title: Detection and quantitation of Agrotis baculoviruses in mixed infections.
abstract: At least four distinct baculoviruses, namely the Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), the A. segetum nucleopolyhedrovirus B (AgseNPV-B), the Agrotis ipsilon nucleopolyhedrovirus (AgipNPV) and the A. segetum granulovirus (AgseGV) have been isolated from larval stages (cutworms) of the species A. segetum and A. ipsilon (Lepidoptera: Noctuidae), which are serious soil pests in agriculture. Cutworms can become infected by at least one of these four baculoviruses and also co-infections of A. segetum larvae with AgseNPV-B and AgseGV are observed under laboratory conditions. Because of their adaption to common hosts and the occurrence in mixed infections, these viruses have a considerable potential as biological control agents of cutworms and are suitable objects to decipher the co-evolution and population dynamics of baculoviruses in mixed infections. However, to facilitate studies on these viruses a reliable tool for detection and identification is essential. A method based on highly specific oligonucleotide primers for multiplex polymerase chain reaction (PCR) that led to the amplification of discriminating fragments of the polyhedrin (polh) and granulin (gran) gene of AgseNPV-A, AgseNPV-B, AgipNPV and AgseGV, was established. Furthermore, the AgseNPV-B and AgseGV specific pairs of primers were applied in real-time PCR (qPCR) for AgseNPV-B/AgseGV ratio determination in samples of mixed infections. It is demonstrated further that for quantifying NPVs and GVs in mixed infections, the method of occlusion body isolation is most crucial and significantly influences the results.
authors.affiliation: Julius Kühn Institute, Federal Research Centre for Cultivated Plants Institute for Biological Control, Heinrichstr. 243, 64287 Darmstadt, Germany.
authors.forename: Jörg T
authors.initials: JT
authors.lastname: Wennmann
authors.affiliation: Julius Kühn Institute, Federal Research Centre for Cultivated Plants Institute for Biological Control, Heinrichstr. 243, 64287 Darmstadt, Germany. Electronic address: Johannes.jehle@jki.bund.de.
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Jehle
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000302:isolation & purification*; D060085:Coinfection / Q000821:virology*; D007915:Lepidoptera / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D000077183:Occlusion Body Matrix Proteins; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods; D019562:Viral Load; D015678:Viral Structural Proteins
chemical_list: D000077183:Occlusion Body Matrix Proteins; D015678:Viral Structural Proteins; C000625703:granulin protein, granulovirus; C009829:polyhedrin protein, Nucleopolyhedrovirus
keywords: Baculoviridae; Co-infection; Cutworms; Quantitative PCR; Virus purification