AU711559B2 - Recombinant Nudaurelia Beta-like virus and vectors - Google Patents

Description

I a..

a a..

a a 1

AUSTRALIA

Patents Act 1990 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION and RHODES UNIVERSITY

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Recombinant Nudaurelia f-like virus and vectors The following statement is a full description of this invention including the best method of performing it known to us:r Field of the Invention: This invention relates to recombinant insect viruses, particularly recombinant Nudaurelia P-like viruses, isolated nucleic acid molecules encoding insect virus genomes, and to the uses thereof. One particular application of the viruses according to the invention resides in their use as biological insecticides.

Background to the Invention: In recent years, considerable effort has been directed to the identification and manipulation of insect viruses for use as biological insecticides. As part of this effort, the present inventors have been working '*with members of the Tetraviridae family of small RNA viruses. This family of viruses first generated attention when spectacular epizotics occurred amongst larval populations of the pine tree emperor moth, Nudaurelia 15 cytherea capensis. Larvae of this moth reached extremely high densities in .the early part of this century in South Africa, completely denuding large areas of plantations of Pinus radiata. In the 1940's Tooke and Hubbard (1941) noted a characteristic disease began to occur that resulted in large S* numbers of larvae dieing and to literally carpet the ground beneath the trees.

Local workers found the diseased insects to be infested by five small RNA viruses they termed y, 6, c, e and p in order of increasing prevalence and physical studies on them were reported (Hendry et al., 1968; Juckes, 1970).

Preceding the reports, however, was a report on a similar virus in Australia that infected the emperor gum moth, Antheraea eucalypti (Grace and Mercer, 1965). Nevertheless, due to the more extensive work on the most prevalent South African virus, the Nudaurelia 3 virus (NpV) became the type virus for the group; it is now the type species of the genus "Nudaurelia P-like viruses" within the family.

Early physical studies combined with in vitro translations determined the group to possess a single positive-sense, single-stranded RNA genome of 1.8 Mda encased in an unenveloped, icosahedral capsid having diameters of 35-40 nM. The studies also established that the group possessed a unique lattice symmetry in their capsid structure and it is this from the Greek tettares, four) that formed the basis for renaming the group the Tetraviridae (Francki et al., 1991).

Later, another tetra-like virus, the Nudaurelia co virus (NoV), was found in Nudaurelia carcasses and was determined to possess a second genomic strand which made it distinct from the others (Hendry et al., 1985).

This virus became the type virus for a second genus, the co-like genus, within the Tetraviridae (Murphy et al., 1995) and was joined by a second similar virus, the Helicoverpa armigera stunt virus (HaSV), found in Australia (Hanzlik et al., 1993).

The two genera of the Tetraviridae are presently distinguished on the basis of their having either a monopartite or bipartite genome as judged by analysis of RNA extracted from purified virus. However, the classification of many P-like viruses was made difficult by the poor state of the RNA and the techniques available at the time. Indeed, recent work on RNA of P-like viruses shows that while it is still correct to classify the two groups on the basis of the number of genomic strands, virus particles from both genera can 15 contain two species of RNA. This confusing picture arises from the identification of a 2.5 kb subgenomic RNA which is coencapsidated with the single 6.5 kb genomic RNA in particles of NpV.

Despite significant serological relatedness, Nudaurelia P-like viruses appear to lack nucleotide sequence homology. For example, the genomic RNAs of Trichoplusia ni (TnV) and Dasychira pudibunda (DpV) were found to lack sequence homology by solution-hybridisation (King and Moore, 1985).

The present inventors have now isolated and sequenced the genomic RNA of NpV. This represents the first available sequence information from the Nudaurelia P-like genus and allows for the manipulation of this virus for, for example, use in biological insecticides and also to enable production of the virus in commercial quantities.

Disclosure of the Invention: Thus, in a first aspect, the present invention provides an isolated nucleic acid molecule of 15 or more nucleotides in length, the nucleic acid molecule comprising a nucleotide sequence which hybridises under medium or high stringency conditions (Sambrook et al., 1989) to the genomic RNA sequence, or a protein-encoding or non-protein-encoding portion thereof, of Nudaurelia p virus (NpV) or other serologically related Nudaurelia P-like virus.

4 Nudaurelia p-like viruses which comprise proteins capable of generating antibodies which are immunologically reactive with the large (60.5 kD) capsid protein of NpV are to be regarded as being "serologically related" to NpV.

Preferably, the nucleic acid molecule according to the first aspect of the present invention, comprises a nucleotide sequence which hybridises to the genomic RNA sequence, or a protein-encoding or non-protein-encoding portion thereof, of Nudaurelia fi virus (NpV) under medium or high stringency conditions. More preferably, the nucleic acid molecule comprises a nucleotide sequence substantially corresponding to all or a proteinencoding or non-protein-encoding portion of the sequence shown at Figure 1 or may contain single or multiple nucleotide substitutions and/or deletions :"and/or additions thereto.

Where the nucleic acid molecule comprises a nucleotide sequence encoding a protein-encoding portion of the genomic RNA sequence of NPV, it is preferred that said nucleotide sequence hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 31-3732, 3884-5788 or 4219-5112 in Figure 1.

Thus, in a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 31-3732 in Figure 1.

In a third aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 3884-5 788 in Figure 1.

In a fourth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 4219-5112 in Figure 1.

The present invention also extends to oligonucleotide primers for the above nucleic acid molecules, antisense molecules and probes for the above nucleic acid molecules and homologues and analogues of said primers, antisense molecules and probes. Such primers and probes are useful in the identification, isolation and/or cloning of the viral genes from NpV and other viruses (whether related or unrelated) carrying similar genes or RNA sequences. They are also useful in screening for NOV or other viruses in the field, especially in order to identify related viruses capable of causing pathogenicity similar to NpV.

The present invention further extends to cloning and expression vectors and host cells insect, yeast and bacteria cells) comprising the above nucleic acid molecules.

The present invention still further extends to a method of producing NpV or other serologically related Nudaurelia p-like virus, comprising culturing a host cell comprising a nucleic acid molecules according to the first aspect under conditions suitable for expression of said NpV or other serologically related Nudaurelia p-like virus.

In a fifth aspect, the present invention provides an infectious, recombinant insect virus vector comprising an expressible nucleic acid molecule comprising a nucleotide sequence corresponding to all or an 15 infectious and/or insecticidal portion of the genomic RNA sequence of N3V or other serologically related Nudaurelia P-like virus.

Infectious, recombinant virus vectors according to the fifth aspect, may comprise a baculovirus, entomopoxvirus or cytoplasmic polyhedrosis virus vector. The vector acts as a carrier for the NpV (or other serologically related Nudaurelia p-like virus) nucleic acid molecule(s) and may be produced by established procedures such as described by Bishop (1992) and International Patent Specification PCT/AU93/00411, the entire disclosure of which is to be regarded as incorporated herein by reference. Upon infection of an insect cell, the virus vector will cause the production of either 25 infectious viral genomic RNA or infectious viral particles.

The expressible nucleic acid molecule referred to in the fifth aspect, may include within a non-essential region(s), one or more exogenous nucleotide sequences encoding one or more substances that are deleterious to insects. Such substances include, for example, Bacillus thuringiensis 6-toxin, insect neurohormones, insecticidal compounds from wasp or scorpion venom or of exogenous origin, or factors designed to attack and kill infected cells in such a way so as to cause pathogenesis in the infected tissue a ribozyme targeted against an essential cellular function). Exogenous nucleotide sequences may be located within a non-essential region(s) by homologous recombination techniques well known to the art.

Thus, in a sixth aspect, the present invention provides a method for controlling the proliferation of a pest insect, comprising applying to an area infected with said pest insect a recombinant virus vector according to the fifth aspect, optionally in admixture with an agriculturally acceptable carrier.

Other modifications which may be made to the infectious recombinant insect virus vector according to the fifth aspect include splitting the RNA genome and cloning the fragments into the insect vector so that it cannot rejoin. One component, preferably the virus RNA replicase, would be expressed from a separately-transcribed fragment, the transcripts of which would not be replicated by the replicase they encode. The remainder or the genome (possibly having insecticidal activity) would be encoded by, for example, a second separately-transcribed fragment, the transcripts of which are capable of being amplified by the replicase. Consequently, whilst the transcripts from the second fragment would effect their insecticidal activity upon the infected insect cell, they would not be able to infect another insect cell, (even if encapsidated) because the replicase or replicase-encoding transcripts would be absent.

This modification would allow an inherent biological containment to be built into the insecticidal vectors, which would allow greater levels of environmental safety.

In a seventh aspect, the invention relates to a method of controlling insect attack in plants by genetically manipulating plants to express all or an infectious and/or insecticidal portion of NpV or other serologically related Nudaurelia p-like virus. Such plants are referred to as transgenic plants.

In an eighth aspect, the invention relates to the transgenic plants per se as described above.

Transgenic plants according to the invention may be prepared for example by introducing a DNA construct including a cDNA or DNA fragment encoding all or a desired infectious and/or insecticidal portion of NpV (or other serologically related Nudaurelia P-like virus, into the genome of a plant. The cDNA or DNA fragment may, preferably, be operably placed between a plant promoter and a polyadenylation signal. The cDNA or DNA fragment may encode all or a desired infectious and/or insecticidal portion of the wild-type, recombinant or otherwise mutated NpV (or other serologically related Nudaurelia P-like virus). For example, deletion mutants could be used which lack segments of the viral genome which are non-essential for replication or perhaps pathogenicity.

The isolated nucleic acid molecules of the invention can be inserted into a plant genome by already established techniques, for example by an Agrobacterium transfer system or by electroporation.

Plants which may be used in this aspect of the invention include plants of both economic and scientific interest. In particular, tomato, potato, corn, cotton, field pea, pine and tobacco.

To enhance the efficacy of infectious genomic RNA or viral particles expressed by transgenic plants according to the invention, the DNA construct introduced into the plants' genome may be engineered to include within a non-essential region or regions of the viral genome one or more exogenous nucleic acid sequences encoding substances that are deleterious to insects such as those described above.

15 The coat proteins from NcoV and HaSV have the ability to form virus like particles (VLPs) when expressed in a baculovirus expression system. It is anticipated that the similar and simple capsid structure of NpV (and other serologically related Nudaurelia p-like viruses) shall also permit formation of VLPs. Such VLPs may be useful as insecticidal agents.

Thus, in a further aspect, the present invention provides a virus-like particle (VLP) prepared from expression of a nucleic acid molecule comprising a nucleotide sequence encoding the capsid protein gene of N3V or other serologically related Nudaurelia p-like virus.

Identification of encapsidation (and replication) signals on virus RNA allows design of RNAs which should be readily encapsidated in VLPs during assembly in a suitable production system. It has, however, been observed by the inventors that on occasion VLPs from the HaSV coat protein encapsidate low molecular weight RNA having no viral sequences. As such, VLPs may be used to administer to insects, one or more substances deleterious to insects such as those mentioned previously in respect to the fifth aspect, as well as nucleotide sequences with insecticidal activities.

Further, the present inventors have identified a central domain within the capsid protein of HaSV which appears to form a structure belonging to the Immunoglobulin (Ig) superfamily. This Ig-like domain is believed to be responsible for the host cell tropism of this virus.

A similar Ig-like domain also appears to be present in the capsid protein of N3V (encoded by sequences within the region of nucleotides 4736- 5182 of the sequence in Figure It is considered that this Ig-like domain may be altered in a manner such that the host cell tropism of the NPV or N3V VLPs, can be modified.

Thus, in a still further aspect, the present invention provides a Nudaurelia virus (NpV) wherein the Ig-like domain within the coat protein has been altered or substituted so as to modify host cell tropism.

Preferably, the Ig-like domain has been altered such that the N3V selectively binds and infects a predetermined cell type which is other than the virus' normal host cell type(s). Such "targeting" enables, for example, the utilisation of the NPV's insecticidal properties in the control of pest insects outside of the normal host species range. It also permits the use of NV as a specific vector agent in gene delivery for, for example, gene therapy.

In yet a still further aspect, the present invention provides a viruslike particle (VLP) prepared from expression of the NpV capsid protein gene, said gene having been altered such that the Ig-like domain of the expressed capsid protein is altered or substituted so as to modify host cell tropism.

Preferably, the gene is altered such that the expressed VLP specifically binds and infects a predetermined cell type(s) other than a host cell type(s) which the VLP, absent the alteration or substitution, would otherwise bind and infect.

By "host cell tropism" we refer to the capacity of viruses and VLPs to bind and infect specific populations of cells within an organism.

Alterations or substitutions of the Ig-like domain may be achieved by replacing the wild-type capsid protein gene(s) with a chimeric gene(s) including nucleotide sequences encoding all or a functional portion(s) of Iglike domains derived from other proteins Fab fragments of IgG's and antibodies, cell adhesion proteins and receptors such as CD4) or non-Ig-like tertiary structures such as peptide loops such as those present on the capsid protein of nodaviruses), small proteins and lectins. Suitable alterations of the Ig-like domain might also be achieved with techniques such as site-directed mutagenesis of the wild-type coat protein gene(s).

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components or features with or without the 9 inclusion of a further, component or feature or group of components or features.

The invention is hereinafter described with reference to the following non-limiting examples and accompanying figure.

Brief description of the Figure Figure 1 provides a nucleotide sequence of a cDNA of 6536 nucleotides encoding the NpV RNA genome.

EXAMPLE 1 Isolation and Sequencing of the NBV genomic RNA.

NV was purified from infected, field-collected larvae of Nudaurelia cytherea capensis as described in Hendry et al., 1985. RNA was extracted as S• described by Hendry et al. (1985), Hanzlik et al. (1993) and in the abovementioned patent application PCT/AU93/00411, and cDNA generated by random-primed reverse transcription of this RNA using methods described in Hanzlik et al. (1995). The double stranded cDNA fragments were made blunt-ended and cloned into the Smal site of the vector pBluescript II KS(-) *(Stratagene) as described in Sambrook et al. (1989) and Hanzlik et al. (1995).

The nucleotide sequence of these resulting cDNA inserts was determined using the ABI PRIS1 T M Dye Terminator Kit; reactions were analysed on an Applied Biosystems Model 373A DNA automated sequencing system. The sequence of a 6.2 kb cDNA is provided at Figure 1.

The nucleotide sequence information shown at Figure 1 reveals a capsid protein precursor (ca 70 kDa) gene encoding a precursor protein which is cleaved at an N/G site into capsid proteins of approximately 60.5 kDa and 8 kDa. Comparison between the coat protein sequences of NpV and of NoV (Agrawal and Johnson, 1992) shows surprisingly little similarity considering that both viruses infect the same host and display the unique T=4 lattice symmetry. Only 20% identity overall (42 similarity) exists between the two coat proteins.

Sequence analysis reveals that the 6.5 kb genomic RNA is dicistronic, encoding both the replicase at the 5'-end and the capsid protein at the 3'-end (Fig. The 5' ORF encodes a 1233 amino acid protein with a calculated molecular weight of 139 kDa, very close to a previous estimate of ca 140 kDa obtained from in vitro translation studies (du Plessis et al., 1991), and containing characteristic sequence motifs found in viral replicases. The initiation codon of this gene is located very near the 5' end of the genomic RNA, consistent with translation of the replicase directly from the genomic RNA as suggested by du Plessis et al. (1991).

The replicase gene is followed by a 154-nucleotide non-coding sequence and the capsid protein precursor gene, which is likely expressed from the 2.5 kb subgenomic mRNA that has been detected in virion RNA. Its translation from this encapsulated RNA is consistent with evidence that the capsid protein was detected among the translation products of total virion RNA (du Plessis et al., 1991). There is no overlapping gene corresponding to the p17 ORF found on HaSV RNA 2. There is, however, an ORF which commences 335 nucleotides downstream from the start of the capsid gene 15 and is in a different reading frame. This ORF encodes a 33 kDa protein which has no obvious counterpart in HaSV or NwoV, nor significant homology to any protein sequences recorded in Genbank; its function, if any, is presently unknown. Although it could possibly be translated from the subgenomic RNA by ribosomes leaking past the start of the capsid gene, there are numerous intervening AUG codons which would probably interfere with its translation.

EXAMPLE 2 Cloning and expression of NBV capsid protein The NPV capsid protein gene was cloned as a PCR product (made using VENT (NEB)) amplified using the following (previously kinased) primers: NBVCAPN: 5'-ACCATGAGCGACCTACACTTGGAT-3' NBVCAPC: 5'-GCCCTACAAGCTAAGGTTCCCTAT-3' The resulting phosphorylated 1.9 kb PCR product was cloned into pBCKS(-) (Stratagene) which had been previously cleaved with EcoRV and dephosphorylated with CIAP. After blue-white selection and mapping of the orientation, the capsid gene was excised as a NotI-HindIII fragment and cloned into compatible sites within the vector pFastbacl (Gibco-BRL) for transfer to a recombinant baculovirus vector as specified by the supplier (Gibco-BRL) for the Bac-to-Bac system. The NPV capsid protein was then expressed in insect cells (Sf9) using this recombinant baculovirus vector as specified by the supplier (Gibco-BRL).

EXAMPLE 3 Recombinant NOV and uses Recombinant virus as a vector for other toxin genes.

"*This involves placing suitable genes under the control of NOV replication and encapsidation signals. Genes which may be suitable include intracellular insect toxins such as ricin, neurotoxins, gelonin and diphtheria toxins. The toxin gene may be placed in the viral gene such that it is a silent (down stream) cistron on a polycistronic RNA, or in a minus strand orientation, requiring replication by the viral polymerase to be expressed.

Standard techniques in molecular biology can be used to engineer these vectors. A suitable recombinant NpV vector may include a reporter gene (or one of the toxin genes mentioned above) inserted in place of the aminoterminal portion of the putative replicase gene, such that the initiation codon used for the replicase that at nucleotides 31-33 of the sequence shown in Figure 1) in used to commence reporter gene translation. The resulting plasmid then carries a complete form of the N3V RNA genome but with the amino-terminal portion of the replicase gene substituted by the reporter (e.g.

GUS) gene. It is desirable to produce a construct with approximately the same size as the NpV genome for encapsidation purposes.

(ii) VLP technology.

Identification of encapsidation (and replication) signals on NpV RNA allows design of RNAs which can be encapsidated in NpV VLPs during assembly in a suitable production system. The VLPs then carry the RNA of choice into the insect host cells where the RNA can perform its intended function. Examples of RNAs which may be encapsidated in this manner include RNAs for specific toxins such as intracellular toxins, such as ricin, neurotoxins, gelonin and diphtheria toxins.

12 (iii) Other uses of VLPs.

The VLPs can be used as vectors for protein toxins. Knowledge of icosahedral particle structure suggests that the amino and especially the carboxy termini are present within the VLP interior. It may therefore be possible to replace or modify the amino acid sequence of the capsid precursor protein such that it encodes a suitable protein toxin which is cleaved of the bulk of the capsid protein during capsid maturation. As with toxin-encoding mRNAs, the N3V VLP delivers the toxin to the host cell of the feeding insect, where it exerts the desired toxic effect.

Use of NfpVin plants.

The use of NpV in the production of insect-resistant transgenic plants is based on the use of either the complete NpV genome or of the replicase 15 gene as a tool for the amplification of suitable amplifiable mRNAs (e.g.

:encoding toxin) or of the capsid protein as a means to deliver insecticidal agents. Suitable strategies are described: 9.

Use of the complete NpV genome.

Fragments of cDNA corresponding to the full-length NpV genome are ~placed in a suitable vector for plant transformation under the control of either a constitutive plant promoter CaMV 35S promoter) or an inducible promoter or a tissue specific leaf-specific) promoter. The cDNA is followed by a cis-cleaving ribozyme (as described in the abovementioned patent application PCT/AU93/00411) and a suitable plant polyadenylation signal. Transcription from the promoter in the vector results in RNAs commencing at the first nucleotide of the NpV cDNA and terminating in the polyadenylation fragments. Self cleavage by the cis-acting ribosomes obtained within the transcripts generates RNA molecules with the 3'-termini corresponding to the natural virus termini. Translations of these transcripts in transgenic plant tissues and cells leads to assembly of fully infectious NpV particles to infect and kill feeding larvae.

Use of portions of the NflVgenome to deliver toxins to insect cells.

This strategy makes use of systems described in above. Plant cells contain an additional transgene encoding a suitable insect-specific, intracellular toxin (as described above). Such a toxin gene is expressed by plant RNA polymerase in either a positive or a negative sense (the latter is preferred) and in such a form that the RNA can be encapsidated by N3V capsid protein and/or replicated by the NOV replicase in infected insect cells.

Transgenic plants would contain two different transgenes, making either unmodified capsid protein precursor or a modified fusion form including a suitable insect-specific toxin. Expression from these two transgenes would be regulated so that only the required amounts of the modified and unmodified forms are made in the plant cell, and assembled in such proportions into the VLPs.

N/V VLPs carrying one or more suitable protein toxins and/or mRNA.

A protein toxin (or toxins) is expressed as a fusion with the capsid protein. The fusion protein then assembles into a VLP carrying the toxin(s).

These VLPs present in the plant tissue exert an anti-feeding effect on insects S. 20 attacking the plant.

EXAMPLE 4 Expression of NBV in other delivery vectors.

Constructs similar to those for plant expression may be introduced into yeast or bacteria by standard techniques. Microbes produced in suitable fermentation or culture facilities and carrying such forms of the virus are then delivered to the crop by spraying. The microbial cell wall provides extra protection for the virus particles produced within the microbes.

Well established techniques exist for culture and transformation of yeast (Ausubel et al., 1989). An example of a yeast expression vector is pBM272, which contains the URA3 selectable markers (Johnston Davies, 1984); Stone and Craig, 1990). Another example of an expression vector is pRJ28, carrying the Trpl and Leu2 selectable markers.

The isolation and sequencing of NPV, together with the simplicity of the tetravirus structure, offer new exciting opportunities to the control of insect pests.

Non-host Production of Tetraviruses On occasion, tetraviruses have proven to be effective insecticides and have certain advantages as such. They exclusively infect lepidopterans, a group of insects responsible for the majority of insect damage to world crops.

Of great concern is that some of these insects, such as H. armigera and H.

zea, have become refractory to the present $4 billion a year worldwide chemical control effort on insect pests. The relatively narrow range of host species for each virus within the tetravirus group is not only desirable for the issue of human safety but also meets modern requirements for low toxicity towards non-target insects. However, for use as insecticides, tetraviruses must counter a biological control agent's major disadvantage compared to chemicals, expense of production. Presently, tetraviruses can only be produced by expensive measures that depend on rearing live insect hosts, an unreliable procedure at times. However, recent work with HaSV has shown that the simple structure of tetraviruses can be exploited in conjunction with genetic engineering to produce the viruses more cheaply in non-host organisms. Furthermore, by having the non-host organism the crop plant itself, a more efficient means of delivering the virus to pest insects is obtained as well.

That HaSV could be made in a plant cell was implied by the ability of at least two of its three components to assemble in a non-host cell coat protein and its mRNA into VLPs) and the specificity of the virus for host midgut cells which suggested the HaSV components would be inert in a plant cell. The hypotheses was tested by somatic expression in plant protoplasts using three synthetic virus genes on DNA plasmids. Upon transcription and expression, these genes, in effect, placed all three components of HaSV, RNA1, RNA2 and coat protein, inside a single plant cell to assemble into the HaSV virion. Two genes were designed to produce exact copies of the two RNAs upon their transcription by the use of a modified CaMV 35S promoter (Mori et al., 1991) to start transcription at the beginning of the 5' virus sequence and cis-acting ribozymes (Altschuler et al., 1992) to cleave immediately after the last 3' virus base on the gene. The third gene was the HaSV coat protein. The plasmids were electroporated into protoplasts which then were incubated for three days and fed to neonate H. armigera larvae.

The larvae displayed the stunting effect caused by HaSV infection and were shown to possess HaSV RNA by Northern blotting. Interestingly, these experiments showed that only two genes, those for RNA1 and the coat protein, were required to stunt the larvae. The Northern blots of these larvae showed no signals for RNA2 or its coat protein mRNA derivative but did show the presence of RNA1. This suggests that RNA1 is able to self-replicate in the midgut cell and that this, not virosis, leads to the toxic lesion that causes the cell to have apoptoses and be shed.

The somatic expression experiments showed that HaSV could be produced without any overt harmful effect in plant cells. This indicated that the same event could be achieved in whole plants with transgenic expression. Towards this end, tobacco and white clover plants were made transgenic with the same three genes via agrobacterium mediated transgenesis and screened for their ability to induce stunting in H. armigera larvae. Tests showed a number were able to do so and the preliminary data (Service, 1996). As is evident, this particular approach, which exploits the simplicity of the tetravirus structure, is not available to more complex viruses such as baculoviruses and appears to bear great promise in the fight against insect pests. It is anticipated that similar manipulation and results can be achieved with NpV.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

References Agrawal, D.K. and Johnson, J.E. (1992). Virology 190, 806-814.

Altschuler, Tritz, R. and Hampel, A. (1992). Gene 122, 85-90.

Ausubel, F.M. et al. (eds), Current Protocols in Molecular Biology. J. Wiley Sons, 1989.

Bishop, D.H.L. (1992) Seminars in Virology 3, 253-264.

Francki, R.I.B. and Fauquet, Knudson, D.L. and Brown, F. (1991).

"Classification and Nomenclature of Viruses: Fifth Report of the International Committee on Taxonomy of Viruses". Springer-Verlag, Vienna, New York.

Grace, T.D.C. and Mercer, E.H. (1965). J. Invertebr. Pathol. 7,241-244.

Hanzlik, Dorrian, Gordon, K.H.J. and Christian, P.D. (1993). J. Gen.

Virol. 74, 1105-1110.

Hanzlik, Dorrian, Johnson, Brooks, E.M. and Gordon, K.H.J.

(1995) J. Gen. Virol. 76, 799-811.

Hendry, Becker, M.F. and van Regenmortel, M.V.H. (1968). S. Afr.

Med. J. 42, 117.

:.Hendry, Hodgson, Clark, R. and Newman, J. (1985). J. Gen. Virol.

66, 627-632.

Johnston, M. Davies, Mol. Cell. Biol. 4, 1440-1448 (1984).

Jukes, I.R.M. (1970). Bull. S. Afr. Soc. Plant Path. Microbiol. 4, 18.

King, L.A. and Moore, N.F. (1985). FEMS Microbiol. Lett. 26, 41-43.

Mori, Kazuyuki, Kobayashi, Okuno, T. and Furusawa, I. (1991). J.

gen. Virol. 72, 243-246.

25 Murphy, Fauquet, Bishop, Ghabrial, Jarvis, A.W., Martelli, Mayo, M.A. and Summers, M.D. (1995). "Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses". Springer-Verlag, Vienna, New York.

du Plessis, Mokhoshi, G. and Hendry, D.A. (1991). J. Gen. Virol. 72, 267-273.

Sambrook Fritsch, E.F. Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).

Service, R. (1996). Science 271, 145.

Stone, D. Craig, Mol. Cell. Biol. 10, 1622-1632 (1990).

Claims (19)

1. An isolated nucleic acid molecule of 15 or more nucleotides in length, the nucleic acid molecule comprising a nucleotide sequence which hybridises under medium or high stringency conditions to the genomic RNA sequence, or a protein-encoding or non-protein-encoding portion thereof, of Nudaurelia fl virus (NPV) or other serologically related Nudaurelia P-like virus.

2. An isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridises under high stringency conditions to the genomic RNA sequence, or a protein- encoding or non-protein-encoding portion thereof, of NpV.

3. An isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence substantially corresponding to all or a protein-encoding or non-protein-encoding portion of the sequence shown at Figure 1.

4. An isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which substantially corresponds to all of the sequence shown at Figure 1.

5. An isolated nucleic acid molecule according to claim 1, wherein the 25 nucleic acid molecule comprises a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 31-3732 in Figure 1.

6. An isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 3884-5788 in Figure 1.

7. An isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which hybridises, under medium or high stringency conditions, or substantially corresponds to that shown at nucleotide 4219-5112 in Figure 1.

8. A method of producing NjV or other serologically related Nudaurelia -like virus, comprising culturing a host cell comprising a nucleic acid molecule according to any one of claims 1 to 4 under conditions suitable for expression of said NPV or other serologically related Nudaurelia P-like virus.

9. An infectious, recombinant insect virus vector comprising an expressible nucleic acid molecule comprising a nucleotide sequence corresponding to all or an infectious and/or insecticidal portion of the genomic RNA sequence of N3V or other serologically related Nudaurelia 3- like virus.

10. A virus vector according to claim 9, comprising material derived from baculovirus, entomopoxvirus, or cytoplasmic polyhedrosis virus.

11. A virus vector according to claim 9 or 10, wherein said vector is capable of infecting insect species including Lepidoptera.

12. A virus vector according to any one of claims 9 to 11, comprising one or more nucleic acid sequences which encode substance(s) which are deleterious to insects. 25 13. A virus vector according to any one of claims 9 to 12, wherein said nucleotide sequence corresponds to all or an infectious and/or insecticidal portion of the genomic RNA of NOV.

14. A method for controlling the proliferation of a pest insect, comprising applying to an area infected with said pest insect a recombinant virus vector according to any one of claims 9 to 13, optionally in admixture with an agriculturally acceptable carrier. A method of controlling insect attack in plants by genetically manipulating plants to express all or an infectious and/or insecticidal portion of NpV or other serologically related Nudaurelia p-like virus.

16. A method according to claim 15, wherein said plant is genetically manipulated to express an isolated nucleic acid molecule according to any one of claims 1 to 7.

17. A method according to claim 15 or 16, wherein said plant is genetically manipulated to express NPV.

18. A transgenic plant resistant to insect attack comprising a genome or subgenome capable of expressing an isolated nucleic acid molecule according to any one of claims 1 to 4 such that the transgenic plant produces NpV or other serologically related Nudaurelia P-like virus, such that insects feeding on the transgenic plant are deleteriously affected. "19. A transgenic plant according to claim 18, wherein said plant produces NpV.. A preparation of NpV or other serologically related Nudaurelia P-like virus, or an insecticidally effective portion of said NpV or other serologically related Nudaurelia P-like virus, suitable for application to plants, said preparation capable of imparting an insect protective effect to plants.

21. A virus-like particle (VLP) prepared from expression of a nucleic acid molecule comprising a nucleotide sequence encoding the capsid protein gene of NpV or other serologically related Nudaurelia P-like virus.

22. A VLP according to claim 21, wherein said VLP encapsidates an insecticidal protein toxin.

23. A VLP according to claim 21, wherein said VLP encapsidates a nucleic acid molecule which is insecticidal or which encodes an insecticidal protein toxin. Dated this Second day of June 1997. COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION and RHODES UNIVERSITY Patent Attorneys for the Applicants: F.B. RICE CO. e S a a