Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties

Kool, Marcel; Voncken, Jan Willem; Lier, F.L.J. van; Tramper, J.; Vlak, Just M.

doi:10.1016/0042-6822(91)91003-y

Cited by 165 publications

(115 citation statements)

References 36 publications

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confidence: 48%

“…The virus resulting from transfection of Sf21 cells was then passaged serially with minimal dilution (1 : 4) in Sf21 cells for 20 passages (final population: P cell 20). RFLP analysis revealed that viruses with large genomic deletions appeared in the population starting at P cell 10, as shown previously (Kool et al, 1991).Viruses of passages P cell 2, P cell 5, P cell 10 and P cell 20 were subsequently amplified in Sf21 cells. After 96 h, the cells were detached by agitation and sedimented by centrifugation (5 min at 2500 g).…”

mentioning

confidence: 63%

“…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).…”

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confidence: 99%

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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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confidence: 48%

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confidence: 63%

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confidence: 99%

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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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“…(i) Baculovirus can harbor very large pieces of DNA. 65,66 (ii) Baculovirus can efficiently infect nondividing cells and is not cytopathic. (iii) More than one recombinant baculovirus can be delivered in a simultaneous infection to a single hepatocyte, thereby making it possible to transfer multiple genes to primary rat hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

et al. 2003

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Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1.Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

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“…Once emerged, the population of FP mutants tends to enrich through successive passages in insect cell cultures, due to its increased capability to produce BVs. The DIPs are generated as consequence of genomic deletions that originate shorter viral genomes (Kool et al, 1990). The DIP genomes can not replicate autonomously, but they can do it with the help of complete genomes.…”

Section: Bvs: the Viral Inoculums In Insect Cell Culturesmentioning

confidence: 99%

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

Claus¹,

Gioria²,

Micheloud³

et al. 2012

Insecticides - Basic and Other Applications

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Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties

Cited by 165 publications

References 36 publications

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

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

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

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