Isolation of a Spodoptera exigua baculovirus recombinant with a 10·6 kbp genome deletion that retains biological activity

Abstract

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25 %. This recombinant lacked 10 593 bp of sequence information, located between 13n7 and 21n6 map units of SeMNPV and including ecdysteroid UDP glucosyl transferase, gp37, chitinase and cathepsin genes, as well as several genes unique to SeMNPV. The result indicated, however, that these genes are dispensable for virus replication both in vitro and in vivo. A mutant with a similar deletion was identified by PCR in the parental wild-type SeMNPV isolate, suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses. The generation of recombinants in vivo, combined with the alternate cloning between insects and insect cells, is likely to be applicable to many baculovirus species in order to obtain biologically active recombinants.

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

“…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).…”

“…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.…”

“…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 al, 1998). 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. Dai et al (2000) isolated an SeMNPV recombinant with a deletion of 10?6 kb from nt 18513 to 29106, encompassing ORFs 15-28 (Fig. 1A).…”

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

“…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.…”