WO2003074714A1 - Baculovirus expression system - Google Patents

Abstract

The present invention relates to novel baculoviruses, baculovirus vectors, transfer vectors and to host cells comprising these viruses or vectors. Furthermore, the present invention relates to methods for producing the novel baculoviruses and baculovirus vectors. The present invention also pertains to a method for producing a protein of interest using the novel baculoviruses and baculovirus vectors.

Description

Baculovirus expression system

Field of the invention The present invention relates to modified baculoviruses comprising in the genome an inactive form of a nucleotide sequence, said nucleotide sequence being enriched in defective interfering virus particles of the baculoviruses. Furthermore, the invention pertains to a method for preparing these baculoviruses and a method for producing a protein of interest using such baculoviruses.

Background of the invention

Baculoviruses are large enveloped, circular dsDNA viruses that can infect over 600 different types of invertebrates, but preferably infect insects. They are widely used as bio-insecticides in agriculture and forestry and can be genetically engineered to improve their effectiveness (Black, B. C, et al, 1997, In L. K. Miller (ed.), The Baculoviruses, pp 341-387. Plenum Press, New York; Inceoglu, A. B., et al, 2001, Pest Manag. Sci. 57:981-987).

More recently, baculoviruses were shown to have potential as gene delivery vectors for gene therapy (Hofmann, C, et al, 1995, Proc. Natl. Acad. Sci. USA 92, 10099-10103; Merrihew, R. V., et al, 2001, J. Virol. 75:903-909) or as vectors for surface display of complex eukaryotic proteins (Grabherr, R., et al, 2001, Trends Biotechnol. 19:231-236). Furthermore, baculoviruses are particularly well-suited for use as eukaryotic cloning and expression vector (King, L. A., and R. D. Possee, 1992, "The baculovirus expression system", Chapman and Hall, United Kingdom; O'Reilly, D. R., et al, 1992. Baculovirus Expression Vectors: A Laboratory Manual. New York: W. H. Freeman.). Advantages of the baculovirus expression sytem are among others that the expressed proteins are almost often soluble, correctly folded and biologically active. Further advantages include high protein expression levels, faster production, suitability for expression of large proteins and suitability for large-scale production. In the large-scale production of baculoviruses as bio-insecticides or in large-scale or continuous production of heterologous proteins using the baculovirus expression system in insect cell bioreactors, the passage effect is a major obstacle. This effect is notable as a significant drop in production by prolonged virus passaging in insect cell culture (Krell, P. J., 1996, Cytotechnology 20:125-137) and is a result of the accumulation of defective interfering particles (DIs) (Kool, M., et al, 1991, Virology 183:739-746). These DIs are rapidly generated in cell culture (Pijlman, G. P., et al, 2001. Virology 283:132-138) and become predominant after prolonged passaging, meanwhile interfering with the replication of intact helper virus and the production of polyhedra. It is known that DIs have retained cis-acting elements important for baculovirus DNA synthesis (Krell, P. J., 1996, Cytotechnology 20:125-137), but what sequences, if any, are involved in the generation of DIs is still enigmatic.

Surprisingly, we have found that inactivating a nucleotide sequence present in the genome of baculovirus, said nucleotide sequence being enriched in defective interfering virus particles, leads to enhanced stability of virus and polyhedra production and therefore prevents the negative consequences of the above-mentioned passage effect.

Description of the invention

Definitions

Herebelow follow definitions of terms as used in the present invention.

Homologous

The term "homologous" is used herein to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell and is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. Additionally, the term "homologous" is also used herein in the context of amino acid sequences (e.g., when one amino acid sequence is said to be X% homologous to another amino acid sequence) is intended to encompass both amino acid identity and similarity between the two sequences. To determine the percent homology of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein). The amino acid residues at corresponding amino acid positions are then compared and when a position in one sequence is occupied by the same or a similar amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position. The percent homology between two sequences, therefore, is a function of the number of identical or similar positions shared by two sequences (i.e., % homology = number of identical or similar positions/total number of positions x 100). Computer algorithms known in the art can be used to optimally align the two amino acid sequences to be compared and to define similar amino acid residues. Preferably, the Basic Local Alignment Search Tool (BLAST) algorithm (described in Altschul, S. F. et al, 1990, J. Mol. Biol. 215, 403-410) is used to compare the two amino acid sequences to thereby determine the percent homology between the two sequences. The term "homologous" as used in the context of nucleotide sequences (e.g., when one nucleotide sequence is said to be X% homologous to another nucleotide sequence) is intended to refer to nucleotide sequence identity between the two sequences. To determine the percent homology of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one nucleic acid molecule for optimal aligmnent with the other nucleic acid molecule). The nucleic acid bases at corresponding nucleotide positions are then compared and when a position in one sequence is occupied by the same nucleic acid base as the corresponding position in the other sequence, then the molecules are homologous at that position. The percent homology between two sequences, therefore, is a function of the number of identical positions shared by two sequences (i.e., % homology = number of identical positions/total number of positions x 100). The Basic Local Alignment Search Tool (BLAST) algorithm mentioned above can also be used to compare the two nucleotide sequences to thereby determine the percent homology between the two sequences.

Heterologous

The term "heterologous" is used herein to refer to a nucleic acid or polypeptide from a foreign cell which does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or which is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which they are introduced, but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed, similarly exogenous RNA codes for proteins not normally expressed in the cell in which the exogenous RNA is present. Furthermore, it is known that a heterologous protein or polypeptide can be composed of homologous elements arranged in an order and/or orientation not normally found in the host organism, tissue or cell thereof in which it is transferred, i.e. the nucleotide sequence encoding said protein or polypeptide originates from the same species but is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognise as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

Inactive form

An "inactive form" of a nucleotide sequence as used herein can be made by deleting the complete nucleotide sequence or a part thereof necessary and/or important for activity and/or functionality or by substituting nucleotides that are necessary and/or important for the activity and/or functionality of the nucleotide sequence. Furthermore, an "inactive form" of a nucleotide sequence can be obtained by disrupting the nucleotide sequence. Disruption of the nucleotide sequence can occur inter alia by insertion of another nucleotide sequence into the nucleotide sequence. Next to that, an "inactive form" of a nucleotide sequence can be obtained by changing the orientation of a nucleotide sequence (inversion of orientation) leading to non-functionality of the nucleotide sequence. Other methods known in the art for inactivating a nucleotide sequence will be apparent for a person skilled in the art and are enclosed herewith.

Replication

As used herein the term "replication" means the process of production of a new DNA strand using a DNA template strand for the copying of the information content of the genome. A baculovirus dsDNA genome which is capable of replication is capable of initiating the reproduction of its genome in the host cell.

Origin of DNA replication The term "origin of DNA replication", usually designated ori, is used herein with respect to the specific site on a DNA molecule where DNA replication is initiated. Bacterial and phage genomes have a single origin of DNA replication, while eucaryotic chromosomes have multiple origins of DNA replication. Anatomy of consensus eukaryotic oris has been described in DePamphilis, M. L., 1993, Annu. Rev. Biochem. 62, 29-63.

Homologous region

As used herein the tenn "homologous region (hr)" relates to a region that is composed of repeated DNA sequences encompassing both direct repeats and imperfect palindromic sequences and has closely related counterparts elsewhere in the baculovirus genome. Between 4 and 17 hrs are found interspersed throughout a single baculovirus genome. Homologous regions (hrs) range in size from 30 to 800 bp and consist of 1 to 30 repeat units (mostly containing repeats centered around an imperfect palindromic sequence) within each hr. The organisation of repeats in hrs in for instance SeMNPV can be found in Broer et al, J. Gen. Virol., 1998, 79, 1563-1572 (see also present figure 9). The nucleotide sequence of one of the hrs presented in the above Broer et al. article is presented in present figure 10. Hrs are cis-acting enhancers of RNA polymerase Il-mediated transcription and may act as origins of DNA replication.

Non-homologous region

As used herein the term "non-homologous region" is any part of the baculovirus DNA genome that is located outside the homologous regions.

Non-hr origin of DNA replication

These particular regions outside the homologous regions (hrs) exhibit activity as origin of DNA replication (ori) and contain unique palindromic and (direct and inverted) repetitive sequences that are not found in hrs or elsewhere on the respective baculovirus genome. Moreover, non-hr oris contain AT-rich regions and putative transcription factor-binding sites and have structural similarities to the consensus eukaryotic origin of DNA replication (see DePamphilis, M. L., 1993, Annu. Rev. Biochem. 62, 29-63). The arrangement of non-hr oris of AcMNPV, OpMNPV and SeMNPV can be found in present figure 11.

Defective interfering particle

Defective interfering particles (DIs) are defective virions, which can grow only in the presence of a helper standard virus. DIs are characterized in that (1) they contain a part of the viral genome, i.e. a defective genome; (2) they may contain normal viral structural proteins; (3) they reproduce only in the presence of helper virus; and (4) they interfere specifically with the intracellular growth of non-defective homologous standard virus. DIs are typically shorter in length than the standard virus. The presence of DIs in cells infected with standard virus reduces the production of standard virus.

A baculovirus genome

The term "baculovirus genome" is herein understood to refer to the nucleic acid that comprises in cis all the genetic elements that are necessary for autonomous replication of the viral genome, assembly of the replicated genome into infective viral particles in an insect host cell and subsequent secretion of the infective viral particle from the host cell. An "infective viral particle" is herein understood to mean a viral particle that is capable of (re)infecting an insect host and completing a full infection cycle without requiring viral functions provided in trans, i.e. other than the host cell's endogenous functions. The term "baculovirus genome" thus refers to a functional viral genome as opposed to the nucleic acids contained in DI's which contain only a part of the baculovirus genome that is defective in the sense that it cannot complete an infection cycle without the presence of a helper virus. The term "baculovirus genome" as used herein expressly includes genomes comprising sequences that are heterologous to the baculovirus. Likewise the term "baculovirus genome" does not necessarily refer to a complete baculovirus genome as the genome may lack viral sequences that are not necessary for completion of an infection cycle. Transfer vector

The term "transfer vector" as used herein refers to a nucleic acid which comprises: (1) a DNA fragment which comprises: (a) at least one nucleotide sequence of interest, the nucleotide sequence being homologous or heterologous to the baculovirus and or to the host cell transfected by the baculovirus and (b) nucleotide sequences from a baculovirus genome flanking the nucleotide sequence of interest on both sides. A transfer vector is used to produce recombinant baculoviruses through double recombination/cross-over events. Preferably, the flanking nucleotide sequences are of sufficient size to allow recombination events to occur between the DNA fragment (1) and a wild type baculovirus genome such that the nucleotide sequence of interest is inserted into the genome and the corresponding nucleotide sequences in the baculovirus genome are replaced by the flanking sequences. Such sizes are known by one skilled in the art. Preferably, the size of the baculovirus flanking sequences are at least about 500 bp on each side of the nucleotide sequence of interest, more preferably, the size of the flanking sequences is at least about 1000 bp, 2000 bp, 4000 bp, 5000 bp on each side. Next to the DNA fragment (1), a transfer vector as used herein comprises a vector fragment (2), i.e. a nucleotide sequence allowing replication and selection in inter alia bacteria, such as E. coli. The vector may be a plasmid, another virus or simply a linear DNA fragment.

Promoter

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

Enhancer The term "enhancer" means a cis-acting nucleic acid sequence which enhances the transcription of a gene and functions in an orientation and position-independent manner.

Infection

The term "infection" refers to the invasion by pathogenic viral agents of cells where conditions are favorable for their replication. Such invasion can occur by placing the viral particles directly on the insect cell culture or by injection of the insect larvae with the recombinant virus or by oral ingestion of the viral particles by the insect. The amount of recombinant virus injected into the larvae will be from 10 to 10 plaque forming units (pfu) of non-occluded virus/larva. Alternatively, larvae can be infected by the oral route using non-occluded budded virions, or more preferably occlusion bodies carrying recombinant viruses. In general, the amount of occlusion bodies fed to the larvae is that amount which for wild-type viruses corresponds to the LD.sub.50 for that species of baculovirus and insect host. The LD₅₀ varies with each species of baculovirus and the age of the larvae. One skilled in the art can readily determine the amount of occlusion bodies to be administered. Typically, the amount will vary from 10-10¹⁰ occlusion bodies/insect.

Transfection

The term "transfection" refers to a technique for introducing purified nucleic acid into cells by any number of methods known to those skilled in the art. These include, but are not limited to, electroporation, calcium phosphate precipitation, lipofection, DEAE dextran, liposomes, receptor-mediated endocytosis, and particle delivery. The chromosomes or DNA can also be used to microinject insect larvae, eggs, embryos or ex vivo or in vitro cells. Cells can be transfected with the chromosomes or with the DNA described herein using an appropriate introduction technique known to those in the art, for example, liposomes.

Operably linked

As used herein, the term "operably linked" refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being "under the control of the promoter. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

Detailed description of the invention In a first aspect, the present invention is concerned with a baculovirus comprising in the genome an inactive form of a nucleotide sequence, said nucleotide sequence being enriched in the genome of defective interfering virus particles (DIs) of that baculovirus. The term enriched is used to indicate that the nucleotide sequence is present more often in the mutant baculovirus (Dl) genome than in the standard baculovirus genome (duplicate, triplicate, etc.) or at a higher density as a consequence of deletions in the baculovirus Dl genome. The nucleotide sequence that is enriched in the genome of defective interfering virus particles (DIs) of that baculovirus will hereafter be called Dl-enriched nucleotide sequence. Enrichment of a particular sequence can be demonstrated for instance by the appearance of super- or hypermolar restriction fragments on a DNA agarosegel, by Southern hybridization, or by quantitative PCR. If the genome of the baculovirus comprises more than one, i.e. 2, 3, 4, 5, 6 etc., Dl-enriched nucleotide sequence preferably all or at least a part, for instance 10, 20, 30, 40, 50, 60, 70, 80, 90%, of these sequences are present in the genome of the baculovirus in an inactive form.

Dl-enriched nucleotide sequences can be of the same type or can be of a different type (see herebelow). They include, but are not limited to, origins of DNA replication of non-homologous regions (non-hr oris), origins of DNA replication of homologous regions (hr oris) and putative packaging signals. Origins of DNA replication of homologous regions preferably contain among others 1 to 30 discrete 30 bp imperfect palindromic homologous repeats interspersed along the genome. Origins of DNA replication of non-homologous regions preferably contain inter alia AT-rich regions, direct and inverted repeats and homologous region-unrelated palindromes.

In a preferred embodiment of the invention the baculovirus comprises in the genome an inactive form of an origin of DNA replication of a non-homologous region (non-hr ori). If more than one non-hr ori is present in a certain baculovirus genome, at least one but preferably more than one non-hr ori is in the inactive form. Nevertheless, the baculovirus is preferably still capable of DNA replication. It is known that other sequences such as for instance promoter regions of early baculovirus genes such as iel can function as non-hr oris (Wu, Y. and Carstens, E. B., 1996, J. Virol. 70, 6967-6972). Non-hr oris in the genome of baculoviruses include, but are not limited to, nucleotides 113912-115178 of the genome of AcMNPV (see Genbank accession no. L22858; see also nucleotides 1-1267 of SEQ ID No. 1), nucleotides 82132-85523 of the genome of SeMNPV (see Genbank accession no. AF169823; see also nucleotides 1-3392 of SEQ ID No. 2), the essential domain thereof consisting of the nucleotides 83284-83934 (see nucleotides 1153-1803 of SEQ ID No. 2), nucleotides 1-3955 of a clone of OpMNPV (see Genbank accession no. D17353; see also nucleotides 1-3955 of SEQ ID No. 3), nucleotides 1-344 of a clone of SpliNPV (see nucleotides 1-344 of SEQ ID No. 4), nucleotides 105528-105678 of the genome of BmNPV (see Genbank accession no. L33180; see also nucleotides 1-151 of SEQ ID No. 5), nucleotides 1170-1890 of a clone BusuNPV (see Genbank accession no. AF045936; see also nucleotides 1-721 of SEQ LD No. 6) and nucleotides 20000-21300 of the genome of CpGV (see Genbank accession no. U53466; see also nucleotides 1-1300 of SEQ ID No. 7).

A baculovirus for use in the present invention can be any baculovirus known in the art. Preferably, this baculovirus comprises in the genome a Dl-enriched nucleotide sequence. Examples of baculoviruses for use in the present invention are, without limitation, AcMNPV (Autographa californica multinucleocapsid nucleopolyhedrovirus), SeMNPV (Spodoptera exigua multinucleocapsid nucleopolyhedrovirus), OpMNPV (Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus), SpliNPV (Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus), BmNPV (Bombyx mori nuclear polyhedrosis virus), BusuNPV (Busura suppressaria single-nucleocapsid nucleopolyhedrovirus) and CpGV (Cydia pomonella granulovirus), HaSNPV (Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus), HzSNPV (Helicoverpa zea single-nucleocapsid nucleopolyhedrovirus), LdMNPV (Lymantria dispar multinucleocapsid nucleopolyhedrovirus), MbMNPV (Mamestra brassicae multinucleocapsid nucleopolyhedrovirus), AgMNPV (Anticarsia gemmatalis multinucleocapsid nucleopolyhedrovirus), XcGV (Xestia c-nigrum granulovirus), PxGV (Plutella xylostella granulovirus), SpltMNPV (Spodoptera litura multinucleocapsid nucleopolyhedrovirus) and CuniNPV (Cunex nigripalpus nucleopolyhedrovirus). In a preferred embodiment of the invention the baculovirus is SeMNPV or AcMNPV, particularly SeMNPV.

As described above, the baculovirus comprises in the genome an inactive form of a Dl-enriched nucleotide sequence, preferably a non-hr ori. An inactive (or nonfunctional) form of such a non-hr ori means that the non-hr ori is incapable of DNA- replication. An inactive (or non-functional) form of a Dl-enriched nucleotide sequence, such as a non-hr ori, can be obtained inter alia by insertion of another nucleotide sequence in the Dl-enriched nucleotide sequence rendering the Dl-enriched nucleotide sequence inactive (or non-functional), by substitution of the Dl-enriched nucleotide sequence or a part thereof that is essential for activity (or functionality) by another nucleotide sequence, by mutation of one or more nucleotides of the Dl-enriched nucleotide sequence rendering the Dl-enriched nucleotide sequence inactive (or nonfunctional), or by inversion of the orientation of the Dl-enriched nucleotide sequence or a part thereof that is essential for activity or functionality. Nucleotides essential for activity or functionality of a Dl-enriched nucleotide sequence such as a non-hr ori can be putative poly-adenylation sites, palindromes, direct and inverted repeats, putative transcription factor binding sites, en ori auxiliary sequenties (see present figure 11). By deleting, such as ter alia deletion of one or more palindromes and/or deletion of one or more direct and inverted repeats, or substituting nucleotides of these sequences or inserting nucleotides and/or other nucleotide sequences therein, these sequences can be inactivated or made non-functional. Preferably, the inactive form of the Dl-enriched nucleotide sequence can be made by deletion of the complete Dl-enriched nucleotide sequence or apart thereof that is essential for activity (or functionality). Such a deleted part can consist of 5, 10, 20, 50, 100, 200, 400, 500, 1000, 2000, 3000, 3750 or more nucleotides depending on the length of the Dl-enriched nucleotide sequence or essential part thereof and on the deletion necessary to make the Dl-enriched nucleotide sequence inactive (or non- functional). In a preferred embodiment of the invention 10%, 20%, 40%, 60%, 80%, 90%, 100% of the Dl-enriched nucleotide sequence is deleted. In a further aspect the invention relates to a baculovirus of the invention further comprising in the genome one or more nucleotide sequences of interest. The nucleotide sequences of interest can be homologous, but are preferably heterologous, i.e. not normally present or expressed by the baculovirus. The nucleotide sequences of interest can be located anywhere in the genome of the baculovirus. Preferably, they are not located in a region that is essential for infection of host cells. For instance one or more of the nucleotide sequences of interest can replace the nucleotide sequence encoding polyhedrin, plO, egt, cathepsin or chitinase. Preferably one or more of the nucleotide sequences of interest are located in any of the above-mentioned Dl-enriched nucleotide sequences. Most preferably, one or more of the nucleotide sequences of interest are located in the non-hr ori(s) present in the genome of a baculovirus. A nucleotide sequence of interest can replace a portion of a Dl-enriched nucleotide sequence or another portion of the baculovirus genome. Alternatively, a nucleotide sequence of interest can be inserted in a Dl-enriched nucleotide sequence or another portion of the baculovirus genome. Combinations of the above substitutions and/or insertions are also enclosed herewith. It is advantageous to replace a Dl-enriched nucleotide sequence with a nucleotide sequence of interest or to insert a nucleotide sequence of interest in such a Dl-enriched nucleotide sequence, since the Dl-enriched nucleotide sequence is rendered inactive (or non-functional) and concurrently the nucleotide sequence of interest is introduced in the baculovirus genome.

The nucleotide sequences of interest according to the invention are preferably in the form of DNA including genomic DNA, i.e. DNA including the introns, cDNA, synthetic DNA, DNA with a backbone modified for stability or for other reasons or DNA comprising unusual bases, such as inosine, or modified bases, such as tritylated bases and may be derived in whole or in part from any source known to the art. The nucleotide sequence may also be an allelic variant of the nucleotide sequence of interest according to the invention. If desired, the nucleotide sequence of interest can be prepared or altered synthetically so the known codon preferences of the intended expression host can advantageously be used. The nucleotide sequence of interest is preferably a full-length nucleotide sequence, but can also be a functionally active part or other part of said full-length nucleotide sequence.

A nucleotide sequence of interest according to the invention can also be operably linked to nucleotide sequences encoding polypeptides that enable rapid detection and purification of the polypeptide encoded by the nucleotide sequence of interest, including, but not limited to, glutathione-S-transferase, maltose-binding protein and metal-binding polyhistidine or to nucleotide sequences encoding detectable markers such as inter alia reporter genes such as genes encoding green fluorescent protein, luciferase, beta-galactosidase, GUS, or chloramphenicol acetyl transferase, or selection genes, i.e. genes encoding enzymes that allow cells to grow on certain media such as media containing antibiotics, such as genes giving hygromycin or neomycin resistance. When using a bacmid, the nucleotide sequence of interest according to the invention can also be operably linked to a gene extending bacterial resistance to inter alia ampicillin, kanamycin, tetracyclin, gentamycin, or chloramphenicol or a gene such as sacB useful for counter-selection in E. coli. Furthermore, the nucleotide sequence of interest can be operably linked to expression-regulating nucleic acid sequences such as a promoter and/or an enhancer showing activity in the host cell of choice that is infected or transfected (or transformed in case of for instance E. coli) by the baculovirus according to the invention. Furthermore, the nucleotide sequence of interest can be operably linked to nucleic acids showing activity in a bacmid host-strain such as E. coli. These expression-regulating nucleic acids can be derived from genes encoding polypeptides, which are either homologous or heterologous to said host cell. The nucleotide sequence of interest may further be operably linked to nucleotide sequences encoding a secretion sequence for the purposes of directing secretion of the polypeptide encoded by the nucleotide sequence of interest out of the host cell that is infected or transfected by the baculovirus according to the invention. Suitable secretion sequences include signal peptides such as the chorion signal peptide, the bombyxin signal peptide and the honey bee prepromelittin signal peptide. The nucleotide sequence of interest may further be operably linked to nucleotide sequences encoding a fusion protein for the purposes of surface display of the polypeptide encoded by the nucleotide sequence of interest at the surface of the baculovirus virion or at the cell membrane of the host cell that is infected or transfected by the baculovirus according to the invention. Suitable baculovirus fusion peptides include GP64, the major envelope glycoprotein of the budded virus genotype of the group I baculoviruses (e.g. AcMNPV, OpMNPV, BmNPV, CfMNPV, ΕpMNPV, AfNPV, AgMNPV), or the envelope fusion protein (F protein) of baculoviruses, preferably of the group II baculoviruses (e.g. SeMNPV, LdMNPV, HaSNPV, HzSNPV, SpltMNPV, SpliNPV, MacoNPV) or granuloviruses (e.g. CpGV, XnGV, PxGV)

The nucleotide sequences of interest may encode any polypeptide, but preferably a polypeptide having industrial or medicinal (pharmaceutical) applications. Examples of proteins or polypeptides with industrial applications include enzymes such as e.g. lipases (e.g. used in the detergent industry), proteases (used inter alia in the detergent industry, in brewing and the like), cell wall degrading enzymes (such as, cellulases, pectinases, beta.-l,3/4- and beta.-l,6-glucanases, rhamnoga-lacturonases, mannanases, xylanases, pullulanases, galactanases, esterases and the like, used in fruit processing wine making and the like or in feed), phytases, phospholipases, glycosidases (such as amylases, beta.-glucosidases, arabinofuranosidases, rhamnosidases, apiosidases and the like), dairy enzymes (e.g. chymosin). Mammalian, and preferably human, polypeptides with therapeutic, cosmetic or diagnostic applications include, but are not limited to, insulin, apolipoprotein A or E, serum albumin (HSA), lactoferrin, hemoglobin α and β, tissue plasminogen activator (tPA), erythropoietin (EPO), tumor necrosis factors (TNF), BMP (Bone Morphogenic Protein), growth factors (G-CSF, GM-CSF, M-CSF, PDGF, EGF, IGF, and the like), peptide hormones (e.g. calcitonin, somatomedin, somatotropin, growth hormones, follicle stimulating hormone (FSH) interleukins (IL- x), interferons (IFN-y), insulin receptor, EGF receptor, tyrosine hydroxylase, glucocerebrosidase. Included are furthermore single chain variable antibody fragments (scFv). Also included are protozoic, bacterial and viral antigens, e.g. for use as vaccines, including e.g. heat-labile toxin B-subunit, cholera toxin B-subunit, envelope surface protein Hepatitis B virus, capsid protein Norwalk virus, glycoprotein B Human cytomegalovirus, glycoprotein S, and transmissible gastroenteritis corona virusreceptors, human T-lymphotropic virus (HTLV-1) p40.sup.x, HTLV-1 env, human immunodeficiency virus (HIV-1) gag, pol, sor, gp41, and gpl20, adenovirus El a, Japanese encephalitis virus env (N), bovine papilloma virus 1 (BPV1) E2, HPV6b E2, BPV1 E6, hepatitis B surface antigen, HIV-1 env, fflV-1 gag, HTLV-1 p40.suρ.x, D. melanogaster Kruppel gene product, bluetongue virus VP2 and VP3, human parainfluenza virus hemagglutinin (HA), influenza polymerases PA, PBl, and PB2, influenza virus HA, lymphocytic choriomeningitis virus (LCMV) GPC and N proteins, Neurospora crassa activator protein, polyomavirus T antigen, simian virus 40 (SV40) small t antigen, SV40 large T antigen, Punta Toro phlebovirus N and Ns proteins, simian rotavirus VP6, CD4 (T4), Hantaan virus structural protein, human B lymphotrophic virus 130-kd protein, hepatitis A virus VPl, VPl and VP2 of Human parvovirus-B19, Classical Swine Fever Virus E2- glycoprotein and the like. Further included are genes coding for mutants or analogues of the said proteins. In yet a further aspect the invention pertains to a baculovirus vector comprising the genome of a baculovirus according to the invention, said baculovirus vector further comprising one or more DNA vector fragments. The DNA vector fragments may be fragments of a plasmid, cosmid or phage. DNA vector fragments preferably comprise sequences that allow that allow the DNA vector (and other sequences linked thereto such as the baculovirus genome) to be replicated in host cells other than insect cells, such as e.g. bacteria or yeasts. The baculovirus vector according to the invention can also be a bacterial artificial chromosome (BAG), a yeast artificial chromosome (YAC), a P-l derived artificial chromosome (PAC) or a yeast centromere plasmid (Yep). The choice of the DNA vector fragment is dependent on the recombinant procedures followed and the host cell used. Expression-regulating nucleic acid sequences such as a promoter, a ribosome binding site, a terminator, a translation initiation signal, a repressor gene, an activator gene or an enhancer may also be present in the DNA vector fragment and can be any nucleic acid sequence showing activity in the host cell of choice that is infected or transfected or transformed (in case of E. coli) by the baculovirus according to the invention and can be derived from genes encoding polypeptides, which are either homologous or heterologous to the host cell and/or the baculovirus. The expression of a nucleotide sequence of interest may also be increased by the expression of other (trans-acting) factors, for example the IE-1 protein of nuclear polyhedrosis viruses.

A baculovirus vector according to the invention may further comprise in the DNA vector fragment one or more nucleotide sequences of interest, wherein each of these nucleotide sequences of interest is either heterologous or homologous to the baculovirus according to the invention and/or the host cell that is infected or transfected by the baculovirus according to the invention. Examples of polypeptides encoded by heterologous nucleotide sequences are listed above. Examples of polypeptides according to the invention that are encoded by nucleotide sequences of interest that are homologous to baculovirus include but are not limited to polyhedrin, plO, GP64, DNA polymerase, p6.9, lef-genes, iap-genes, bro-genes, ie-genes, structural ODV- and BV- genes, ptp, ctl, protein kinases, egt, super oxide dismutase, fgf, ubiquitin, gta, ets, etm, pena, 25k, gp37, vlf-1, gp41, pnk, eg30, vp39, helicase, 38k, vp80, HE65, gρl6, pρ34, alk-exo, ρ94, 35k, ρ26, p74, me53, ρe38. Yet a further aspect of the invention includes a transfer vector, comprising a DNA vector fragment and a part of a baculovirus genome said part comprising an inactive form of a Dl-enriched nucleotide sequence according to the invention, preferably an origin of DNA replication of a non- homologous region of a baculovirus genome. Preferably, the part of the baculovirus genome is a part of the genome of a baculovirus according to the invention, in particular AcMNPV and SeMNPV, with SeMNPV being most preferred. The part of the genome of the baculovirus present in the transfer vector according to the invention is preferably of sufficient size to allow recombination events to occur between the transfer vector and a wild type baculovirus genome such that the part of the baculovirus genome present in the transfer vector is inserted into the genome of the wild-type baculovirus. The size of the baculovirus flanking sequences necessary to allow recombination events are known in the art and preferred sizes are disclosed in the definitions section of the present application. In another embodiment of a transfer vector according to the invention, the transfer vector further comprises one or more nucleotide sequences of interest according to the invention. The nucleotide sequence(s) of interest can be present in the DNA vector fragment of the transfer vector. Preferably, the nucleotide sequences of interest are present in the part of the baculovirus genome of the transfer vector. Preferably, the nucleotide sequences of interest are flanked on both sides by nucleotide sequences of the baculovirus genome. Thereby, they can be inserted in the baculovirus genome and can inactivate a nucleotide sequence of the baculovirus genome, preferably a Dl-enriched nucleotide sequence such as a non-hr ori. The DNA vector fragment of the transfer vector according to the invention preferably comprises specific transcriptional and/or translational elements that direct expression in a suitable host. Such elements include, but are not limited to, (viral) promotors such as the polyhedrin promotor, origins of DNA replication, antibiotic selection markers facilitating maintenance of the transfer vector in for instance bacteria such as E. coli, a suitable multiple cloning region, viral sequences for homologous recombination with the baculovirus genome, leader sequences with identification and purification tags, leader sequences for signalling secretion and fusions with reporter proteins.

A method for preparing a recombinant baculovirus, comprising the steps of: a) preparing a transfer vector according to the invention, b) transferring said transfer vector together with a genome of a baculovirus in a suitable host, c) culturing the host so as to produce the recombinant baculovirus, and optionally d) collecting the recombinant baculovirus, is also a part of the invention. The transfer vector according to the invention can be prepared by cloning apart of the baculovirus genome (flanking sequences of the nucleotide sequence to be substituted) in a suitable vector (plasmid, cosmid, etc) by standard cloning techniques or PCR. Subsequently, one or more nucleotide sequences of interest, optionally with suitable (viral) promoters, are inserted in the cloned baculovirus sequence, so that these nucleotide sequences of interest are flanked by baculovirus sequences needed for homologous recombination (at least 500 bp flanking sequence on either side). Thereafter, the transfer vector can be transferred together with a genome of a baculovirus or a baculovirus vector into a suitable host. Preferably, the transfer vector and the genome of a baculovirus or a baculovirus vector are co- tranfected into a suitable host. An alternative is transfection of the transfer vector in the host, followed by a super-infection with the baculovirus or baculovirus vector.

Preferred methods for efficiently transferring the transfer vector and the genome of a baculovirus are among others CaPO , electroporation and lipid-mediated transfection. Suitable hosts include insects, preferably Lepidopteran insects such as for example, without limitation: Autographa californica; Bombyx mori; Spodoptera frugiperda; Spodoptera exigua; Spodoptera litura, Spodoptera littoralis, Choristoneura fumiferana; Heliothis virescens; Heliothis zea; Helicoverpa armigera, Helicoverpa punctigera, Anagrapha falcifera, Helicoverpa zea; Helicoverpa virescens; Orgyia pseudotsugata; Lymantria dispar; Plutella xylostella; Malacostoma disstria; Trichoplusia ni; Pieris rapae; Mamestra configurata; Mamestra brassica and

Hyalophora cecropia. Preferred hosts are cells such as inter alia insect cells including Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, S£21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, Ha2302, Hz2E5 and High Five from Invitrogen. Insect cells are cells from the insect species which are subject to baculovirus infection. Cells of other species that can be used are Drosophila S2, CHO and HeLa cells. Baculoviruses can enter these cells and use them for protein expression, but can not infect these cells properly. The host of choice are then cultured so as to produce the recombinant baculovirus. Culturing methods for cells and insects are known in the art, see for instance King, L. A. and R. D. Possee, 1992, The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow, 1992, Baculovirus Expression Vectors: A Laboratory Manual, New York; and W. H. Freeman and Richardson, C. D., 1995, Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39. Furthermore, the produced recombinant baculovirus can be collected from the cells or insects, but prefeably from the culture medium. Said culture medium can be collected by for instance centrifugation, filtration or ultrasonic perfusion and recombinant baculovirus can be isolated by ter alia plaque assays known in the art and succesive end-point dilution assays, and subsequent verification that the recombinant baculovirus contains the desired feature by SDS-PAGE, followed by Western blot, ELISA or immunofluoresence if antibody is available, Northern hybridisation, Southern hybridisation analysis, PCR, RT-PCR, or detection of markergene expression (e.g. GFP, B-galactosidase, GUS, luciferase). If a recombinant baculovirus with a desired feature, preferably a baculovirus according to the invention, has been identified, collected and isolated this baculovirus is expanded through a high titer stock, i.e. a master stock for future expansion. Usual titers range between 5x10 and 5x10 PFU/ml (plaque assay) or TCID50/ml (endpoint dilution assay) and they are stored at preferably -70°C. Other methods of producing recombinant baculovirus are described in Kitts, P.A., 1996, Cytotechnology 20, 111-123, which is incorporated by reference herein. An alternative to the use of a transfer vector is the use of linear transfer DNA, synthetized by PCR using for instance PCR primers (60-100 bp) with 5' ends containing flanking sequences (40-80 bp) needed for homologous recombination with a baculovirus or baculovirus vector, and 3' ends to amplify a (positive and/or negative) selectable marker gene for selection in a host strain for a baculovirus such as insect cells, or a host strain for a baculovirus vector such as E. coli.

The PCR product containing viral flanking sequences is transferred, preferably cotransfected with the viral DNA into insect cells. Recombination between a transfer vector and linear viral DNA reduces the background of non-recombinant parental baculovirus. Recombination between a transfer vector and linear viral DNA with a "lethal deletion" prevents background of non-recombinant parental baculovirus. Recombinant baculoviruses can also be constructed in vitro. Using the Cre-loxP recombination system in vitro with a baculovirus vector with a copy of loxP in its genome. A transfer vector with a loxP site and nucleotide sequences of interest are mixed together and incubated with Cre in vitro, resulting in a baculovirus containing the desired nucleotide sequences of interest. Another way is direct cloning of DNA fragments into linear baculo viral DNA, followed by transfection of insect cells. A recombinant baculovirus produced according to any of the above methods is also a part of the present invention.

Another aspect of the invention concerns a method for preparing a baculovirus vector, comprising the steps of: a) preparing a first baculovirus vector comprising one or more DNA vector fragments of interest and the genome of a baculovirus or a part of that genome, b) preparing a transfer vector according to the invention, c) transferring said transfer vector with the first baculovirus vector in a suitable host, d) culturing the host so as to produce a second baculovirus vector, and optionally e) collecting the second baculovirus vector.

The first baculovirus vector can be produced by inserting one or more DNA vector fragments, preferably DNA vector fragments comprising elements as described before, in a baculovirus genome, for instance by direct cloning or by homologous recombination (in insect cell culture or in insects) using a transfer vector comprising one or more DNA vector fragments. Such a baculovirus vector can also be constructed using homologous recombination in E. coli (e.g. ΕT-cloning, described by Muyrers, J. P., et al, 1999, Nucleic Acids Res. 27:1555-1557). In case of direct cloning the preferred host for maintaining the first baculovirus vector are bacterial cells such as E. coli, less preferably, insect cells or even less preferably, yeast. When the first baculovirus vector is prepared by homologous recombination in insect cells using a transfer vector comprising one or more DNA vector fragments the preferred hosts are insect cells such as the insect cells described above, or alternatively, bacterial cells, or alternatively but less preferably, yeast. When the first baculovirus vector is prepared by homologous recombination in E. coli (e.g. ΕT-cloning) using a linear transfer DNA comprising one or more DNA vector fragments the preferred hosts is E. coli. The transfer vector according to the invention can be prepared as described above. Thereafter, the transfer vector according to the invention can be transferred together with the first baculovirus vector into a suitable host. Preferably, the transfer vector and the first baculovirus vector are co-transfected into a suitable host. Alternatively, the suitable host can also be transfected/electroporated with the transfer vector and subsequently the suitable host can be infected with baculovirus vector. Preferred methods for efficiently transferring the transfer vector and the first baculovirus vector are among others CaPO₄, electroporation and lipid-mediated transfection, particle bombardment. Suitable hosts are insect cells as described above (thus also including bacterial cells). Next, the suitable hosts can be cultured so as to produce a second baculovirus vector, and when desired the second baculovirus vector is collected. For culture conditions and collection/isolation methods see above. Preferably, the second baculovirus vector comprises an inactive form of a Dl-enriched nucleotide sequence, preferably this nucleotide sequence is an origin of replication of a non-homologous region of a baculovirus according to the invention, but more preferably of a baculovirus selected from the group consisting of AcMNPV, SeMNPV, OpMNPV, SpliNPV, BmNPV, BusuNPV and CpGV, preferably AcMNPV and SeMNPV, particularly SeMNPV. A baculovirus vector produced according to the invention is also a part of the present invention. Besides that, the invention relates to a method for producing a protein of interest comprising the steps of: a) transferring a baculovirus according to the invention comprising one or more nucleotide sequences of interest or a baculovirus vector according to the invention comprising one or more nucleotide sequences of interest, wherein at least one of the nucleotide sequences of interest encodes the protein of interest, in a suitable host, or co-transfecting a baculovirus genome together with a transfer vector according to the invention comprising one or more nucleotide sequences of interest, wherein at least one of the nucleotide sequences of interest encodes the protein of interest, in a suitable host, b) culturing the host so as to produce the protein of interest, and c) collecting the protein of interest.

The baculovirus according to the invention comprising one or more nucleotide sequences of interest according to the invention or the baculovirus vector according to the invention comprising one or more nucleotide sequences of interest according to the invention can be transferred to the suitable host by injection (insects), infection or transfection/electroporation/particle bombardment. Suitable hosts are insects and insect cells as described above. Alternatively, mammalian cells can also be used. Baculoviruses do not replicate in these cells, but proteins can be expressed therein when promoters suitable for mammalian expression are used. Insects and insect cells are preferably cultured under conditions disclosed above.

Collecting the protein of interest depends on the expressed protein or polypeptide and the host cells used but can comprise recovering the protein or polypeptide through isolation. When applied to a protem/polypeptide, the term "isolation" indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins. It is preferred to provide the protein in a greater than 40% pure form, more preferably greater than 60% pure form. Even more preferably it is preferred to provide the protein in a highly purified form, i.e., greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE. It can be very helfull to express the protein of interest as a fusion protein to facilitate protein purification and protein detection on for instance Western blot and in an ELISA. Suitable fusion sequences include, but are not limited to, the sequences of proteins such as for instance glutathione-S-transferase, maltose-binding protein, metal-binding polyhistidine, green fluorescent protein, lucif erase and β- galactosidase. The protein may also be coupled to non-peptide carriers, tags or labels that facilitate tracing of the protein, both in vivo and in vitro, and allow for the identification and quantification of binding of the protein to substrates. Such labels, tags or carriers are well-known in the art and include, but are not limited to, biotin, radioactive labels and fluorescent labels.

A cell comprising a baculovirus according to the invention, a baculovirus vector according to the invention or a transfer vector according to the invention is also a part of the present invention. The cell may be prokaryotic or eukaryotic. If the cell is eukaryotic, it is preferably an insect cell or a mammalian cell or a yeast cell. If the cell is prokaryotic, it is preferably a bacterial cell such as E. coli.

A kit comprising a baculovirus according to the invention, a baculovirus vector according to the invention or a transfer vector according to the invention or a combination thereof is also a part of the present invention. Optionally, said kit further comprises one or more suitable hosts, preferably insects, insect cells or bacterial cells or a combination thereof, culture medium for one or more of the suitable hosts, one or more baculovirus strains and instructions for using the kit components. Said kit is preferably useful for producing a protein of interest, preferably a heterologous protein according to the invention.

Description of the figures Figure 1 shows a restriction profile of intracellular DNA of wildtype SeMNPV-

US1 upon passaging (Pl-25) in Se301 insect cells. (A) DNA digested with^bαl and run in a 0.6 % agarose gel. Passage numbers are indicated above the lanes and the viral genomic Xbal-A and -F fragments on the left. Lane M contains a λ/EcoRI/5α HI/HtndIII DNA size marker. Sizes (kb) of the hypermolar novel bands (2.6 to 7.0) and the novel 9 kb fragment are indicated on the right. (B) Southern blot using the SeMNPV non-^r ori (nt 83122 - 84048) as a probe. The viral genomic 6.6 kb Xbal-F (containing the non-Ar ori) and an additional hybridizing 5.3 kb band are indicated on the left.

Figure 2 represents a schematic overview of the genetic organization of hypermolar and other non-hr ori hybridizing bands compared to the complete SeMNPV genome. (A) Genetic organization of the genomic DNA with nucleotide positions according to the complete SeMNPV genome (16). Block arrows represent the respective ORFs. Grey and light-grey boxes refer to sequences on either side of Xbaϊ (Xb) 83132, containing Sspl (S), Pstl (P), EcoRI (Ε), and Xhol (Xh) sites. The non-Ar ori is presented as a cross-hatched box between the two Sspl sites (11). (B) Genetic arrangement hypermolar 2.6, 3.0 and 4.1 kb fragments of SeMNPV-USl (Sewt) and non-hypermolar cohybridizing 5.3 kb fragment (genomic fragment of a SeMNPV deletion mutant) in the Southern blots. Nucleotide positions and sequence overlaps/insertions are indicated at the junction sites. (C) Genetic arrangement hypermolar 3.0 kb fragment of SeBAClOph, containing two junctions.

Figure 3 shows the replicative form of the hypermolar 2.6 and 3.0 kb Xbal fragments by partial digestion of ICV SeMNPV-USl DNA of P10, using increasing amounts of Xbal. On the right the genomic 6.6 kb Xbal-F and the additional 5.3 kb band as well as the hypermolar Xbal bands of 2.6 and 3.0 kb are indicated. On the left the multimers of the 2.6 and 3.0 kb Xbal fragments are indicated by arrows.

Figure 4 shows the abundancy hypermolar 2.6 and 3.0 Xbal fragments in BV as compared to ICV SeMNPV-USl DNA of P12. The genomic 6.6 kb Xbal-F and the hypermolar 2.6 and 3.0 kb Xbal fragments are indicated with arrows. (A) Agarose gel with &αl-digested intracellular viral (ICV) and budded virus (BV) DNA. (B) Southern blot probed with the non-hr ori (nt 83122 - 84048) of SeMNPV.

Figure 5 A represents the outline of the strategy for the construction of a complete SeMNPV bacmid by direct cloning. The bacmid backbone vector was amplified from the AcMNPV bacmid (Gibco BRL) by PCR using primers DZ113 and DZ114 with Bsu36l (B) restriction sites. The product was cloned into PCR-XL-TOPO (Invitrogen) and was circularized by Bsu36l digestion and subsequent self-ligation. SeMNPV-USl DNA and the bacmid backbone vector were linearized with SaήDl (S), ligated, and transformed to E. coli DHlOβ. SeBAClO contains the complete SeMNPV genome. Figure 5B represents an overview of the strategy to construct a non-hr ori deletion mutant bacmid by "ΕT-recombination" (34) in E. coli DHlOβ. Competent cells harboring SeBAClO and an arabinose-induced pBADαβγ were transformed with a PCR product composed of the chloramphenicol (Cm^R) resistance gene from pBeloBACll, and 50 bp 5'-overhangs flanking the non-hr ori of SeMNPV, needed for homologous recombination. Transformants were selected on plates containing kanamycin (Kan) and chloramphenicol (Cm). The non-hr ori deletion mutant was designated SeBAClOΔnonhr.

Figure 6A shows the restriction profile (Pstl) of parental SeMNPV bacmid SeBAClO and the non-hr ori deletion mutant SeBAClOΔnonhr. The genomic non-hr ori containing fragment Pstl-l (7017 bp) and with Cm^R gene insertion (7303 bp) are indicated.

Figure 6B shows a schematic representation of the non-hr ori and flanking regions in SeBAClO and SeBAClOΔnonhr. Primers DZ127 and DZ128 used for identification are indicated as well as the sizes of the expected PCR products.

Figure 6C shows PCR on SeBAClO and SeBAClOΔnonhr using primers DZ127 and DZ128 to confirm the identity of both bacmids.

Figure 6D shows the outline of the strategy to insert an intact polyhedrin gene in the SeMNPV bacmids SeBAClO and SeBAClOΔnonhr. First the SeMNPV polyhedrin gene with its own promoter was amplified by PCR using primers with H dTII and Sm l restriction sites. The product was cloned into a HindϊlUSndBl linearized pFastBACl vector (Gibco BRL) generating pFBlSepol. Donor plasmid pFB IS epol was used to restore the SeMNPV polyhedrin gene, generating SeMNPV bacmids SeBAClOph and SeBAClOphΛnonlir.

Figure 7 shows the restriction profile (Xbal) of ICV DNA of serially passaged SeMNPV bacmids SeBAClOph (A) and SeBAClOpl Δnonhr (B) in Se301 insect cells. Passage numbers (top) and the hypermolar band of 3.0 kb (SeBAClOph) are indicated. Figure 8 A shows the titers of serially passaged BV of SeMNPV-USl (♦), SeBAClOph (Δ), and SeBAClOphΔnonhr (0).

Figure 8B shows pictures of infected Se301 insect cells with SeBAClOph and SeBAClOphΔnonhr at P2 and P20, respectively. Figure 9 shows the organisation of palindromic repeats (P), direct repeats (DR) and other motifs within Se-hrl to Se-hr6 (see Broer et al, J. Gen. Virol., 1998, 79, 1563-1572).

Figure 10 representsa nucleotide sequence of the 1,3 kb Sα/I-EcoRI fragment within fragment pSeEcoRI-2.2 encompassing SeMNPV hr6. Restriction sites are indicated in italics. Palindromic repeats PI to P4 are underlined. An asteriks (*) represents a mutation in PI that disrupts the BglH-sitQ. The direct repeats DRla and DRlb are doubly underlined. The CGATT-motif is denoted in bold and marked with a ♦ , above or below the sequence depending on whether the motif is present on the forward or complementary strand. Putative poly-A signals are in bold. The CANNTG (MLTF?USF)-motif are marked as ♦ . The stopcodons of ORF xh 135 and xb 187 are boxed (see Broer et al, J. Gen. Virol., 1998, 79, 1563-1572).

Figure 11 shows a comparison of the arrangement of AcMNPV, OpMNPV and SeMNPV non-hr like oris. Arrows (P1-P6) represent palindromic sequences, black boxes (AT) represent putative poly-adenylation signals, small triangles (R1-R4) represent repeated sequences and asterisks represent putative transcription factor binding sites. Open boxes indicate the ori auxiliarry sequences, boxes marked ore and due represent origin recognition element and double stranded unwinding element respectively.

Examples

Materials and methods Cells, insects and viruses The Spodoptera exigua cell line Se301 (8,9) was donated by Dr. T. Kawarabata (Institute of Biological Control, Kyushu University, Japan) and was propagated at 27°C in Grace's supplemented medium (Gibco BRL) containing 10% foetal calf serum (FCS; Gibco BRL). Fourth instar S. exigua larvae were infected by contamination of artificial diet with 4xl0⁵ SeMNPV-USl (Gelernter, W. D., and B. A. Federici, 1986, J.

Invertebr. Pathol. 48:199-207) polyhedra per larva (Smite, P. H., and J. M. Vlak, 1988, J. Invertebr. Pathol. 51:107-114). Haemolymph was collected as previously described (Ukel, W. F., M. et al, 2000, Virology 275:30-41) and was defined as the passage zero (P0) budded virus (BV) inoculum to initiate serial passage in cultured Se301 cells. Serial undiluted passaging was carried out as previously described (Pij nan, G. P., et al, 2001 Virology 283:132-138). Infectious budded virus titers were determined using the endpoint dilution assay (Vlak, J. M., 1979, J. Invertebr. Pathol. 34:110-118).

DNA isolation, Southern hybridization, colony lift, molecular cloning, and sequencing Intracellular viral DNA and budded virus DNA was isolated as previously described (Summers, M. D., and G. E. Smith, 1987. "A manual of methods for baculovirus vectors and insect cell culture procedures", Texas Agricultural Experiment Station Bulletin No. 1555). Digested viral DNA was run overnight in ethidium bromide stained 0.6% agarose gels, and Southern blotting was performed by standard capillary upward blotting (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. "Molecular

Cloning: A laboratory Manual", 2^nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) using Hybond-N (Amersham Pharmacia) filters. As a DNA size marker, λ-DNA digested with EcόBl/HindϊlVBamHl was used. Randomly primed DNA probes for Southern hybridization were made using the DIG non-radioactive nucleic acid labeling and detection system (Roche). PCR products (927 bp) of the SeMNPV non-/zr ori were made with reverse primer DZ127 5'-CATCGATGCGTACGTGACTTTC-3' (nt 84027 - 84048; SΕQ ID No. 8) and forward primer DZ128 5'- CCTTGCGTTCCTTTGGTG-3' (nt 83122 - 83139; SΕQ ID No. 9), purified using the High pure PCR purification kit (Roche), and DIG-labeled overnight. Hybridization and colorimetric detection with NBT-BCIP (Gibco BRL) were performed according to the manufacturer's recommendations. Hypermolar viral Xbal bands were cut from the gel, purified with Glassmax (Gibco BRL), and cloned into pUC19 by electrotransformation of E. coli DH5α using standard methods (Sambrook, J., Ε. F. Fritsch, and T. Maniatis. 03

26 1989. "Molecular Cloning: A laboratory Manual", 2^nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). A colony lift assay (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. "Molecular Cloning: A laboratory Manual", 2^nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was used to isolate the cloned submolar 5.3 kb fragment using the same probe as described above. Automatic sequencing was performed using an ABI prism 310 genetic analyzer (Perkin Elmer) at the laboratory of Molecular Biology, Wageningen University. Sequence analyses were performed using BLAST (Altschul, S. F., et al, 1997, Nucleic Acids Res. 25:3389-3402) from the UWGCG computer programs (release 10.0).

Construction of bacmid cloning vector

The bacmid vector for direct cloning of SeMNPV was constructed by PCR using the Expand long template PCR system (Roche). Custom made primers (Gibco BRL) were designed using DNAstar Primerselect and were based on the sequence of AcMNPV transfer plasmid ρVL1393 (Luckow, V.A., and M. D. Summers, 1988, Bio- technol. 6:47-55), which was the backbone of the transfer vector pMON14272 used to construct the AcMNPV bacmid bMON14272 (Luckow, V. A., et al, 1993, J. Virol. 67:4566-4579). Primers DZ113 (5'- CCTTCCΓG GGTACCTTCTAGAATTCCGGAG-3 ') (SEQ ID No. 10) and DZ114 (5'-CCTTCCTG4GGCCGGGTCCCAGGAAAGGATC-3 ') (SEQ ID No. 11) were oppositely directed to sequences flanking the Bglϊ cloning site of pVL1393, and contained additional Bsu36l restriction sites (italics) for circularization at their 5' end. DZ114 also contained an internal SanDl restriction site (underlined) for direct cloning into S nDI-linearized SeMNPV-USl DNA. The template for PCR was purified AcMNPV bacmid bMON14272 DNA from the Bac-to-Bac Kit (Gibco BRL). The resulting 8.5 kb PCR product was cloned into the 3.5 kb pCR-XL-TOPO vector (Invitrogen), digested with Bsu36l, self-ligated, and cloned into electrocompetent DHlOβ E. coli cells. The obtained bacmid cloning vector was designated BAC-Bsu36I and its identity was verified by restriction analysis.

Direct cloning SeMNPV-USl as bacmid

SeMNPV-USl DNA for direct cloning was purified using alkaline treatment of polyhedra and by previously described methods (O'Reilly, et al, 1992, Baculovirus Expression Vectors: A Laboratory Manual. New York: W. H. Freeman). Two μg of viral SeMNPV-USl DNA was linearized by digestion with 10U of SαnDI (Stratagene) for 16h at 37°C. The restriction enzyme was heat-inactivated for 15 min. at 65°C. One μg bacmid cloning vector BAC-Bsu36I was digested with 10U of SαraDI in a total volume of 35μl for 3 h at 37°C. The 8.5 kb vector was dephosphorylated using 1U HK™ Thermolabile Phosphatase (Epicentre). The enzymes were heat-inactivated for 15 min. at 65°C prior to gel-purification of the linearized cloning vector DNA with Glassmax (Gibco BRL). Ligation was performed for 16h at 15°C with approximately 500ng linearized SeMNPV DNA and 25 ng linearized vector DNA in a total volume of 20 μl using 6U T4 DNA ligase (Promega). Electrocompetent E. coli DHlOβ cells (Gibco BRL) were transformed with 2 μl ligation mix at 1.8 KVolt using a Biorad Gene Pulser. The transformed cells were recovered in SOC medium for 45min at 37°C and spread on agar plates containing kanamycin. This procedure resulted in 111 putative SeMNPV bacmid clones, designated SeBACO-110.

Deletion SeMNPV non-/zr ori by ET-recombination in E. coli.

For deletion mutagenesis of the active essential domain of the non-hr ori of SeMNPV-USl bacmid SeBAClO, 68-70 bp long primers were designed with 50 bp 5'- ends flanking the deletion target region on the SeMNPV genome. Forward primer DZ153 was 5'

CATTTACTCGAAAACACTGTACACTTCGTCAAAATAAATGACGCAATATTTT TAAGGGCACCAATAACTG 3' (SEQ ID No. 12), with a viral flanking sequence from nt 83237 to 83286 according to the SeMNPV complete genome sequence (IJkel, W. F., et al, 1999, J. Gen. Virol. 80:3289-3304). Reverse primer DZ154 was 5' ATTTCAAAAATTAGAATCAAAACCCAATTTGCCGGCAACGTTTTAATATTTT CCTGTGCGACGGTTAC 3' (SEQ ID No. 13), with a viral flanking sequence from nt position 83981 to 83932. The locus to be deleted, which is the essential domain of the SeMNPV non-hr ori, is defined by two Sspl sites. These Sspl restriction sites are included in the primers (underlined). The 3'ends of the primers anneal to the chloramphenicol gene of pBeloBACl 1 from nt position 735 until 1671. PCR on pBeloBACl 1 was performed using the Expand long template PCR system (Roche) according to the manufacturer's protocol, giving a product of 1036 bp. The PCR product was purified using the High pure PCR purification kit (Roche), cut with Dpήl to eliminate residual pBeloBACll template, phenol/chloroform extracted, and ethanol precipitated. Approximately 0.5 μg PCR product was used for transformation of electrocompetent E. coli DHlOβ containing both SeBAClO and homologous recombination helper plasmid pBAD-αβγ. DHlOβ containing SeBAClO were heat-shock transformed with pBAD-αβγ and subsequently made electrocompetent according to Muyrers, J. P., et al, 1999, Nucleic Acids Res. 27:1555-1557. Briefly, 70 ml of LB medium was inoculated with 0.7 ml of an overnight culture. At an OD600 of 0.1-0.15, ET-protein expression from pBAD-αβγ was induced by the addition of 0.7 ml 10% L-arabinose. The cells were harvested at an OD600 of 0.3-0.4 and made electrocompetent by 3 subsequent washes with ice-cold 10%) glycerol. The cells were transformed with the purified PCR product in 2mm electroporation cuvets (Eurogentec) using a Biorad Gene Pulser (2.3 kV, 25 μF, 200 Ω). The cells were resuspended in 1 ml LB medium and incubated for lh at 37°C, and subsequently spread on agar plates containing kanamycin and chloramphenicol. Colonies were picked and screened by restriction analysis and PCR, and the non-hr ori deletion mutant was designated SeBAClOΔnonhr. PCR was performed with forward primer DZ127 and reverse primer DZ128 as previously described.

Reconstitution SeMNPV polyhedrin gene by pFastBACl donor plasmid To reconstitute the polyhedrin gene in SeMNPV bacmids SeBAClO and

SeBAClOΔnonhr, a donor plasmid pFBlSepol was constructed. The pFastBacl vector (Gibco BRL) was digested with Sn Bl and HmdiJI to delete the AcMNPV polyhedrin promoter and the MCS. The SeMNPV polyhedrin gene with its own promoter and the first putative transcription termination signal was amplified by the Expand long template PCR system (Roche) using forward primer DZ 138 5'-

CCCCCGGGTATATACTAGACGCGATTAC-3' (nt 135475-135494; SEQ ID No. 14) and reverse primer DZ139 5'-CC^4GCnTGTAATACTTACCTTTTGTG-3' (nt 757- 776; SEQ ID No. 15), containing Smal andHmdIII restriction sites (italics), respectively. The resulting 930 bp fragment was cloned into a pGEM-Teasy vector (Promega), sequenced and subsequently cloned as a Smal/Hindϊll fragment into the pFastBacl vector to generate pFBlSepol. The protocol from the Bac-to-Bac manual (Gibco BRL) was followed to transpose the SeMNPV polyhedrin gene from pFBlSepol into the αttTn7 rransposon integration site of SeMNPV bacmids SeBAClO and SeBAClOΔnonhr to generate SeBAClOph and SeBAClOphΔnonhr, respectively.

Transfection of SeMNPV bacmids Se301 cells were seeded in a 6-wells tissue culture plate (Nunc) at a confluency of 25% (5x10⁵ cells). Transfection was performed with approximately 1 μg SeBAClOph or SeBAClOphΔnonhr DNA using 10 μl Cellfectin (Gibco BRL). As a positive control, 1 μg SeMNPV-USl DNA was transfected as well. After 5 and 7 days, polyhedra were formed by the cells transfected with SeMNPV-USl and the bacmids, respectively. Budded virus containing supernatant (defined as PI) and infected cells were harvested 14 days post transfection (90% polyhedra containing cells).

Results

Serial passage SeMNPV in Se301 insect cells.

SeMNPV-USl was serially-passaged 25 times in the S. exigua cell line Se301 using budded virus (BV) from infectious hemolymph, defined as passage 0 (P0) inoculum. A decrease of polyhedra production was observed after less than five passages, indicating a dramatic passage effect. Intracellular viral (ICV) DNA was purified and subjected to Xbal (Fig. la), Pstl, EcoRI and Xfiol (not shown) digestion. A rapid reduction of the major genomic Xbal- A fragment was observed (Fig. la), coinciding with disappearance of the Pstl-C and -D fragments (not shown). These fragments are located within the Xbal-A fragment. At the same time, a novel Xbal- fragment of about 9 kb became more abundant and was cloned and sequenced. This fragment was aheady present in the PI DNA and appeared to consist of the remnants of the Xbal-A fragment as a result of a 26.5 kb deletion (from nt 15301 to 41759), according to the complete genome sequence of SeMNPV. The occurrence of mutants with deletions in this particular genomic region is a common phenomenon of SeMNPV infection in cell culture, but these deletions do not compromize BV or protein production (Dai, X. J., et al, 2000, J. Gen. Virol. 10:2545-2554). In vivo, such deletion mutants also exist and can act as parasitic genotypes (Munoz, D., et al, 1998, Appl. Environ. Microb. 64:4372-4377). Analysis hypermolar bands.

Hypermolar fragments accumulated in Se301 cells from P10 onwards and they were visualized as Xbal restriction fragments of 2.6 and 3.0 kb in agarose gels (Fig. la). From P15 onwards also bands of 4.1, 5.5, 7.0 kb and higher became hypermolar. Bands of 3.0 kb were also found in Pstl and Xhol digests, whereas the smallest but predominant 2.6 kb fragment was also observed in an EcoRI digest (not shown). The abundant 2.6 and 3.0 kb Xbal bands were cloned and sequenced and it was found that the Xbal sites on either side of the cloned inserts corresponded to the SeMNPV Xbal restriction site at position 82132, according to the complete sequence of SeMNPV. Most interestingly, both the 2.6 and 3.0 kb fragments contained the entire SeMNPV non-Ar origin of DNA replication (nt 83286 - 83932) and a junction of sequences flanking this non-hr ori (Figure 2).

In contrast to the 3.0 kb fragment, the 2.6 kb fragment contained an EcoRI but not a Pstl or Xhol restriction site, explaining its presence in an EcoRI digest and its absence in the Pstl and XJiol restriction patterns. Noteworthy is the presence of an overlapping stretch of 9 bp at the junction site in the 2.6 kb fragment (Fig. 2b), which in the complete SeMNPV genome is present on either side of the non-hr ori, leaving 2.6 kb in between. Because of the presence of a junction site and the same Xbal (position 82132) on either side of the fragments, it was concluded that these fragments must exist in the ICV DNA preparation either as DNA minicircles or as tandem repeats in a larger concatenated form.

To investigate whether the other hypermolar bands of 4.1, 5.5, 7.0 and higher also contained the non-Ar ori, a Southern blot of the Xbal digest was made, with a non- radioactive DIG-labeled probe of the non-hr ori (nt 83122 - 84048). The result (Fig. lb) showed that these fragments hybridized strongly to the probe and therefore it was concluded that a range of molecules of different sizes containing the SeMNPV non-hr origin of DNA replication predominated upon serial passage.

The 4.1 kb band was also cloned and sequenced and it was found that the borders of this fragment and the junction overlap of 9 bp were identical to those of the 2.6 kb fragment (Fig. 2b). The difference in size was explained by a duplicated non-hr ori present in this 4.1 kb fragment.

Replicative form hypermolar 2.6 and 3.0 kb oαl-fragments To investigate whether the abundant Xbal fragments of 2.6 and 3.0 kb exist as minicircles or as tandem repeats in a larger concatenated form, ICV DNA of PI 0 (at a stage that only the 2.6 and 3.0 bands were abundant) was subjected' to partial digestion wit . Xbal, using increasing amounts of restriction endonuclease during digestion for 20 min. Hybridization was performed with the same non-Ar ori probe as described above (Fig. 3). The partial Xbal digests of P10 viral DNA showed a "step-ladder" of multimers of the 2.6 and 3.0 kb bands. This suggests that the accumulation of the SeMNPV non-Ar ori occurs via high molecular weight concatemers of tandem repeats of different sizes. Not only is this likely to be the case for the 2.6 and 3.0 kb fragments, but also for the 4.1, 5.5, 7.0 kb and larger fragments from P15 onwards.

Identification of an additional 5.3 kb fragment

In addition to the non-Ar ori containing genomic Xbal-F fragment of 6.6 kb, an unexpected additional band of approximately 5.3 kb also hybridized to the non-Ar ori probe (see Figs, lb and 3). Similar to the Xbal-F fragment, it became submolar from PI 5 onwards. In order to identify and analyze this additional band, we cloned Xbal- digested intracellular SeMNPV DNA of P10 from Se301 cells of around 5.3 kb. Since the majority of the clones obtained were expected to contain either the comigrating Xbal-H or -I fragments of 5.2 kb, over 200 clones were screened by a colony lift, using the same non-Ar ori probe as described before. Several positive clones were picked up and two of these were sequenced terminally and by primer walking. The sequence, identical for both clones, revealed that the 5.3 kb fragment consisted of two joined, but distantly located sequences from the SeMNPV genome (Fig. 2b). The ends of the fragment corresponded to Xbal sites at positions 82132 and 119846, respectively. The junction between the two fragments was formed by an overlapping sequence stretch of 19 bp, containing multiple GTC-repeats, located at positions 86426-86446 and 118807- 118780. The presence of this 5.3 kb band in the wildtype SeMNPV DNA was confirmed by Southern hybridization.

Abundancy of the SeMNPV non-Ar ori concatemers in BV versus ICV

Since the non-Ar ori concatemers appeared to accumulate in ICV DNA, it was investigated whether these concatemers were equally abundant in DNA preparations from B V. Successive viral infections in cell culture causing the passage effect go through BVs. Viral DNA preparations from ICV and BV were digested with Xbal, analyzed on an agarose gel (Fig. 4a), and Southern-hybridized with a non-Ar ori probe (Fig. 4b). The restriction profile indicated that the concatemers made up of multimers of the 2.6 and 3.0 kb fragments were abundantly present in ICV, but in BV they were only visible by Southern hybridization. This indicated that concatemeric viral DNA was packaged in BVs.

Construction SeMNPV bacmid by direct cloning

To investigate whether concatemers of the SeMNPV non-Ar ori could be generated de novo in Se301 insect cells rather than selected from a pre-existing pool of genotypes, a full-length infectious clone of SeMNPV was obtained though E. coli (bacmid). A SeMNPV bacmid was constructed by direct cloning, utilizing a convenient unique SanOl restriction site in the polyhedrin gene (Fig. 5a). The bacterial part of the bacmid cloning vector was obtained by PCR using the commercial AcMNPV bacmid as a template. After circularization of the PCR product and subsequent linearization with SanΩl, the linearized SeMNPV DNA was cloned into the vector, resulting in the generation of 111 SeMNPV bacmid clones, designated SeBACO through 111.

After a first analysis with Xbal, five putatively complete clones (SeBAClO, 35, 72, 92 and 110) were selected for more extensive analysis. Expected restriction profiles were generated using the complete sequence of SeMNPV and the cloning vector (Life Technologies). Using DNAstar Editseq and Mapdraw, and Mapsort (GCG) a restriction map was constructed. Restriction profiles of the five bacmids were investigated in detail by digestion with Bglll, EcoRI, Pstl, Xbal, and Xhol (not shown). Due to Dam- methylation in the bacmid host E. coli DHlOβ, 5 of the 20 Xbal restriction sites were blocked, resulting in a dissimilar pattern compared to the wildtype SeMNPV Xbal digest. Ultimately, SeBAClO, 92, and 110 were found to contain all predicted restriction fragments and therefore were presumed to contain the complete SeMNPV genomic DNA. SeBAC35 and SeBAC72 both contained small deletions in the Xbal-A fragment and were not further considered.

Construction of a non-Ar ori deletion mutant by ET-recombination in E. coli.

Since non-Ar ori sequences accumulated upon serial passage, we investigated whether the stability of SeMNPV in Se301 insect cells could be enhanced by deletion 03

33 of the non-Ar ori. As the generation of SeMNPV recombinants by classical cotransfections and alternate cloning (Dai, X. J., et al, 2000, J. Gen. Virol. 10:2545- 2554) is complicated, time-consuming, and often results in major genomic deletions, the non-Ar ori was specifically deleted from the full-length infectious "bacmid" clone of SeMNPV-USl (SeBAClO) by "ET-recombination"( Muyrers, J. P., et al, 1999, Nucleic Acids Res. 27:1555-1557). This novel technique allows homologous recombination to occur in the BAG host strain, E. coli DHlOβ. A linear transfer DNA was amplified by PCR with primers, consisting of 50bp 5 '-ends, flanking the target region to be deleted (non-Ar ori, nt 83283 - 83934) on the SeMNPV genome, and of 20 bp 3' ends to amplify a chloramphenicol resistance gene (Cm^R) to be able to select for recombinant clones (Fig. 5b). The 1037 bp PCR product was used to electrotransform E. coli DHlOβ containing SeBAClO and an arabinose-induced plasmid pBADαβγ. Recombinant bacmids were selected by kanamycin and chloramphenicol resistance. This resulted in the recombinant SeMNPV bacmid SeBAClOΔnonhr, in which the 651 bp non-Ar ori was replaced by a chloramphenicol resistance (Cm^R) gene. The altered genotype of the recombinant bacmid was confirmed by Pstl digestion and PCR (Fig. 6). The genomic Pstl-I fragment of SeBAClO (7017 bp) was anticipated to be 286 bp bigger in SeBAClOΔnonhr, giving a fragment of 7303 bp (Fig. 6a). PCR with primer pair DZ 127/128 (Fig. 6b) was also carried out to confirm that recombination had been successful. The PCR product of 1213 bp (Fig. 6c) was cloned into pGEM-Teasy (Promega) and completely sequenced, revealing that recombination had occurred precisely at the anticipated locus via the 50 flanking nucleotides.

Serial passage SeMNPV bacmids in Se301 insect cells The availability of a genetically homogeneous SeMNPV bacmid (SeBACl 0) and a derived non-Ar ori deletion mutant (SeBAClOΔnonhr) now allowed to determine whether non-Ar ori concatemers are generated de novo in cell culture or pre-exist and become selectively amplified, and whether virus stability might be enhanced by deletion of this non-Ar ori. Prior to serially passaging the bacmid-derived BVs in cell culture, the polyhedrin gene was reintroduced in the (polyhedrin negative) bacmids by transposition from pFBlSepol as described in Materials and Methods, giving SeBAClOph and SeBACl OphΔnonhr (Fig. 6d). After transfection of Se301 cells, the BV containing supernatant was defined as the passage 1 (PI) virus stock and was used to initiate serial undiluted passage. ICV DNA was purified and digested with Xbal. Similar to SeMNPV-USl, deletions in Xbal-A occurred for both bacmids SeBAClOph and SeBAClOphΔnonhr (Fig 7). The deletion in SeBAClOphΔnonhr was mapped as a junction overlap of 3 nt (AAC) from 20162 to 36396, spanning ORF17-35. From P6 onwards a small hypermolar Xbal fragment of 3.0 kb was visible in DNA preparations of SeBAClOph (Fig. 7a). This fragment was cloned and sequenced and appeared to contain the non-Ar origin of DNA replication and a junction sequence (Fig. 2c) also observed with SeMNPV-USl wildtype (Fig. 2b). In contrast, the analysis of ICV DNA from SeBACl OphΔnonhr-infected cells did not reveal any accumulation of hypermolar fragments (Fig. 7b), whereas BV titers remained at high levels (Fig. 8a) and polyhedra production levels remained constant for at least 20 passages (Fig. 8b). These results demonstrated that absence of the non-Ar ori strongly increased the stability of the SeMNPV genome in Se301 insect cells.

SEQUENCE LISTING

<110> Stichting voor de Technisc e etenschappen

<120> Baculovirus expression syste

<130> Baculovirus expression system

<140> <141>

<160> 15

<170> Patentln Ver. 2.1

<210> 1

<211> 1267

<212> DNA

<213> Autographa californica nucleopolyhedrovirus

<400> 1 aagcttgtca atttcttcgg cttgcaaagg gctgccaacg cgttcgtttt gaatgcgcgt 60 aatccggttt acggtattgt tggcgcgaac aataaactcc tcaactggca aattaacaat 120 tttgtttgcg tactcattgt gcactgcggc caggttttgt agaatgtttt cgggaaaaat 180 ggcaattcta ttaaatttga catgtttttg attgtataca tagttttgat attcttccag 240 cgtaggatat ttgtttaaac tcttgacgca ttcaatgtac aatttgtgca gtgacaaaat 300 tctgttaaaa tccaaacgag aacatttctc aaaagttatt tcttgaccgt tgaaatgtac 360 actttgcaat tgtttcaata aactgtcgta aaaagttttt ccttcttcaa gcacaaacgc 420 ggggcgcatc gtgttatcta caacgcttat gtacttgtca aaatcttcaa ttatatgata 480 gaaatacaaa tatctctccg cgtttatgga cgtgtcgttt aaaacatgtt cgtcaacaac 540 tccgttatga tttactttca aaaatttcaa atcttgcaaa gcgtccgcgt tggtcaactt 600 gttgataata aatttgtctt tgcattcaaa cgctctgttt gcaatccact ccacagcgtc 660 caaaacggac atgcgtttaa acatgttgat acgttttaga caatacgctc gtttttttac 720 cgcctcaacg ttcacgtccg tgtagtcgca ccattgcagg atttgcaaca tgtcctcggc 780 aaaatgcgcg aactgccgca gcttttcctt tccaaaatgt tgattgtcgt gtttaaaaag 840 caacgttgaa atttccgaga cataccacaa agccgtgggc aattttactt tgatcagcgg 900 ctccatagcc aggttgctga acccgatcat gcattccgtg ttgttaatgc ggtaaatgac 960 atagcgttta aagtagtcct ttacattatc gtcaatgtat tctgcgtcgt ttatgtgctt 1020 gtacagcaaa tagtacataa ggcccgcgtt aaacgcgacc tttttagcgt caaaatacgt 1080 gcacgccaac acgtaatcgt tgtattcgtc gaattgctcg ttgggcacta tggcgcccgt 1140 aaaagggcgt ctgctgcgcg gtgacaaacg cgttccatgc tgaatcaact gcttcaaact 1200 ttccaaatta taacaatatt caattgaatt tttaatctct ttattttggc tccataaaag 1260 aggaaac 1267

<210> 2

<211> 3392

<212> DNA

<213> Spodoptera exigua nuclear polyhedrosis virus

<400> 2 tctagacaac tatacgtttc gtcgataatt tttttgataa ttcaatttca acgaaacgat 60 cctcatcttc gtcaaagtga catgttaaat ggataaactt ttaaattcgt tttgacgagt 120 gtcattgaaa ttacaaaatt gatttcgtcg aaaacttcaa ttttttgtca attttcaccg 180 acaacagcac gcatttcgtt gaaattacat taacattatg tttgtaactg cgcaatagat 240 ttttcgtcga aattaatgct agtttcgtca aagtgtcgta attatcaatt tccacgacaa 300 tgatatgttt ttcgtccaag atatagcaga aatgtttaaa tactagcgtc gtcgaaaata 360 tcgcttcatt cgagaacaca acgcgacatg cgtatcgact ttgacaaaaa tatggttgct 420 gaacaacatc atcataatcg tacgcaacca tactaccgta gtaccaaaaa caacaccgcc 480 accataacat tcgacgatct ggccgacatg tcaaagttga ctctgacgcg catgcgggcg 540 tttatcaaga aaaacacaaa cattgtcggc gtcaatagat acgatcgggc tagactggaa 600 cgaatactgc cgagagcggt caaggaggcg gaaacgaaaa aacgcgccgc cgagttaaag 660 aacgccgccg cagccgcagc ttgcgacgtc gttgaaacac ctcgaacatt cgtgatcgaa 720 gtgaatagaa tgttggtcga ttgtagtagg acacgatcga aagcaatagc cgcgttggag 780 aaacgataca caaagatggt cgtgtgttcg agtaaagtga atataccaga aacgacgtgt 840 cgcgtctgca aaaagttcat ggaaagttgg agaagcgtca cgttcggagc gtgeggtcat 900 agcgtcgtgt gtgcatcgtg cgatgaagat ggcaatgaaa aaaagtgtcc teaatgeaaa 960 caagtcataa tgtacacttt tgcgtttaaa ccttgcgttc ctttggtggc eggctttcaa 1020 taaaattttg tattacattt aaatgttttt ttattgactt tttgtcgtcg aaactggcac 1080 tagtttcgtt gaaattaaaa aaatgcattt actcgaaaac actgtacact tegtcaaaat 1140 aaatgacgca atattctgtt tttattgcat catctttaac atgtttcgac atgtatgatt 1200 tgcgtcatat ggtttctgcg aaatacacat ccctttcatc gatatcatat tgttttgaca 1260 aagatgatgc aataggtgat atgatatcat cataaattaa ctgegcaata gattttgtaa 1320 ctgcgcaatt ttctaactgc gcaatagatt ttacatcact tcttcgacaa ttgeatacat 1380 ttcgatgaaa acaatatgtg tattttgtcg aaaaggctat gtttttcgtc gaaacgtaaa 1440 ttacatttcg atgaaatgtc attggtttcg ttttacaagg ttaatttcac cgacaatacc 1500 gtctgtttct tcgaaaacgc attgaaaaaa aaaatggttt actcaaagat tcttagactt 1560 actcgacgat aacgcatttt tcgtcgaaac cgactttatg aaacgtttat taaaaattta 1620 gccaacctca ctcgttgtcg aaagtggccc ttgtttcgct tgaaatgaaa ttgaaaaatt 1680 tttgtcgcaa acattcgact cggaccgctt catcgatatt ataggegtet ttcgtcgaaa 1740 tctatattaa attcgatagc acgacgactt tgtggagatt cacatttgat tcacagttac 1800 aatattaaaa cgttgccggc aaattgggtt ttgattctaa tttttgaaat agtttcacca 1860 ttgtatatta catcgacgta cacgttcttt ttgtcgaaag tcacgtacgc ategatgegg 1920 ggaacgattt gtatcgtgtg gtatgtgagt ttgtcctggg gateggtaac gtatacgaac 1980 ccgtcgagac ataaactgcc atgcgtgcct cccgcgtctc cgctgacggg cacgatgeaa 2040 tgattatcgt cgttgacata gtagtcgctg atcaccgata ttagagcgcc gttgtcgtca 2100 aagatgggcg cgctcacgta cagttgggcc atgaattcgg caatgttgct cttctgcact 2160 accgccggaa cctgaccgta cacgtaatac tttccataat gatgatgegt gtagtacatt 2220 cgatcctgaa ctcgcatcct ccgaagatat gcctttttgt tcgaatcgaa gagcataatg 2280 ttcacatacg aatttttctt cagcttggcg aacttgacgt cgctagccac geceggatat 2340 tggtatttga tatgctcaaa ttccggcgcg gtgtcgtgct ggecgaaaac gtgaacgegg 2400 accgatcggt ccttttcgct gatgagatac actcgtcgcg cgacatggtt caccgaatag 2460 atgacagact tcatgtgttc gacgcgggcg gcgaaagtag agggtttggc gacatcgttg 2520 gtcgcaaccg aattaataca catcgacatt gaaaatgatg tegtttcgat gatcgttcaa 2580 ggttttatat agagagcaaa ttatacacac acgcatcgtt tactgtaaat atatttcaag 2640 tttattttta attttctctc ggcaaataca acatacttta caacgttttg cgcattcggc 2700 acacgtcgaa acgtgacgac acggtagaaa gcacgtgtct ctctcgcgct cgaaacatat 2760 cttgcacatg atgtcatcga tattgctggc agccgagttt gttattttgt cattgetaaa 2820 aatagaaatg tcatcatcat cattatccaa tttgtcgtcg tttagatttg gataaatgtt 2880 tgcgtattgg ttattttttc ttgtagtgac attgttatta atgtcgatat aatcgtcgtc 2940 gtcaatatct tcccaattag gagcggaagg tgcgttgaag tgacagttgg gcgagtaatg 3000 agcgtgtata atccgtacgt cttcgtcggc tttcagtttg acgatgacaa atttgeaega 3060 tgagcagcga atttcaaatt ttttgccgta atggtagaaa ccgttcttgg cgagcgtctc 3120 gcgattgtcc ctgtagtgtg tacgtgaagt tttaaatttg ggaaaggatt gtttgegtag 3180 cgcttcgttt aggaatagcc gctcgttggc catgggacac gtcgagaacg tatgatactt 3240 gaggcgacgt gcgtcgagct ttttgatgta tagcgaacaa tatgegcatt tgtaattgea 3300 taacgcgtct cggtagatac ccccggcgac gagatcgtcg atgtattctg gagtcaggca 3360 actgttttca aaggtttgga ggcgatcgcg eg 3392

<210> 3

<211> 3955

<212> DNA

<213> Orgyia pseudotsugata nuclear polyhedrosis virus

<400> 3 atgtctctat ccagtaagct tcttgtgtac gcctactacg gctcctacaa tttgccgcac 60 gaccgctacg gcgagtccta ccatttgtac agaatagtgc acgagcacct gaccaacacg 120 tacgtgagca aegegtegtg cgtgcgccgc gacatcgcca cggcgcgctg cttgaacagc 180 gggcatcttt gettcgaegt ggcgcgccag ttgctggacg taagcgaggt ggccgcgcgc 240 ttgtcggcct ggttccgctg cggcgacgcc accggcctgt gcgcagacat gcagcgcgcg 300 ctggccgaca ttgaccgcca cgcgccgctt gccaggcgcg ttggccgaag ggccaacatc 360 tttgcgctgg acgcgatcgc cgacattccg agcgacgtaa caaacaacct gcagggcatc 420 attggccggt tcatgcaσtt tccgcgctgc agcggcctgg cgcgggtcgc cgacgtgttt 480 gatccggaca tcagagccga cggttggtgg taccacaaat tttgcgtgtt gacgtacatg 540 caccttgtag cctgcggtgc agtgcccgcc ggatcggcca cgcggttgcg cgacgcggtc 600 gcaaaacata ttgggcccaa cgacgagggc aactgcgcgc cggcgattgc ggcggtgtac 660 ggccgattct gcgccattgg acgggagcac tttgcgσacc acaaaacggc ctgcatgcac 720 atcctgttcc agtttatgcg caacgacctg acgccggcgg acgagcggca cccctgtttt 780 ggcgtgatca aggattttgg ccggcagtgc aaggacacgt acaccgacct gcgcacacac 840 gccgacgcgc tctacatcca cggcacgacg gaccgacaaa aaaacgcgtt gtttgacctg 900 ttgtgctgcg tgaacgcgtc ggacattgac gccgactgct acgactgtgt tgtaaacaaa 960 ttttatgcaa cccaaaataa aaagtataaa atgtaaacgt tgttttgtta gttcacccaa 1020 tcgtttgtga agcgtggtcg cgcgcaacat gcagccgtat tctttgcgag aacaagcgcc 1080 gcggcagttt gtcaagctgg aggctcttgt gctggaagac gcggtggcgc tcaattcgaa 1140 cggcccgttg cgtttgtaga agcaaacaac cattgcggtg cttagttgac cggcggctga 1200 ttgcgcaact gctggttgcg cactgttgga ttatgagtct gtttgtgaaa gccaagctgt 1260 aatggagcgc gccatcgtgt ttttgttaat gtttgaacgc gcctacatgc tacatctgaa 1320 cattgttagg tcgtatactc aataaaatgt caaaataaat gttatgtttt atttaaaata 1380 gcctttttta caatgtcctt tgccacgttc acggcgσagt cgacttcgat cacttttttg 1440 ggcagccggc gaatcttgtg cccgccgttg gcaaattctt ccttgatgca atgcacggcc 1500 agcagcgggt tgggtgcacg ccctcgaaca tcaacgtcca tgccttcctc aaactccagc 1560 ttgcgcttgc gaaagtgctc ttcctgaccg cgcgcaaacg cgatctgcgt aacgccgcgg 1620 tcttcaacgc gacccatgaa cacggccaaa tgcgggtgct tggtcacgtc gcgcggaaac 1680 accacgccgg gcgccgtccg tgtgggcgcc gcgcactgca acgcgttaag tttcgtgtcc 1740 aatgtttgca ggcgctgctc aaattgctcg aaccgggcgc cgacctcgag tttaaacgac 1800 ttgtgcgcgc taataaactt gtcgctgttg gtgttgagcg ttttgacgtg gttcagcacc 1860 gcctcaattt tatccaacaa attgtcgcga tcgtggtcct tgtttgctcc aacggcgagc 1920 agtacctcgt ttttgttggc aaacgaaaat tgctgcaaca actggaccag cccgtgcttg 1980 gtggcaaaca agctgcccag agaatcgggt tgggcaggcg gcgcgcccaa caccaactca 2040 ttcagctggc gcacgtgttg ccggtcaaag ttgcagcgct gcaacaaatc gtggctaatc 2100 ttcaatcctt tgcaaaaatc cacgacgctg cagcagcggc cggccgtttc gtccaacacg 2160 taccggaact tgaacacgaa cgggtggcag cgaaacacgt cgcggcgcac caaatccgag 2220 tgtgcacgtt ggtcgaccgc gtaccgtctg taggcttgtt cgtcaaattc ggcgtttact 2280 tgctcgtcgg cggcgttcca ccagccaaac atccagctga acatgctttg atgcgttgtt 2340 gcaattgctg agtcaaacag cgcgagtagt cgcccccctt ataattgtaa ctaaacggcg 2400 cgcggatttg cttgtttgcg ttgcaaaaca cgtctcggtc cacgtcgggc cagtaacgca 2460 ataccaggtc ctcgcgcgcg tccccgatat acatgtgcac tgcgcgccgg acgcaatcgg 2520 cgaagctgcc cggccggatg tcgcccgaaa ccagctttgc gggttgcttg aacacgttga 2580 accagtgttc gcgtaaactt ttgggcgcgt ccattttaaa cttgccgcaa aacttgagcc 2640 acaaatggaa cccgcggtta ccgctgaaca tgatgcgctg cacggcgtct tctttgcctt 2700 caaaaaacaa cataaacgcg gtggcgccca cgtttacctt tagcataagt tcggctttgt 2760 cggcgcaatc tttgaaatcg gcgtcgatca cccattcgcg gccgccctcc tccagcggct 2820 tcacgtgcac gtcactgatc gagtttttaa caatgtatgc aaacagctgc gcggcgctgt 2880 cgaaaaaatt gtgcgcgtgc acccatcgtg gccggtctgt catgaacgcc agccggcgac 2940 tgtcgttgta cgcgatggcg tcccacatca tctgaacccg ctccggggtg tacttgcacg 3000 gcgccatcgg cggctgctat aaaaaacggg cgaggccgta ggggcacaca ctcttgattc 3060 cccaaacaat tgcaatggtt tttctaatta ttgcgctcac gttactcgcg actggcgcgc 3120 gggccgcaag tattctagcg gtgctgccca cgccggctta tagccaccac gtcgtgtaca 3180 gggcgtacgt gcacgcgctt gtgaaaaact gccacaacgt gaccgtgatc aagccacagc 3240 tgctcgacta cgccgtgcag gatgaatgcg gtcgtgtgga gcaaatcgac gccgacatgt 3300 ccgcgcaaca atacaagaag ctggtggcca gttcgggcgt gttccgcaag cgcggcgtgg 3360 tggccgacga gaccaccgtc accgccgaca actacatggg cctaatcgaa atgttcaagg 3420 accagtttga caacgccaat gtgcgtcgct ttctctccac taaccgcaca tttgacgccg 3480 tggtggtcga ggcctttgcc gactacgcgc tggtgtttgg gcacctgttt cgccccgcgc 3540 ccgtgattca aattgcgccg ggctacggcc tggccgaaaa ctttgaacgc cgccgcgccg 3600 tggcgcggca cccgctgcac tacccaacat ttggcgcagc agctttgacg cggcgcggcg 3660 gcgcgctcag tgaatggcgc ttgctgaacg agttcgagct gctggcgcgg cggtccgacg 3720 aactgctaaa acaacaattc ggaaaaagca cgcccaccat caggcagctg cgcgacaacg 3780 tgcagctgct gctgcttaat ttgcaccctg tgtacgacaa caaccggccg gtgccgccca 3840 gcgtgcagta cttgggcggc ggactgcact tggcgcaggc gctgccgcaa cggctggacg 3900 ccccgctcga acgccgactc aacgagtctg tcgacggggc ggtttacgta agctt 3955 <210> 4

<211> 344

<212> DNA

<213> Spodoptera littoralis nuclear polyhedrosis virus

<400> 4 aattcggacg atggtgcttt cgtcggcgat tgaggcggcg acggcgatgg tgcttcggcg 60 ggaggaggac gcgtgatggg cgcgatcgaa cgttctcact tgatcgttcg ttataattta 120 atactgcatc atgtgatgag cgtgttgtaa tttgtctcaa tgtcccgtat gacgagagtc 180 tctaatttca ccacagacgg aggctcggtg atctggccag atcgccgttc gacgtgagat 240 ttcattgatc agacggttct cgatttctac aacgaatcct gcgatccgtc gccgtcgtcg 300 tggtatctgt tcagcatcga tacagattca taaagttcat tctg 344

<210> 5

<211> 151

<212> DNA

<213> Bombyx mori nuclear polyhedrosis virus

<400> 5 aaacgtgttc gtcaacaact ttgttataat ttactttcaa aaatttcaaa tgttgcaaag 60 cgtccgcgtt ggtcaatttg ttgataataa atttgtcttt gcattcaaac gctctgtttg 120 caatccactc cacagcgtcc aaaacggaca t 151

<210> 6

<211> 721

<212> DNA

<213> Busura suppressaria nuclear polyhedrosis virus

<400> 6 tcagtaaaag tttattttat ttgtatgaca accaatcaaa acgtttcaat aattacggtc 60 aaaagaaatc aagttttcag cgctgacgct aataaattgt tggccgccaa gccgtcgaac 120 gataaaacgt tgtttttcgt ttgaataatt taccaaaaaa atgtcgttgt caaaaattca 180 atttggcgat aagcaagttt gagtgaagca aatagcaatc tcatcgtggc aaacaaagga 240 ttgttacaag cgtttgatat aattaactcg caaaaaaacc gcccaattag caaatcgtat 300 ggccgacatc gctcaggacg tgaatagcca aatcgtccga tccgcaattg ttccatttgt 360 tagcggttcg cgcgttgaga aacaaagaat acgcgttttt gcgcacacaa aaacgcagct 420 tcaaatcgta gtcttaagcg cttgggcgac aacaatgtgg tgtacaaatt cgactatgta 480 cccaacgcga tgaacgtgtt taacaaagtg aaagaaaatc ttcccaaaga caaatacaag 540 gccagacaca acaaaatcac actgttggaa aacttaactc gagaccagct tatcgaagcg 600 gtgtaatcat ccatgatgga aagacaaatt ataaagacat taatttcaaa agaaaataaa 660 taaaaatcat taaattgtag atgtgtattt tatttataaa tttctcaaaa tgttacactt 720 g 721

<210> 7 <211> 1300 <212> DNA

<213> Cydia po onella granulovirus

<400> 7 cttaagataa gctagccaag gacatgtgag ctgtcacgta ggccatattg gtgtgtaagt 60 cacgtggggt aatgtgatta gggtttgtaa taaaatattt aaagtttaat atttttcttt 120 acaacaataa tataatataa tacaattatt ataatacatt tttataatta taatacattt 180 ttataatata atacattttt ataatacatt tttataatac attgagttaa caagtaacaa 240 ttaatcaaaa tttgcaagct catgaaactg ctgtaactga ctaaagtaag aggtgctgtg 300 tttagagttg aagattgagc actcagggtc atgtatgtag tggttcagac agaatataca 360 ctatggctca tcttcatcat caataatgtc atcatcatct tcatcatcaa taatgtcatc 420 atcatcttca tcttcatctt catcttcatc atcaacttca tcatcaataa tgtcatagtc 480 atcatcatca tcatcgacct cattaatcac atagtcatca tcatcatcga cctcattaat 540 catctcccgg tccacggtcg tgtcatgttc ctcagccttg ttggttgtaa ggtcactccg 600 ctcaaacacc atcagaatgt tgcgcagctg tgtttttcat cctcttgtat tatgcctttt 660 tcaaacaaaa agcactcagg gtcatgtttg tagtggttca gacagaatat acactgtggc 720 ccttcatcat catcatagtc atcatcatca tcgacctcat taatcatctc ccggtccacg 780 gtcgtgtcat gttcctcagc cttgttggtt gtaaggtcac tccgctcaaa caccatcaga 840 atgttgcgca gctgttgttt ttcatcctct tgtattatgc ctttttcaaa caaaaagcac 900 tcagggtcat gtttgtagtg gttcagacag aatatacact gtggcccttc atcatcatca 960 tagtcatcat catcatcgac ctcattaatc atctcccggt ccacggtcgt gtcatgttcc 1020 tcagccttgt tggttgtaag gtcaatttca tcatcatcca catccaccac aggcgcagca 1080 tcttcaataa taacgtcatt atcagccacc ttcacctctg gcaaaccctt cagctccacc 1140 atgtacctca ctaccgcctc gtttgctcca ctgtccccat catcactccg ctcaaacacc 1200 atcagaatgt tgcgcagctg ttgtttgtca tcctcttgat atggcttttt caaacaaaac 1260 tggtaaagct ggcggttggt gtactgggga cggatacaca 1300

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: rimer

<400> 8 catcgatgcg tacgtgactt tc 22

<210> 9 <211> 18 <212> DNA <213> Artificial Sequence

<220> <223> Description of Artificial Sequence:primer

<400> 9 ccttgcgttc ctttggtg

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence .-primer

<400> 10 ccttcctgag gtaccttcta gaattccgga g 31

<210> 11 <211> 31 <212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence:primer

<400> 11 ccttcctcag gccgggtccc aggaaaggat c 31

<210> 12

<211> 70

<212> DNA

<213> Artificial Sequence

<220> <223> Description of Artificial Sequence: primer

<400> 12 catttactcg aaaacactgt acacttcgtc aaaataaatg acgcaatatt tttaagggca 60 ccaataactg 70

<210> 13 <211> 68 <212> DNA <213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: rimer <400> 13 atttcaaaaa ttagaatcaa aacccaattt gccggcaacg ttttaatatt ttcctgtgcg 60 acggttac 68

<210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence : primer

<400> 14 cccccgggta tatactagac gcgattac

<210> 15 <211> 28 <212> DNA <213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: primer <400> 15 ccaagctttg taatacttac cttttgtg

Claims

Claims

1. A baculovirus comprising in its genome an inactive form of a nucleotide sequence, the nucleotide sequence being enriched in genomes of defective interfering virus particles derived from a baculovirus.

2. A baculovirus according to claim 1, wherein the nucleotide sequence is an origin of DNA replication in a non-homologous region.

3. A baculovirus according to claim 1 or 2, wherein the nucleotide sequence or a part thereof is deleted.

4. A baculovirus according to any of the claims 1-3, wherein the baculovirus is selected from the group consisting of AcMNPV, SeMNPV, OpMNPV, SpliNPV, BmNPV, BusuNPV and CpGV.

5. A baculovirus according to any of the claims 1-4, further comprising in the genome one or more nucleotide sequences of interest.

6. A baculovirus vector comprising the genome of a baculovirus according to any of the claims 1-5, further comprising one or more DNA vector fragments.

7. A baculovirus vector according to claim 6, wherem the DNA vector fragments comprise one or more nucleotide sequences of interest.

8. A transfer vector, comprising a DNA vector fragment and a part of a baculovirus genome comprising an inactive form of a nucleotide sequence of a baculovirus genome, the nucleotide sequence being enriched in defective interfering virus particles of the baculovirus.

9. A transfer vector according to claim 8, wherein the nucleotide sequence is an origin of DNA replication of a non-homologous region.

10. A transfer vector according to claim 8 or 9, wherein the baculovirus genome is selected from the group consisting of the genomes of AcMNPV, SeMNPV, OpMNPV, SpliNPV, BmNPV, BusuNPV and CpGV.

11. A transfer vector according to any of the claims 8-10, further comprising one or more nucleotide sequences of interest.

12. A method for preparing a recombinant baculovirus, comprising the steps of: a) preparing a transfer vector according to any of the claims 8-11, b) transferring said transfer vector together with a genome of a baculovirus in a suitable host, c) culturing the host so as to produce the recombinant baculovirus, and optionally d) collecting the recombinant baculovirus.

13. A method for preparing a baculovirus vector, comprising the steps of: a) preparing a first baculovirus vector comprising one or more DNA vector fragments of interest and the genome of a baculovirus or a part of that genome, b) preparing a transfer vector according to any of the claims 8-11, c) transferring said transfer vector together with the first baculovirus vector in a suitable host, d) culturing the host so as to produce a second baculovirus vector, and optionally e) collecting the second baculovirus vector.

14. A method for producing a protein of interest, comprising the steps of: a) transfecting a baculovirus according to claim 5 or a baculovirus vector according to claim 7, wherein at least one of the nucleotide sequences of interest is encoding the protein of interest, in a suitable host, or co-transfecting a baculovirus genome together with a transfer vector according to claim 11, wherein at least one of the nucleotide sequences of interest is encoding the protein of interest, in a suitable host b) culturing the host so as to produce the protein of interest, and c) collecting the protein of interest.

15. A cell comprising a baculovirus according to any of the claims 1-5, a baculovirus vector according to claim 6 or 7 or a transfer vector according to any of the claims 8-11.

16. A kit comprising a baculovirus according to any of the claims 1-5, a baculovirus vector according to claim 6 or 7 or a transfer vector according to any of the claims 8-11 or a combination thereof.