Isolation of a Spodoptera exigua baculovirus recombinant with a 10.6 kbp 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 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 10593 bp of sequence information, located between 13.7 and 21.6 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.
Dai
Xiaojiang
X
State Key Laboratory for Biocontrol and Institute of Entomology, Zhongshan University, Guangzhou 510275, People's Republic of China2.|Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
Hajós
József P
JP
Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, PO Box 7, 1518 Budapest, Hungary3.|Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
Joosten
Nina N
NN
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
van Oers
Monique M
MM
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
IJkel
Wilfred F J
WFJ
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
Zuidema
Douwe
D
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
Pang
Yi
Y
State Key Laboratory for Biocontrol and Institute of Entomology, Zhongshan University, Guangzhou 510275, People's Republic of China2.
Vlak
Just M
JM
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1.
D000818:Animals; D016367:Baculoviridae / Q000302:isolation & purification* / Q000472:pathogenicity; D015153:Blotting, Western; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D016679:Genome, Viral; D016297:Mutagenesis, Site-Directed; D021281:Quantitative Structure-Activity Relationship; D018411:Spodoptera / Q000821:virology*
D004274:DNA, Recombinant; D004279:DNA, Viral