Isolation of a Spodoptera exigua baculovirus recombinant with a 10 super(.)6kbp genome deletion that retains biological activity

When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity...

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Bibliographic Details
Published in:Journal of general virology 2000-10, Vol.81 (10), p.2545-2554
Main Authors: Dai, X, Hajos, J P, Joosten, N N, Van Oers, MM, IJkel, WFJ, Zuidema, D, Pang, Y, Vlak, J M
Format: Article
Language:English
Subjects:
Baculovirus
Multicapsid nucleopolyhedrovirus
Noctuidae
Nuclear polyhedrosis virus
p10 gene
Spodoptera exigua
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Summary:When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25kbp 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 as 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 10593bp of sequence information, located between 13 super(.)7 and 21 super(.)6 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.
ISSN:0022-1317