Characterization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome : a (phylo)genetic study

Baculoviruses are attractive biological alternatives to chemical insecticides for insect pest control. So far, more than 600 baculoviruses have been isolated from different insect species which provide a rich resource for developing new viral insecticides. Most baculoviruses are host specific. Baculovirus nomenclature is based on the host from which the virus was first isolated and the taxonomy is based on the morphology of the occlusion body, polyhedra (nucleopolyhedroviruses = NPVs) or granula (granuloviruses = GVs). Until 1995, the NPVs had been further subdivided into single-nucleocapsid (S) and multiple-nucleocapsid (M) NPVs. Phylogeny studies based on the polyhedrin gene indicated that lepidopteran NPVs can be classified into two groups (I and II). Group II NPVs were found to encompass both SNPVs and MNPVs, whereas group I NPVs were composed of only MNPVs. Three Group I NPVs, Autographa californica (Ac) MNPV, Orgyia pseudotsugata (OpMNPV) and Bombyx mori (Bm) NPV, have been entirely sequenced and their genome organization was found to be very similar. Such detailed sequence information from other baculoviruses is scare particularly from SNPVs and GVs. The subject of this thesis is the Buzura suppressaria (Busu) NPV which was isolated from a Geometrid insect (tea moth) and has been used successfully as a bio-insecticide in China. Significant amount of sequencing was conducted on a genomic library of this virus which allowed us to conduct comparative investigations on the genome organization and phylogeny.

First, the most conserved baculovirus gene, the polyhedrin gene, was identified by hybridization and then sequenced (Chapter 2). The location of the polyhedrin gene was used to set the zero point of the physical map of the viral genome. Phylogeny research based on the polyhedrin sequence placed BusuNPV into the Group II baculoviruses (see Chapter 8).

Ecdysteroid UDP-glucosyltransferase (EGT) is an enzyme encoded by baculoviruses which delays larval molting and allows the virus to extend the production of large numbers of progeny virus. Deletion of the egt gene from the genome of baculoviruses results in an increased speed of kill and a reduced larval feeding activity and therefore enhances viral insecticidal activity. The strategy of deleting egt is now being adopted for the engineering of baculoviruses of economically important insect pests. In Chapter 3, the identification, sequencing and comparative analysis of the egt gene of BusuNPV is reported. This is the first egt gene characterized in a SNPV. Maximum parsimony analysis based on the amino acid sequence of baculoviruses EGTs confirmed that BusuNPV belongs to Group II baculoviruses.

In order to study the genome organization of BusuNPV, a plasmid library covering the entire genome was constructed and the inserts were terminally or completely sequenced. In Chapter 4, the sequence analysis of the Hin dIII-J region (3.2 kb) of the BusuNPV genome revealed five ORFs ( pep , ORF117 , ctl , iap and sod ). The genomic arrangement of these ORFs in BusuNPV turned out to be significantly different from that of AcMNPV and other characterized baculoviruses. This indicates that BusuNPV may have a gene arrangement distinct from other baculoviruses identified so far.

To further investigate the genomic organization and phylogenetic status of BusuNPV, a detailed physical map was constructed by extensive restriction enzyme analysis and Southern blot hybridization (Chapter 5). The size of the viral genome was estimated to be approximately 120.9 kb. About 43.5 kb of dispersed sequence information was generated from the plasmid library. Fifty-two ORFs, homologous to those of other baculoviruses, were identified and their location on the BusuNPV genome determined (Chapter 5). Although the gene content of BusuNPV, based on these 52 ORFs, is similar to that of AcMNPV, BmNPV and OpMNPV the arrangement is, however, significantly different from the latter, highly co-linear Group I NPVs. A new approach (GeneParityPlot) was developed to represent the differences in gene order among baculoviruses when limited sequence information is available. The method is useful to identify potential conserved gene clusters. The data obtained show that BusuNPV is a distinct baculovirus species and suggest that group II viruses may have a genome organization distinct from Group I viruses. It is concluded that the gene distribution along baculovirus genomes may be used as an independent parameter to study baculoviral phylogeny.

Protein P10 is a major component of the fibrillar structures found in the cytoplasm and nucleus of infected insect cells. This small protein is involved in the release of polyhedra from infected cell-nuclei late in infection and plays a role in morphogenesis of the occlusion body. A putative p10 gene was identified for the first time in a SNPV (BusuNPV) based on its size and domain structures. Its function was determined in a 'swap assay' by using an AcMNPV p10 deletion mutant as an acceptor for the putative BusuNPV p10 gene (Chapter 6). The AcMNPV recombinant expressing the BusuNPV P10 formed fibrillar structures in the cytoplasm of Spodoptera frugiperda cells, but was unable to induce nuclear disintegration. This supports the current hypothesis that additional viral factors are required for this process.

Polyhedrin is the major component of the occlusion body, which encapsulates the virions and protects them against physical and chemical decay. The gene swap assay was applied to investigate the specificity of the polyhedrin gene in the generation of occlusion bodies (Chapter 7). The BusuNPV polyhedrin gene expressed by AcMNPV resulted in the formation of normal occlusion bodies containing multiple-capsid virions. The occlusion bodies were as infective as normal AcMNPV suggesting that the occlusion process is not SNPV or MNPV specific. However, the recombinant AcMNPV expressing the polyhedrin gene of Spodoptera exigua (Se) MNPV, had an altered polyhedron morphology, pirmidal in shape, with few occluded virions. The infectivity was also lower than that of wild-type AcMNPV. These data suggest that the occlusion process is fine-tuned and may be dependent on one or more factors in addition to polyhedrin, that determine the size, shape and occlusion of virions.

The genetic analysis compiled in this thesis shows that BusuNPV is a distinct baculovirus species. Alignment of various BusuNPV ORFs with baculovirus homologues and phylogenetic analyses indicated that SNPVs do not form a monophyletic group within the phylogenetic trees. The gene content of BusuNPV is very similar to that of AcMNPV, BmNPV and OpMNPV, suggesting that baculoviruses may have most of their genes in common. In addition to gene homology genome organization may also reflect the evolutionary history of baculoviruses. The molecular genetic studies of BusuNPV and their impact to future engineering of the virus for improved insecticidal activity are discussed (Chapter 8).