Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure

The role of biotic mechanisms in baculovirus (BV) dispersal was investigated using three Lepidopteran pests; Plodia interpunctella (Hubner), Ephestia cautella Walker and Mamestra brassicae L. and their respective BVs. The first two, pyralid moths, are pests of stored products, and the third is a pest of brassicas. Detailed host biology and virus mortality studies were undertaken to provide background information for the main investigation of virus dispersal using P.interpunctella. BV infectionincreased larval activity and thus BV dispersal in all three species until the disease at an advanced stage caused sluggish behaviour and mortality. However, larva to larva virus transmission was limited, especially as the integuments of diseased pylarids did not rupture and cannibalism was rare when a suitable food supply was available. P.interpunctella adults which received a sub-lethal BV dose in the larval stages appeared normal but the proportion of eggs oviposited, the viability of the eggs and the survival of the progeny were reduced. However, no BV was detected in the adult stage. Transovum transmission occurred if the adults were externally contaminated with BV. Scavangers, predators and adult parasitoids which fed on diseased prey voided viable BV in their faeces. This contaminated the larval medium and resulted in larval infection. The predators tested readily fed on diseased prey but the parasitoids were less able to compete with BV for hosts, Bracon hebetor Say preferentially avoided diseased hosts. There was little evidence to indicate that BV can be mechanically vectored between host larvae on the ovipositor of a parasitoid or the mouthparts of a predator. The relative potential of the host and other biotic mechanisms to cause BV dispersal is discussed.

Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.

From Introduction: The pine emperor moth, Nudaurelia cytherea capensis Stoll is an insect which, during the larval stage, causes extensive defoliation of the pine tree, Pinus radiata in the Cape province. These insects are susceptible to a virus disease, which on occasions causes large scale mortality. Five nonoccluded viruses have been shown to infect the pine emperor moth, and of these, one found in the greatest concentration, Nudaurelia capensis β virus (NβV) has been characterised to the greatest extent. This virus has been shown to contain RNA, to be isometric with a diameter of 36 mm, and to have a molecular weight of 16 million. The virus occurs in all stages of the insect's development, and by fluorescent antibody staining has been shown to develop in the cytoplasm of the host's cells. There have in recent years been a number of reports describing nonoccluded RNA viruses which appear to be similar to NβV. These are the viruses isolated from the moths Gonometa podocarpi and Antheraea eucalypti, and the one from the citrus red mite, Panonychus citri. These viruses have not been as extensively characterised as NβV, so the extent of the similarity between them and NβV is not known. However it would appear as if their discovery collectively heralds the emergence of a distinct new grouping within the nonoccluded RNA viruses of insects. This work reports the isolation and further characterisation of N. capensis β virus, its protein and nucleic acid.

Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.

After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.

Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.

No description available.

A systematic survey of the microbial and invertebrate natural enemies of Phyllophaga spp. was conducted from 1979 to 1981 in 45 localities in southern Quebec. Chronic but low (nonepizootic) rates of biotic regulation were found in all host life stages except eggs. The natural enemies included 36 species of predatory and parasitic insects, 15 mite species, six fungal species, five bacterial species, three different nematodes, one virus, one gordian worm, and one sporozoan. Microorganismal pathogenicities to white grubs were demonstrated by infectivity tests and laboratory bioassays using four methods of inoculation. Field-type microplot studies on the fungus Metarhizium anisopliae and the nematode Mikoletzkya aerivora were undertaken in 1982. The fungus and the nematode showed potential as biological suppressants of Phyllophaga grubs by causing 91% and 68% mortality, respectively. Twenty-seven chemicals were tested as attractants against P. anxia adults in 1981 in four localities in southern Quebec. Hexanoic acid was consistently the most attractive chemical to beetles of both sexes.

The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.