In vivo isolation of baculovirus genotypes

Smith, Ian R.; Crook, Norman E.

doi:10.1016/0042-6822(88)90165-1

Cited by 149 publications

(104 citation statements)

References 27 publications

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“…was still so low that it had effectively not changed. This is another indication that the number of founders of infection is small, as has previously been suggested (Smith & Crook, 1988).…”

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confidence: 48%

“…was still so low that it had effectively not changed. This is another indication that the number of founders of infection is small, as has previously been suggested (Smith & Crook, 1988).Deletion mutants missing ie-1, or with multiple copies of the non-HR ori, were rapidly purged from the populations during passaging in vivo (Fig. 2b).…”

mentioning

confidence: 55%

“…As the number of viruses initiating infection appears to be small (e.g. Smith & Crook, 1988), initially the m.o.i. is extremely low.…”

mentioning

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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“…was still so low that it had effectively not changed. This is another indication that the number of founders of infection is small, as has previously been suggested (Smith & Crook, 1988).…”

mentioning

confidence: 48%

mentioning

confidence: 55%

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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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“…Comparison with a baculovirus isolate from Buzura thibtaria indicated that many of the submolar bands observed for BusuNPV were equimolar in ButhNPV (data not shown). In vivo cloning techniques (Smith & Crook, 1988) or plaque purification techniques using cultured cells (King & Possee, 1992) are required to isolate and characterize the genotypic variants of BusuNPV in more detail.…”

Section: Resultsmentioning

confidence: 99%

Distinct gene arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome.

Wang

Arif

Jin

et al. 1998

Journal of General Virology

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“…The original virus also contained a GV, which was separated and removed by using glycerol and sucrose gradients following methods described previously (Crook & Payne, 1980). A cloned genotype of AdorNPV was obtained through three successive rounds of in vivo cloning, using the limiting dilution method described by Smith & Crook (1988a). Absence of the GV was confirmed by PCR using AdorGV-specific primers.…”

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confidence: 99%

Genomic sequence and biological characterization of a nucleopolyhedrovirus isolated from the summer fruit tortrix, Adoxophyes orana

Hilton

Winstanley

2008

Journal of General Virology

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Adoxophyes orana nucleopolyhedrovirus (AdorNPV) was isolated from overwintering larvae from an orchard in the UK. The nucleotide sequence of the AdorNPV DNA genome was determined and analysed. The genome contains 111724 bp and has a G+C content of 35.0 mol%. The analysis predicted 121 ORFs of 150 nt or larger. Of these putative genes, 118 were homologous to genes identified previously in the Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) genome (83.3-100 % aa identity), and three AdorNPV ORFs were unique. There were four small homologous regions that consisted of a similar core sequence and at the same relative positions in the genome as AdhoNPV, but they differed in the number of repeats and orientation. Some genes that have been reported to have major roles in baculovirus biology were either absent or truncated in the AdorNPV genome. These included chitinase, which is involved in the liquefaction of the host, and the C-terminal of the ecdysteroid UDP-glucosyltransferase (egt) protein, which was truncated by 149 aa compared with AdhoNPV, with essential amino acids absent. The AdorNPV genome encoded two inhibitor of apoptosis (iap) genes compared with three in AdhoNPV and three bro genes compared with four in AdhoNPV. The susceptibility of A. orana larvae to AdorNPV was evaluated in laboratory bioassays using inoculation by microdroplet feeding and applied dose assays. LD 50 for neonates was 56 occlusion bodies rising to 2.3¾10 4 for fifth instar larvae. Median survival time values using an LD 80 dose were 8.8 days for neonates and 7.0 days for fifth instar larvae. INTRODUCTIONThe family Baculoviridae consists of invertebrate viruses with large circular, covalently closed, double-stranded DNA genomes with enveloped nucleocapsids and an occluded form of the virus embedded in proteinaceous occlusion bodies (OBs) . There are two genera of baculovirus, the Nucleopolyhedrovirus (NPV) and the Granulovirus (GV) based on OB morphology . The NPVs are further classed into group I and II NPVs (Bulach et al., 1999;Zanotto et al., 1993). The majority of the sequenced NPVs infect insects of the Lepidopteran order, but NPVs that infect dipteran or hymenopteran insect species like the mosquito-infecting Culex nigripalpus NPV, or the NPVs from three Neodiprion species, have been sequenced (Afonso et al., 2001;Garcia-Maruniak et al., 2004;Lauzon et al., 2004; Duffy et al., 2006). Baculoviruses are used worldwide as biological control agents of agricultural and forest pests. The summer fruit tortrix moth (Adoxophyes orana) is a pest of apples and pears in most of Europe and Japan.During the past 25 years, there has been increased interest in the use of a naturally occurring GV as a control agent for the summer fruit tortrix (Cross et al., 1999). Recently, a mixture of baculoviruses was recovered from larvae in the UK (Hilton & Winstanley, 2008). These were separated and found to comprise an NPV and a GV. The GV (Adoxophyes orana GV) has been characterized and completely sequenced (Wormleaton et al., 2003;Hilton & Winstanley, 2...

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In vivo isolation of baculovirus genotypes

Cited by 149 publications

References 27 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

Distinct gene arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome.

Genomic sequence and biological characterization of a nucleopolyhedrovirus isolated from the summer fruit tortrix, Adoxophyes orana

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