Mamestra configurata nucleopolyhedrovirus (MacoNPV) : potential chitin-binding proteins and their role in oral infectivity
The bertha armyworm (Mamestra configurata) is a major pest of canola and other oilseed crops. A promising control agent for this species is the baculovirus Mamestra configurata nucleopolyhedrovirus (MacoNPV). Baculoviruses are insect-specific viruses. Infections initiate in the host midgut following ingestion of virus particles called occlusion bodies. For a productive infection to occur, the occlusion bodies must dissolve to release the infectious occlusion-derived virions. These virions must pass through the peritrophic matrix, a protein-chitin meshwork that lines the midgut of most insects and provides protection against abrasion and pathogen invasion. The mechanism by which the baculovirus virions transit the peritrophic matrix is unknown. Following the initial infection of midgut cells, a second virion phenotype, the budded virus, is released from infected cells and establishes a systemic infection within the insect. The 11K group of genes, which are conserved among baculovirus species and other insect-infecting viruses, encode proteins with a predicted chitin-binding domain. The degree of conservation of these genes among insect-infecting viruses suggests that they may play a role in insect infectivity. It is possible that the gene products could be involved in an interaction between the baculovirus occlusion-derived virions and the peritrophic matrix or the chitin-secreting cells of the midgut epithelium, and therefore may be involved in initial oral infectivity. The two 11K genes from MacoNPV (ORF 118 and ORF 164), and their homologues in a second species of baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV [ORF145 and ORF150]) were expressed in a baculovirus expression system. The ability of the proteins, Maco118, Maco164, Ac145, and Ac150, to bind to chitin was assessed in vitro using chitin-coated beads. Each of the four proteins binds to chitin, and hydrophobic interactions mediate the binding. Other binding mechanisms are likely involved, but were not determined in this project. To determine the function of these proteins, a series of gene knockout and repair constructs was produced for AcMNPV ORF 145 and ORF 150 using an established bacmid system. An analysis of the knockout and repair constructs using quantitative real-time polymerase chain reaction showed that deletion of either ORF 145 or ORF 150 had no effect on the rate of budded virus production or viral DNA replication. Oral and injection bioassays were performed in Trichoplusia ni larvae to determine if there were differences in infectivity between the knockout, repair, and wild type constructs. Injection assays, in which budded virus from each construct was injected directly into the insect haemocoel, therefore bypassing the midgut and peritrophic matrix, indicated that there was no statistical difference in infectivity between the knockout, repair, and wild type constructs at a dose of 15 TCID50 U per larva. Oral bioassays, in which larvae were fed occlusion bodies from each virus construct, indicated that there was no statistical difference in mortality rates between the knockout, repair, and wild type constructs. The results from this study indicate that although the baculovirus 11K genes are highly conserved among baculovirus species, and the 11K gene products from MacoNPV and AcMNPV interact with chitin, they are not required for oral infectivity in T. ni larvae, and likely serve another function in the baculovirus infection cycle.
DegreeMaster of Science (M.Sc.)
CommitteeHegedus, Dwayne; Gillott, Cedric
Copyright DateDecember 2012