Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo

Heldens, J.G.M.; Strien, E.A. van; Feldmann, Angelika; Kulcsár, Péter István; Muñoz, Delia; Leisy, Douglas J.; Zuidema, D.; Goldbach, Rob; Vlak, Just M.

doi:10.1099/0022-1317-77-12-3127

Cited by 44 publications

(59 citation statements)

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“…Large-scale production of SeMNPV for biological control is carried out in vivo using insect larvae, since SeMNPV infection in cell culture leads to the rapid generation and predominance of deletion mutants. These mutants, with deletions up to 25 kb, often lack the ability to infect S. exigua larvae by oral ingestion of OBs (Heldens et al, 1996). Therefore, the genetic engineering of SeMNPV via recombination in cell culture is complicated (Dai et al, 2000) and precludes the in vitro production of biologically active SeMNPV in insect cell bioreactors.…”

Section: Introductionmentioning

confidence: 99%

“…Heldens et al (1996) described a SeMNPV mutant with a deletion in XbaI-A of about 25 kb, spanning open reading frames (ORFs) 14-41 (IJkel et al, 1999). This mutant replicated efficiently in cell culture but lacked bioactivity in vivo.…”

Section: Introductionmentioning

confidence: 99%

“…required for virus replication in cultured insect cells or in vivo after intrahaemocoelic injection. In previous studies involving SeMNPV deletion mutants generated via cell culture or in vivo (Heldens et al, 1996;Dai et al, 2000), it could never be excluded that a minor amount of intact helper virus was still present enabling predominant replication of deletion mutants. With the current strategy using bacmids, pure baculovirus mutants were generated from single-copy bacterial artificial chromosomes maintained in E. coli (Luckow et al, 1993;Pijlman et al, 2002).…”

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confidence: 99%

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Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Pijlman

Pruijssers

Vlak

2003

Journal of General Virology

127

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Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29-35. In this paper we have shown that SeMNPV ORFs 15-35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15-35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviridae. INTRODUCTIONSpodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infects the single insect species S. exigua (Smits & Vlak, 1988) and belongs to the group II NPVs. Baculoviruses of this group do not contain a GP64 homologue, but have a functionally homologous F protein as a structural element of the budded virus (BV) . BVs are required for the systemic spread of infection throughout the insect, whereas occlusion-derived viruses (ODVs) are involved in the horizontal spread of the virus in insect populations (Blissard & Rohrmann, 1990). Occlusion bodies (OBs) are ingested orally and the alkaline environment of the midgut causes the release of the ODVs. The ODVs first pass the peritrophic membrane and subsequently fuse to the midgut epithelial cells, thereby causing the initial infection (Funk et al., 1997). Large-scale production of SeMNPV for biological control is carried out in vivo using insect larvae, since SeMNPV infection in cell culture leads to the rapid generation and predominance of deletion mutants. These mutants, with deletions up to 25 kb, often lack the ability to infect S. exigua larvae by oral ingestion of OBs (Heldens et al., 1996). Therefore, the genetic engineering of SeMNPV via recombination in cell culture is complicated (Dai et al., 2000) and precludes the in vitro production of biologically active SeMNPV in insect cell bioreactors.Deletions in the SeMNPV genome predominantly occur within the XbaI-A restriction fragment and these deleted genotypes are present in SeMNPV wild-type isolates where they may act as parasitic genotypes (Muñoz et a...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Pijlman

Pruijssers

Vlak

2003

Journal of General Virology

127

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“…1a; F 1,10 56.937, P50.025). Unlike S. exigua MNPV (Heldens et al, 1996), passaged populations of AcMNPV retain infectivity -albeit reduced -through to passage 20. It was thus possible to use polyhedra of the P cell 20 population to reinfect insect larvae.…”

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confidence: 99%

“…Populations with a high frequency of these deletion mutants can completely lose infectivity for insect larvae (Heldens et al, 1996). The presence of such deletion mutants is therefore likely to be deleterious to the fitness of a virus population in vivo, although deletion mutants that can increase fitness are found in some natural baculovirus isolates (Ló pez-Ferber et al, 2003).…”

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confidence: 99%

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

Zwart

Erro

Oers

et al. 2008

Journal of General Virology

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The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.The passage of baculoviruses in cultured insect cells leads to the rapid accumulation of deletion mutants, some of which have been demonstrated to have defective interfering properties (Kool et al., 1991;Pijlman et al., 2001;Wickham et al., 1991). Populations with a high frequency of these deletion mutants can completely lose infectivity for insect larvae (Heldens et al., 1996). The presence of such deletion mutants is therefore likely to be deleterious to the fitness of a virus population in vivo, although deletion mutants that can increase fitness are found in some natural baculovirus isolates (Ló pez-Ferber et al., 2003). Dai et al. (2000) found that alternate passaging of baculoviruses in insect cells and larvae resulted in sustained infectivity for insect larvae, suggesting that purifying selection (Li, 1997) occurs in these animals. In other words, there may be stabilizing selection for a particular trait, in this case the ability to replicate autonomously. Consequently, individuals that do not possess this trait are removed from the population. However, the fate and dynamics of these cell culturederived deletion mutants after reintroduction into insects have not been systematically addressed. Therefore, a clonal baculovirus was first serially passaged in insect cells to enrich for deletion mutants, and the resulting population was reintroduced into insect larvae. The effects of both in vitro and subsequent in vivo passaging were investigated by determining the virulence and occlusion-derived virion (ODV) content of polyhedra, and the frequency of occurrence of deletion mutants by quantitative real-time PCR (qPCR).In order to generate a clonal population of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), bacmid technology (Luckow et al., 1993) was employed. A bacmid with restored polyhedrin (polh) expression was generated using the pFastBac-DUAL/Polh construct (Zwart et al., 2008). This bacmid was used to transfect Spodoptera frugiperda Sf-AE-21 (Sf...

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Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect‐cell bioreactors

Pijlman¹,

Vrij²,

End³

et al. 2004

Biotech & Bioengineering

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Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred. These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmid-derived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmid-derived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed.

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Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo

Cited by 44 publications

References 21 publications

Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect‐cell bioreactors

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