Characterization of Nuclear Polyhedrosis Viruses obtained from Adoxophes orana and from Barathra brassicae

ln infectivity experiments some A. orana larvae died after being inoculated with an inoculum containing NW isolated from B. brassicae. The polyhedra formed upon infection occluded single virus particles, whereas the inoculum contained polyhedra with bundles of virus particles. This change could be explained either by activation of a virus in A. orana, which is singly embedded, or the inoculum from B. brassicae had infected A. orana and consequently the inclusion of virus particles in outer membranes is controlled by the hosts. This thesis describes studies performed to discriminate between both possibilities. Therefore, the first task was to characterize the virus particles from B. brassicae and A. orana NPV and their polyhedra by different techniques (Chapter 1).The properties of the NW of A. orana and of B. brassicae as observed with the electron microscope and polyacrylamide gel electrophoresis are similar to those found for many other NPVs. The polyhedra of both NPVs differ in size and shape. Most of the A. orana polyhedra are globular and range in diameter from 1-2 μm. Most of the B. brassicae polyhedra are hexagonal or pentagonal in outline and range in diameter from 1.5-4 μm. Analysis of polyhedral protein by polyacrylamide gel electrophoresis shows the presence of two polypeptides of molecular weight 28,000 and 54,000 Daltons.Treatment of the polyhedra of both viruses with sodium carbonate ruptures the polyhedral membrane and the virus particles and polyhedral proteins are released. The virus particles of A. orana polyhedra are singly embedded in the polyhedral matrix and have a size of 250 x 60 nm. The multiply embedded virus particles of B. brassicae have a size of 347 x 113 nm. Analysis of the viral proteins by SDS-polyacrylamide gel electrophoresis showed that NW of A. orana has 5 polypeptides of 68,000, 48,000, 39,000, 32-34,000, and 28,000 Daltons, respectively. Those of the NPV of B. brassicae were 69,000, 57,000, 46,000, 34-39,000, and 28,000 Daltons, respectively.In the polyhedral membrane fractions of both polyhedra one polypeptide of molecular weight of 28,000 Daltons as estimated by polyacrylamide gel electrophoresis, was found.Due to proteolytic activity associated with the polyhedra, which is evident after dissociation of the polyhedra, it was difficult to establish the number of polyhedral proteins and their molecular weight (Chapter 2). The electrophoretic pattern of polyhedral proteins of A.orana and B. brassicae polyhedra dissociated in alkali differed from those proteins obtained by other means. Six to seven polypeptides with molecular weights between 28,000 and 8,000 Daltons were found after incubation at pH 10.5. After inactivation of the enzyme only two polypeptides with molecular weights of 28,000 and 26,000 Daltons were observed. When the polyhedral proteins were analysed without incubation at pH 10.5 also two proteins were found, but their molecular weight was 54,000 and 28,000 Daltons.On the basis of the results described in Chapters 1 and 2 it can be concluded that the virus particles of B. brassicae and A.orana NPV differ with respect to size, the way of occlusion, and the form and size of the polyhedra involved. Protein analysis by polyacrylamide gel electrophoresis reveal some difference in molecular weight of viral protein but no significant difference in the protein composition of their polyhedra. Further analyses of amino acid composition and sequence of these proteins is necessary to elucidate possible differences.To differentiate further between both viruses their genomes were analysed (Chapter 3). Both genomes are circular double-stranded DNA molecules. The molecular weights of A.orana and of B. brassicae NPV-DNAs are 6.7 x 10 <sup><font size="-1">7</font></SUP>and 8.9 x 10 <sup><font size="-1">7</font></SUP>Daltons, respectively as determined by electron microscopy and by renaturation kinetic analysis. The renaturation also indicated that both genomes contain only unique sequences. The buoyant density in CsCl of the NPV-DNA of A. orana and of B. brassicae is 1.694 and 1.696 g/cm <sup><font size="-1">3</font></SUP>, respectively. These values are in good agreement with (G+C) contents of 34.5 and 37%, respectively as determined by thermal denaturation. The digestion of the A. orana and of the B. brassicae NPV-DNA with endonuclease Eco RI resulted in completely different electrophoretic patterns. Also in experiments oncompetition hybridization no homology between these genomes was found. The conclusion of these studies is that these two NPVs can be clearly differen tiated by their DNA properties.In order to study the occurrence of viral DNA in uninfected larvae the DNA of A. orana and B. brassicae was isolated and the complexity studied (Chapter 4). The genomes of A.orana and of B. brassicae differ in their kinetic complexity as estimated from the reassociation data on hyperchromicity, but they are both relatively small and show remarkable similarity in the extent of intragenome homology. A haploid cell of A. orana has a DNA equivalent of 4.2 x 10 <sup><font size="-1">10</font></SUP>and that of B. brassicae of 8.4 x 10 <sup><font size="-1">10</font></SUP>Daltons. The intragenome homology was estimated to be 10 and 9% for A.orana and B. brassicae genome, respectively. The (G+C) content, estimated by thermal denaturation, was found to be 36.2% for the A. orana genome and 35.8% for the B. brassicae genome.The results obtained during rearing of insects from surface-sterilized eggs and from untreated eggs showed that the NPV of A.orana and of B. brassicae can be transmitted to the progeny of these insects on the outside of the eggs (transovum) as well as inside the eggs (transovarially) (Chapter 5). Evidence for transovarial transmission was also obtained from reassociation of viral DNA with the host DNA of homologous insects reared from surface-sterilized eggs. These experiments revealed the presence of viral sequences in host DNA: 0.03 and about 2.5 viral copies for the diploid quantity of the A. orana and of the B. brassicae host DNA, respectively.Results obtained in infectivity experiments with insects in various developmental stages showed that transstadial transmission is a prerequisite for generation-to-generation transmission.The presence of a latent virus infection in both insects could also be demonstrated in cross-inoculation experiments (Chapter 6). When the larvae of A. orana and of B. brassicae were inoculated with polyhedra of the reciprocal species, the number of larvae containing polyhedra increased compared with that of the control. Comparison of the restriction endonuclease Eco RI pattern of DNA isolated from polyhedra used as inocula with that from polyhedra obtained after cross-inoculation indicated that both viruses are not cross-infective but that they activate a latent virus infection in both insects. Because the cross- inoculation experiments were done under laboratory conditions (as aseptic as possible), it could be concluded that the B. brassicae NPV is not suitable for biological control of A. orana in the field, because this virus is not cross-infective