Molecular and biological studies of fast and slow killing granuloviruses

Abstract

The aim of this project was to examine the relationship between granuloviruses (GVs) and factors or genes governing host range, speed of kill and tissue tropism. Hybridisations performed between "fast-killing" (fast) and "slow-killing" (slow) GVs showed that fast GVs appeared to be closely related, as did slow GVs. Fast GVs showed low DNA similarity and only partial collinearity to slow GVs. The exception was Adoxophyes orana GV (AoGV), which is a slow GV but showed relatively high DNA similarity and collinearity to fast GVs. This implied that the differences in speed of kill were not necessarily due to the large variations within the genome between fast and slow GVs. Dose and time mortality studies were performed using AoGV and the genome was physically mapped. This would allow easier access to areas of interest on the genome such as regions that did not hybridise to other GVs, that could be unique to AoGV, and would allow the initiation of a sequencing project of the whole genome. The granulin-containing area of the genome was sequenced for comparisons to other GVs. From these data, it is proposed that the relatedness of GVs is dependent on the family of Lepidoptera they infect rather than the tissue tropism of the virus or its speed of kill. An attempt to expand the host range of Cryptophlebia leucotreta GV (C1GV), to include Cydia pomonella, was undertaken by recombination with Cydia pomonella GV (CpGV). In one experiment C1GV replication was rescued in C. pomonella cells. This was either by complementation of the genome by CpGV or recombination with a gene or factor involving host range and will require further studies to identify the gene/factor responsible. Any recombinant GVs need to be assessed thoroughly for possible expansion of host range to non-target insects. Therefore, transfer vectors were constructed for the production of recombinant GVs containing a reporter gene (lacZ). A recombinant CpGV expressing ß-galactosidase was produced and will need to be cloned before further work is performed. The course of CpGV infection in hosts differing in permissivity to CpGV could also be studied using these recombinant viruses. Another part of this research investigated speed of kill. It involved the study of a 2.45 kbp region in CpGV-M1, which was not present in the genotype CpGV-R3. CpGVM1 is faster killing than CpGV-R3. A recombinant CpGV-R3 that contained this extra region from CpGV-Ml was produced and was compared to CpGV-R3 and CpGV-M1 by bioassay to assess its speed of kill. The speed of kill was not increased indicating that this region was not responsible. However, the study revealed some putative origins of replication of the CpGV genome