A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment.

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

A rapid bioassay screen for quantifying nucleopolyhedroviruses (Baculoviridae) in the environment.

A quantitative bioassay method for the detection of Helicoverpa armigera (Lepidoptera: Noctuidae) singly encapsulated nucleopolyhedroviruses (HaSNPVs) in soil is described. Calibration curves used for estimating soil virus titres in environmental samples were generated by incorporating a sterilised soil into a semi-synthetic insect diet then inoculating known concentrations of an HaSNPV into the soil-diet mixture. Calibration curves were constructed for soil diets containing varying proportions of soil: 0, 1, 5, 10 and 25% soil (w/v). Their accuracy was assessed in a series of blind tests in which the actual soil virus concentration fell within the estimated mean 95% confidence region for each of three samples. The five soil-diet incorporation rates were compared in terms of larval survivorship and growth rate. There was no significant difference in larval survivorship after 10 days (i.e. for the duration of the bioassay period). The stage structure of bioassay larvae at 10 days and pupal weight at 20 days was significantly different for individuals reared on 25% soil-diet in terms of both a slower growth rate and a lower mean pupal weight compared to individuals reared on 10, 5 and 1% soil diets. This did not, however, appear to lead to greater variability in bioassay response at the high soil rate at 10 days. The level of sensitivity of virus detection achieved using this method was extremely good with the LC10 value for mid-first instar H. armigera larvae reared on the 25% soil-diet estimated at 26 polyhedral inclusion bodies (PIBs) per gram of soil. The suitability of using this approach for quantifying Helicoverpa NPVs in Australian soils was assessed by comparing percent bioassay infection across a range of five isolates known to be present in Australia. The effect of soil pH and soil management (cultivated versus non-cultivated) on percent bioassay infection was also examined. In both cases, no significant differences were observed. Finally, percent idopathic mortality, percent NPV infection and estimates of Helicoverpa SNPV concentration in a selection of samples from the Australian environment are presented.

The establishment of heliothine cell lines and their susceptibility to two baculoviruses.

A total of eight cell lines were established from Helicoverpa armigera (3) and H. punctigera (5) embryos and ovaries. Cell lines were established and grown in TC100 and/or TC199-MK containing 10% fetal bovine serum. The serum-free medlium ExCell 400 was also used, with and without 10% supplemental fetal bovine serum, but failed to generate cell lines from fat bodies, embryos, or ovarian tissues. Cell lines consisted of heterogenous cell types ranging from oval to fibroblast-like. This is the first report on the successful establishment of cell lines from H. punctigera. Cell lines from the two species were distinguishable from each other by DAF-PCR, and noticeable differences in minor bands were observed among cell lines from the same species. All of the established cell lines from both species were susceptible to HzSNPV but did not replicate more virus than that of a H. zea cell line (BCIRL-HZ-AM1-A11). However, an H. punctigera cell line (HP1) replicated AcMNPV to the highest titer (1.0 X 10(8) 50% tissue culture infective dose/ml), and only one of the H. armigerm cell lines (HA1) was susceptible to this virus.

Molecular characterization of Drosophila C virus isolates.

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Taq I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family.

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

A molecular taxonomy for cricket paralysis virus including two new isolates from Australian populations of Drosophila (Diptera: Drosophilidae).

Two new isolates of cricket paralysis virus, TAR and SIM, are described that were originally isolated from laboratory colonies of Drosophila melanogaster and Drosophila simulans respectively. Using a combination of biological, serological and molecular characters it was possible to distinguish the SIM isolate from all other isolates and it is thus described as a new strain; CrPVSIM. The TAR isolate however, was indistinguishable from the CrPV reference isolate CrPVVIC/GM/D2(2)/Gm/D2(2) (Teleogryllus commodus, Victoria, Australia, 1968). The molecular characters used in the present study were obtained by combining PCR and restriction endonuclease digestion of the amplified fragments. This work demonstrates that such molecular characters, when used in combination with others, provide a powerful set of taxonomic characters for classifying CrPV isolates and strains and assessing their genetic relatedness.

A suggested taxonomy and nomenclature for the cricket paralysis and Drosophila C virus complex.

Biophysical, biological, and serological characters are presented which in combination allow isolates of cricket paralysis virus (CrPV) and Drosophila C virus (DCV) to be separated and seven distinct strains of CrPV to be defined. Reference isolates for CrPV and DCV are suggested along with a system of nomenclature that allows the passage history of a particular isolate to be easily described.

A novel small RNA virus isolated from the cotton bollworm, Helicoverpa armigera.

A small RNA virus with novel characteristics has been isolated from laboratory-bred larvae of Helicoverpa armigera. Infection by the H. armigera stunt virus causes severe retardation of larval development and subsequent death. Its particles are isometric, 38 nm in diameter, and have a buoyant density of 1.296 g/ml in caesium chloride. The viral capsid has two major non-glycosylated protein components with M(r)s of 65,000 and 6000, and contains a genome composed of two non-polyadenylated single-stranded RNA molecules with lengths of 2.4 kb and 5.5 kb. The 5' termini of these RNAs are capped; their 3' termini are unblocked. In vitro translations of the viral RNAs showed synthesis of large proteins of sizes near the maximum coding capacity of each strand along with synthesis of numerous smaller proteins; no evidence for processing of precursors was seen. The physicochemical properties of the virus are most similar to those of the Nudaurelia omega virus, a provisional member of the Tetraviridae, although no antigenic relationship was observed between the two viruses. The bipartite genome and distinct capsid structure of these two viruses indicate the existence of a previously unrecognized virus group.

A simple vacuum dot-blot hybridisation assay for the detection of Drosophila A and C viruses in single Drosophila.

Specific cDNA clones were constructed from the single stranded RNA genome of Australian isolates of both Drosophila A and C viruses. These clones were used to develop a nucleic acid hybridisation assay capable of detecting reliably 3.9 ng of DAV and 19.3 ng of DCV virus particles, respectively. The sensitivity of the assays were largely unaffected by soluble host material. Single Drosophila naturally infected or artificially inoculated with DAV or DCV were found to contain in excess of 130 ng of virus. The results presented here demonstrate that the vacuum dot-blotting protocol and the hybridisation assays developed are capable of detecting DAV and DCV in single Drosophila and may therefore be applied to the study of DAV and DCV in natural Drosophila communities.