expression of reporter genes. Further, recombinant AcMNPVs have been constructed which contain synthetic promoters (Miller, 1990), modifications of existing promoter sequences (Kang, 1990) and heterologous promoters (Miller, 1991). It has also proven possible to add more copies of a promoter (Emery and Bishop, 1987; Takehara et al., 1988), in some instances up to three additional promoters, to allow the co-expression of up to four heterologous proteins (Belyaev and Roy, 1993).
Due to their high pathogenicity to insects, and the large range of insects from which baculoviruses have been isolated, several NPVs have been employed as biological insecticides. In a few cases these viruses have been applied over large areas to achieve insect pest control, and a proportion of these have been commercially or semi-commercially produced. Even though they are high infectious to their hosts, many baculoviruses can not be used for insecticidal purposes in their wild-type form as they take a relatively long time to kill an infected individual. Considerable interest has therefore been shown in the manipulation of baculoviruses, especially NPVs, to improve their speed of action. To achieve this, recombinant viruses have been generated that express heterologous DNA sequences encoding proteinaceous agents that are toxic or otherwise deleterious to their insect host. Such manipulations in AcMNPV include the insertion of genes encoding proteinaceous toxins from the straw itch mite
Pyemotes tritici
(Tomalski and Miller, 1991) and the scorpion
Androctonus australis
(Stewart et al, 1991). In these examples the expression of the heterologous DNA sequences led to significant reductions in the LT
50
(time at which 50% of the test organisms had died/been paralysed). In other examples, improved insecticidal properties have been achieved with heterologous genes that express proteins that disrupt the normal physiology and hormonal control mechanisms of the host. In one instance, a gene encoding juvenile hormone esterase (JHE) was expressed by a recombinant AcMNPV (Hammock et al. 1990) giving some improvement in insecticidal potential. In a later example, a recombinant AcMNPV expressing an inactive form of JHE gave further improvement in insecticidal potential. Further, Maeda (1989) demonstrated that a recombinant BmSNPV expressing a synthetic gene encoding the diuretic hormone of
Manduca sexta
reduced the time to death by about 20%.
It is noteworthy that not all toxin encoding genes that have been inserted into NPVs lead to the generation of recombinant viruses with improved insecticidal potential. Insertion of the gene encoding the delta-endotoxin of the entomopathogenic bacterium
Bacillus thuringiensis
subsp. kurstaki did not generate AcMNPV recombinants with improved insecticidal potential. Likewise, insertion of a gene synthesised from the known protein sequence of a proteinaceous toxin from the scorpion
Buthus europeus
did not improve the insecticidal potential of AcMNPV (Carbonell et al., 1988).
In most of the above examples, the virus is produced in cell culture and is genetically polyhedrin negative (pol−), with the inserted gene disrupting expression of the polyhedrin gene. Despite the fact that these viruses are incapable of occlusion, it has been found that pre-occluded viruses (POVs), that is viral progeny produced for inclusion into polyhedra, are infectious per os to their host organisms (Wood et al, 1993).
The insect genus Helicoverpa/Heliothis include a number of species which are major pests of broadacre crops. For the development of recombinant virus-based insecticides for these species, it is highly desirable that suitable viruses be identified which are infectious to most, if not all, of the major pest species but are not infectious to non-Helicoverpa/Heliothis insects. In this regard, it has been found that AcMNPV does not have an appropriate infectivity range.
For instance, Vail et al. (1978) demonstrated that
Helicoverpa zea
was between 12.9 and 68.9 times less susceptible to AcMNPV than was
Heliothis virescens.
Furthermore, we ourselves have shown that
H. armigera
is over a thousand (1,000) times less susceptible than
H. punctigera
to AcMNPV. In contrast, HzSNPV shows infectivity to most Helicoverpa/Heliothis species (with the notable exception of
H. subflexa
which is about 1000 fold less susceptible than any other species), but has not been found to infect any species outside of the subfamily Heliothinae. Consequently, HzSNPV appears to be a suitable candidate for development for use as a Helicoverpa/Heliothis specific insecticide.
The present invention have now identified a further candidate virus for the development of recombinant virus-based insecticides for Helicoverpa/Heliothis, in
Helicoverpa armigera
SNPV.
Accordingly, in a first aspect of the present invention relates to a recombinant HaSNPV characterised in that heterologous DNA is located in one or more non-essential regions of the viral genome, in a manner to permit the expression of the heterologous DNA, and wherein said recombinant HaSNPV is pol
+
.
Preferably, the recombinant HaSNPV is prepared from an HaSNPV isolate with a polyhedrin gene including a nucleotide sequence greater than 95% (more preferably, greater than 99%) homologous to any of the nucleotide sequences shown in FIG.
4
. Alternatively, the recombinant HaSNPV is prepared from an HaSNPV isolate having a Bam H
1
restriction fragment size profile as shown in Table 1, or includes a nucleotide sequence greater than 95% (more preferably, greater than 99%) homologous to the nucleotide sequence shown in FIG.
3
.
Recombinant HaSNPV according to the invention may be used as biological insecticides, optionally in adm
Commonwealth Scientific and Industrial Research Organisation
McDermott & Will & Emery
Mosher Mary E.
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