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Seed lots of tomato and capsicum ( Solanum lycopersicon and Capsicum annuum , respectively) are required to be free of quarantine pests before their entry to Australia is permitted. Testing of samples from 118 larger seed lots in the period 2019-2021 revealed that 31 (26.3%) carried one or more of four Tobamovirus species, including tomato mottle mosaic virus (ToMMV), which is a quarantine pest for Australia. Testing of samples from a further 659 smaller seed lots showed that 123 (18.7%) carried a total of five Tobamovirus species, including ToMMV and tomato brown rugose fruit virus (ToBRFV), which is also a quarantine pest for Australia. Estimated prevalence of contamination by tobamoviruses ranged from 0.388% to 0.004% in contaminated larger seed lots. Analyses of these data allow us to estimate probabilities of detection of contamination under different regulatory settings.

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.

Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism., (Copyright 2000 Academic Press.)

An antigenic 30 K virion protein of Heliothis armigera entomopoxvirus (HaEPV) has been identified as a homologue of the chordopoxvirus (ChPV) VP8 major virion core protein. Like its homologue in vaccinia virus, the mature HaEPV 30 K protein is derived by post-translational cleavage of a precursor at a conserved AGA motif. The HaEPV 30 K protein is the first EPV structural virion protein to be described, and elucidation of its characteristics provides evidence for the assumption that morphological similarities observed between virions of the sub-families Entomopoxvirinae and Chordopoxvirinae by microscopy reflect corresponding similarities at a molecular level. Sequencing of the HaEPV genome adjacent to the 30K locus identified an ORF encoding a homologue of the regulatory sub-unit of the ChPV poly(A) polymerase enzyme; the conceptual product of this ORF showed 25-31% aa sequence identity to those of various ChPVs. The presence of this gene in the HaEPV genome supports the hypothesis that there is a substantial correspondence in basic metabolic processes of members of the two poxvirus sub-families, despite their utilization of divergent host groups. In contrast, the relative positions of the 30 K and poly(A) polymerase loci in the HaEPV genome provide further evidence of substantial genomic re-arrangement subsequent to divergence of these viral taxa.

The genome of Heliothis armigera entomopoxvirus (HaEPV) has been mapped with four restriction endonuclease enzymes (BamHI, HindIII, PstI and XhoI), and its length estimated at 233 kbp. An EcoRI-generated HaEPV genomic fragment hybridized to all fragments identified as genomic termini, providing the first experimental evidence for the presence of terminal repeat elements in an EPV genome. The HaEPV spheroidin and nucleoside triphosphate phosphohydrolase I (NPHI) genes have been cloned and sequenced, and their deduced products shown to possess high levels of identity with homologues from other Genus B entomopoxviruses (EPVs). The genomic locations of these and other HaEPV genes and open reading frames have been determined; comparison of their locations with those of homologues in the Amsacta moorei EPV genome largely supports an hypothesis that the Genus B EPVs share a conserved genomic organization which differs from that of chordopoxviruses. It is proposed that genes of EPVs can be assigned to five actual or predicted homology-based groups, a categorization which is useful for directing and interpreting investigations of EPV gene functions and relationships.

The Heliothis armigera entomopoxvirus (HaEPV) genome encodes a predicted 68 kDa polypeptide related to the 'rifampicin resistance' protein of vaccinia virus (with 30 % identity), and an homologous swinepox virus protein (27% identity). We were unable to isolate an HaEPV genotypic variant encoding a predicted C-terminal truncated form of the protein, suggesting that the C terminus of the molecule may be essential to protein function, and, in turn, that this function may be essential to viral replication. HaEPV replication was substantially reduced in host cells exposed to rifampicin, but the observed cytotoxic properties of the drug made it impossible to determine the specific cause of that inhibition. We suggest that possession of a gene encoding a member of this polypeptide family might represent a defining molecular characteristic of the Poxviridae.

Continuously replicating cell lines have been established from embryonic tissue and circulating hemolymph cells of the melolonthine Antitrogus parvulus Britton. Isozyme analyses demonstrated that cell lines from both tissue sources expressed essentially the same isoforms of enzymes as A. parvulus larvae and thus confirmed the species of their origin. Karyotype analyses showed that cells from both tissue sources had accumulated changes in chromosome number and morphology during culture. Availability of melolonthine-derived cells should assist in vitro studies of the pathogens of this important group of beetles.

A gel retardation assay was used to demonstrate binding of wound tumor virus transcripts by a protein component of leafhopper vector cell extracts. Comparative binding studies employing terminally modified and internally deleted transcripts established that the segment-specific inverted repeats present in the terminal domains of the viral transcripts were necessary but not sufficient for optimal binding. An additional involvement of internal sequences in either the formation or the stabilization of the binding complex was indicated. Results of competitive binding experiments confirmed the sequence- and structure-specificity of the protein-RNA interaction and revealed apparent differences in the ability of individual viral transcripts to form a stable binding complex. Possible implications of structure-specific interactions between wound tumor virus transcripts and a host component and the role of the terminal inverted repeats are discussed.

A circular, viroid-like satellite RNA (sat RNA) was detected in lucerne transient streak virus (LTSV), from lucerne in South Australia. It was larger than the previously reported sat RNA of LTSV, being similar in size and sequence homology to the 388 nucleotide sat RNA previously shown to be encapsidated by subterranean clover mottle virus (SCMoV) isolated from subterranean clover in Western Australia. This indicates that under field conditions, very similar sat RNAs can be associated with two distantly related sobemoviruses, LTSV and SCMoV. The natural hosts of these viruses are lucerne and subterranean clover, respectively.

A theory is presented which can explain the interaction of the major factors known to influence in vitro extrusion of the microsporidian polar filament. It is proposed that the pH, and concentration and species of cation in the external medium influence the activity of carboxylic ionophore molecules in spore membranes in the following manner: (1) Alkaline environmental conditions establish a proton gradient across the spore plasma membrane, and facilitate the activation of ionophore molecules in this membrane. (2) This proton gradient drives an ionophorically-mediated cation/proton exchange across the plasma membrane. (3) As protons are lost from the sporoplasm its alkalinity increases, so that ionophore molecules in organelle membranes (i.e. in the polaroplast and posterior vacuole) are activated. This initiates a cation/proton exchange between sporoplasm and organelles. (4) Continued movement of cations into organelles in the spore causes major osmotic imbalance across spore membranes. This leads to a rapid inflow of water into the spore and swelling of the polaroplast and posterior vacuole. The associated pressure increase in the spore causes the explosive discharge of the polar filament through the polar cap. This model is used to explain previously published results from the literature, and methods of testing predictions generated by this hypothesis are outlined.

Sequence analysis of the genomic segments which encode the five wound tumor virus nonstructural polypeptides has been completed. The complete nucleotide sequence of segments S4 (2565 bp), S6 (1700 bp), S9 (1182 bp), and S10 (1172 bp) are presented in this report while the sequence of segment S12 (851 bp) has been described previously (T. Asamizu, D. Summers, M. B. Motika, J. V. Anzola, and D. L. Nuss, 1985, Virology 144, 398-409). Comparison of the only published sequence for another member of the genus Phytoreovirus, that of rice dwarf virus segment S10, with the combined available wound tumor virus sequence data revealed similarity with WTV segment S10: 54.9 and 30.6% at the nucleotide and amino acid level, respectively. Although wound tumor virus and rice dwarf virus differ in plant host range, tissue specificity, vector range, and disease symptom expression, the level of sequence similarity shared by the two segments suggests a common origin for these viruses. The potential use of a phytoreovirus sequence database for predicting functions of viral encoded gene products is considered.