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Abstract

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing beta-galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh(-)/LacZ+ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of beta-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus, on A. gemmatalis (Lepidoptera: Noctuidae) larvae

“…The presence of AgMNPV-SF and/or EpapGV viral DNA in the treated larvae, was determined using specific primers designed to amplify the AgMNPV polyhedrin ORF (Arana et al, 2001) and EpapGV granulin ORF (Parola et al, 2002).…”

Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus

“…Transfer vector pAg-I -Ppo I was constructed as described by McCarthy and Romanowski [ 13 ] ( Figure 1 ). Briefly, linkers containing the I -Ppo I recognition sequence (CTCTCTTAA'GGTAGC) were introduced flanking the lacZ gene in pAgPHZ [ 14 ]. AgMNPV-2D DNA was then co-transfected with pAg-I Ppo I in UFLAg-286 cells using Tfx-20™ (Promega, Fitchburg, WI, USA) ( Figure 1 ).…”

Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

“…Stocks of AgMNPV-SF [38,39], were grown on monolayers of the UFL-Ag-286 cell line in plastic tissue culture flasks, tittered by plaque assay [40] and maintained as culture supernatants. The UFL-Ag-286 cell line was kindly provided by Bergmann Morais Ribeiro (Laboratorio de Virología e Microscopia Eletrônica, Universidade de Brasilia, Brasil).…”