Peter D. Christian

Evolutionary and taxonomic implications of conserved structural motifs between picornaviruses and insect picorna-like viruses

Summary. A comparison of the recently determined structure of an insect picorna-like virus, Cricket paralysis virus (CrPV), with that of the mammalian picornaviruses shows that several structural features are highly conserved between these viruses. These conserved features include the topology of the coat proteins, the conformation of most loops, and the general arrangement of the internally located N-terminal arms of the coat proteins. The conformational conservation of the N-termini of the three major coat proteins between CrPV and the picornaviruses suggests a putative ancestral T = 3 virus. Comparisons of the genome structure and amino-acid sequence of the coat proteins of CrPV with a number of other insect picorna-like viruses show that most of them belong to a novel group, recently given the interim name Cricket paralysis-like viruses. Two other insect picorna-like viruses, Infectious flacherie virus (IFV) and Sacbrood virus (SBV), for which the genome sequences have recently been determined, have very different coat protein sequences and a genome organization more like the picornaviruses. However, the position of the small VP4 protein in the structural protein polyprotein as well as the mechanism for its cleavage from VP3 upon assembly strongly suggests an evolutionary link to the “Cricket paralysis-like viruses”. We propose that the picornaviruses, Cricket paralysis-like viruses and IFV/SBV group are a natural assemblage. The ancestor for this assemblage had a structure based upon the CrPV/picornavirus paradigm and a genome encoding a single major coat protein; gene duplication and rearrangements have subsequently to produced the viruses that we observe today. We also discuss the possible relatives of the proposed assemblage and the likely implications of future structural studies that may be carried out on the putative relatives.

A comparison of techniques for detecting Invertebrate iridescent virus 6.

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.

Picornalike Viruses of Insects

Over the last 30 years, a large number of small (less than 40 nm in diameter) RNA-containing viruses (SRVs) have been isolated from insects and insect cell cultures, the vast majority of which have undetermined relationships with other animal or plant viruses. Nevertheless, two groups of insect SRVs have been sufficiently well characterized to merit their own families, namely, the nodaviruses and the tetraviruses (Murphy et al.,1995; see also Chapters 8 and 9, this volume). In addition to these two well-characterized families of insect SRVs, there are a number of known birnaviruses from insects (Murphy et al., 1995) and a single calicilike virus (Kellen and Hoffman, 1981; Hillman et al., 1982). The remaining 65 or so viruses are to a large extent relatively uncharacterized (Appendix). However, among these a reasonably large 〞group〞 of viruses exist that have characteristics that superficially resemble those of the mammalian picornaviruses. The mammalian picornaviruses are classically defined by having isometric, unenveloped virions of around 30 nm in diameter, with buoyant densities between 1.30 and 1.46 g/ml, depending on the genus (Table I). The virions contain three major capsid proteins of around 30,000 Da (30 kDa) and another small protein of around 7 kDa. The RNA genome is positive sense, polyadenylated at the 3′ end and has a small protein (VPg) covalently linked to its 5′ end.

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.

Differential Adsorption of Occluded and Nonoccluded Insect-Pathogenic Viruses to Soil-Forming Minerals

ABSTRACT Soil represents the principal environmental reservoir of many insect-pathogenic viruses. We compared the adsorption and infectivity of one occluded and two nonoccluded viruses, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) (Baculoviridae), Cricket paralysis virus (CrPV) (Dicistroviridae), and Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae), respectively, in mixtures with a selection of soil-forming minerals. The relative infective titers of HaSNPV and CrPV were unchanged or slightly reduced in the presence of different minerals compared to their titers in the absence of the mineral. In contrast, the infective titer of IIV-6 varied according to the mineral being tested. In adsorption studies, over 98% of HaSNPV occlusion bodies were adsorbed by all the minerals, and a particularly high affinity was observed with ferric oxide, attapulgite, and kaolinite. In contrast, the adsorption of CrPV and IIV-6 differed markedly with mineral type, with low affinity to bentonites and high affinity to ferric oxide and kaolinite. We conclude that interactions between soil-forming minerals and insect viruses appear to be most important in nucleopolyhedroviruses, followed by invertebrate iridescent viruses, and least important in CrPV, which may reflect the ecology of these pathogens. Moreover, soils with a high content of iron oxides or kaolinite would likely represent highly effective reservoirs for insect-pathogenic viruses.

Persistence of Invertebrate iridescent virus 6 in soil

Soil represents an important reservoir for mostentomopathogenic viruses. Invertebrateiridescent viruses (IIVs) (Iridoviridae) arenon-occluded DNA viruses that infectagriculturally and medically important insectspecies, especially in damp or aquatichabitats. We used virus extraction and insectbioassay techniques to determine the effect ofsoil moisture and soil sterility on thepersistence of Invertebrate iridescentvirus 6 (IIV-6) in a soil over a 90 day periodin the laboratory. Loss of activity of IIV-6in dry soil (6.4% moisture, −1000 kPa matricpotential) was very rapid and was not studiedbeyond 24 h. Soil moisture did not affect therate of inactivation of virus in damp (17%moisture, −114 kPa matric potential) or wetsoil (37% moisture, −9.0 kPa matricpotential). In contrast, soil sterilizationsignificantly improved the persistence of IIV-6activity, both in damp and wet soil. Controlvirus suspensions retained 0.72–0.87% oforiginal activity after 90 days, which wassignificantly more than the activity retainedin soil. These figures represent half lives of4.9 days for IIV-6 in non-sterile soil, 6.3days in sterilized soil (data pooled formoisture treatments), and 12.9 days for thecontrol virus suspension. We conclude thatextra-host persistence in soil habitats may bean important aspect of the ecology of IIVs.

Insect Viruses: New Strategies for Pest Control

For the last 50 Years, insect control programs have relied heavily on the use of chemical insecticides. Today, however, awareness of the unintended biological consequences of the use of broad-spectrum insecticidal compounds and changing attitudes toward this ecological cost are now coupled with the ever-increasing problem of target-pest resistance. On the other hand, it now seems possible that advances in biotechnology will lead to novel uses of entomopathogens, and that such approaches might have the potential to circumvent a majority of these problems. In this chapter we outline the possibilities for integration of insect viruses, or their constitutive parts, into novel insect control agents and strategies.

Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment.

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.