- Volume 83, Issue 4, 2002

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (<10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

The nucleotide sequence of the Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) genome has been determined and analysed. The circular dsDNA genome contains 118584 bp, making it the smallest group I NPV sequenced to date. The genome has a G+C content of 40·7% and encodes 136 predicted open reading frames (ORFs), five homologous repeat regions and one unique repeat region. Of the genome, 92·9% encodes predicted ORFs and 2·2% is in repeat regions; the remaining 4·9% of the genome comprises nonrepeat intergenic regions. EppoMNPV encodes homologues of 126 Orgyia pseudotsugata MNPV (OpMNPV) ORFs and 120 Autographa californica MNPV ORFs, with average identities of 64·7 and 53·5%, respectively. Between the four sequenced group I NPVs, 117 ORFs are conserved, whereas 86 ORFs are conserved between all fully sequenced NPVs. A total of 62 ORFs is present in all baculoviruses sequenced to date, with EppoMNPV lacking a homologue of the superoxide dismutase (sod) gene, which has been found in all other fully sequenced baculoviruses. Whole genome phylogenetic analyses of the ten fully sequenced baculoviruses using the sequences of the 62 shared genes, gene content and gene order data sets confirmed that EppoMNPV clusters tightly with OpMNPV in the group I NPVs. The main variation between EppoMNPV and OpMNPV occurs where extra clusters of genes are present in OpMNPV, with sod occurring in one such cluster. EppoMNPV encodes one truncated baculovirus repeated ORF (bro) gene. The only repeated ORFs are the four iap genes. Eight, randomly distributed, unique ORFs were identified on EppoMNPV, none of which show any significant homology to genes in GenBank.

Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein (‘60K’) plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-β, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.

North American and Eurasian isolates of Wheat streak mosaic virus (WSMV; genus Tritimovirus) and Oat necrotic mottle virus (ONMV; genus Rymovirus) were examined. Nine WSMV isolates differentially infected oat, barley, inbred maize line SDp2 and sorghum line KS56. The WSMV isolates clustered into groups based on phylogenetic analyses of the capsid protein (CP) cistron and flanking regions. WSMV isolates from the United States (US) and Turkey were closely related, suggesting recent movement between continents. Although more divergent, WSMV from Iran (WSMV-I) also shared a most recent common ancestor with the US and Turkish isolates. Another group of WSMV isolates from central Europe and Russia may represent a distinct Eurasian population. Complete genome sequences of WSMV from the Czech Republic (WSMV-CZ) and Turkey (WSMV-TK1) were determined and comparisons based on complete sequences yielded relationships similar to those based on partial sequences. ONMV-Pp recovered from blue grass (Poa pratensis L.) in Germany displayed the same narrow host range as ONMV-Type from Canada. Western blots revealed a heterologous relationship among CP of WSMV and ONMV. Phylogenetic analyses of the capsid protein cistron and flanking genomic regions indicated that WSMV and ONMV are related species sharing 74·2–76·2% (nucleotide) and 79·2–81·0% (amino acid) identity. Thus, ONMV should be removed from the genus Rymovirus and designated a definitive member of the genus Tritimovirus. Phylogenetic analyses further suggest that Sugarcane streak mosaic virus is not a tritimovirus, and may represent a new genus within the family Potyviridae.

Ageratum yellow vein disease is caused by the whitefly-transmitted monopartite begomovirus Ageratum yellow vein virus and a DNA β satellite component. Naturally occurring symptomatic plants also contain an autonomously replicating nanovirus-like DNA 1 component that relies on the begomovirus and DNA β for systemic spread and whitefly transmission but is not required for maintenance of the disease. Here, we show that systemic movement of DNA 1 occurs in Nicotiana benthamiana when co-inoculated with the bipartite begomovirus Tomato golden mosaic virus and the curtovirus Beet curly top virus (BCTV), but not with the mastrevirus Bean yellow dwarf virus. BCTV also mediates the systemic movement of DNA 1 in sugar beet, and the nanovirus-like component is transmitted between plants by the BCTV leafhopper vector Circulifer tenellus. We also describe a second nanovirus-like component, referred to as DNA 2, that has only 47% nucleotide sequence identity with DNA 1. The diversity and adaptation of nanovirus components are discussed.