Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo

1 Department of Virology, Wageningen Agricultural University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands 2 Institute for Plant Protection Research, Binnenhaven 5, 6709 PD Wageningen, The Netherlands 3 Departmento Produccion Agraria, Universidad Pública de Navarra, Campus de Arrosadia, 310006 Pamplona, Navarra, Spain 4 Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331, USA The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus ( Se MNPV) has high potential for development as a bio-insecticide for control of the beet armyworm ( S. exigua ). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in SeUCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNa from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases Xba I, Bam HI, Bgl II, Pst I, Sst I, Hin dIII and Spe I. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p 10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12·9 and 32·3. Present address: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary Received 18 June 1996; accepted 13 August 1996.