Host Range and Population Survey of Spodoptera frugiperda Rhabdovirus.

The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.

Detection and survey of coffee ringspot virus in Brazil.

Coffee ringspot virus (CoRSV) a member of the proposed genus "Dichorhavirus", was surveyed on commercial and research farms spanning an area responsible for the majority of Coffea arabica production in Brazil. Virus-infected plants were found at one hundred percent of locations (n = 45) sampled. All cultivars, regardless of cherry color, were found to serve as hosts, suggesting that there is limited resistance in commercially employed germplasm. Reverse transcription PCR analysis revealed that the virus is contained within symptomatic lesions, with little systemic spread throughout leaves. Phylogenetic analysis based on the ORF1 (nucleocapsid) gene identified a strong geo-spatial relationship among isolates, which clustered into three clades. Despite low genetic diversity among isolates, variation in symptom expression was observed in the experimental host Chenopodium quinoa. Our analyses support the hypothesis that the spread of CoRSV is constrained by the clonal expansion of thelytokous populations of Brevipalpus phoenicis. The widespread occurrence of this virus suggests that it is much more prevalent than previously thought.

Characterization of Coffee ringspot virus-Lavras: a model for an emerging threat to coffee production and quality.

The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a member of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified.

Specific and common changes in Nicotiana benthamiana gene expression in response to infection by enveloped viruses.

Microarrays derived from Solanum tuberosum expressed sequence tags were used to test the hypothesis that genetically distinct enveloped viruses elicit unique changes in Nicotiana benthamiana gene expression. The results of our study, which included Sonchus yellow net virus (SYNV), a plant rhabdovirus that replicates in the nucleus of infected cells, and Impatiens necrotic spot virus (INSV), a plant bunyavirus that replicates in the cytoplasm, were consistent with this hypothesis. Statistically significant changes (P< or =0.01) in the expression of 275, 2646 and 4165 genes were detected in response to INSV at 2, 4 and 5 days post-inoculation (d.p.i.), respectively. In contrast, 35, 665 and 1458 genes were expressed differentially in response to SYNV at 5, 11 and 14 d.p.i., respectively. The microarray results were verified by Northern hybridization using a subset of these genes as probes. Notably, INSV, but not SYNV, induced expression of small heat-shock protein genes to high levels. In contrast to SYNV, infection by INSV resulted in downregulation of all histone genes, of which the downregulation of histone 2b expression to very low levels was confirmed by Northern hybridization. The expression of a putative WRKY transcription factor at 11 d.p.i., but not at 5 or 14 d.p.i., in SYNV-infected tissue suggested that the temporal response to virus infection was identified readily using our experimental design. Overall, infection by INSV resulted in larger fold changes in host gene expression relative to infection by SYNV. Taken together, the present data demonstrate differential responses of a common host to two genetically distinct viruses.

Vesicle-associated double-stranded ribonucleic acid genetic elements in Agaricus bisporus.

Double-stranded ribonucleic acids (dsRNAs) were isolated from fruit bodies of commercial strains of the cultivated mushroom (Agaricus bisporus) by polyethylene glycol-NaCl precipitation, differential centrifugation, rate-zonal centrifugation in sucrose, and equilibrium centrifugation in cesium sulphate. In all seven of the mushroom isolates examined, three dsRNAs were identified: two major dsRNA segments of > 13.1-kb (L-RNA) and 2.4-kb (S-RNA) and a minor segment of 5.2-kb (M-RNA). L-, M-, and S-RNAs co-purified with spherical fungal vesicles measuring approximately 75 nm in diameter. The three dsRNAs were intimately associated with the vesicles as suggested by their lower buoyant density in cesium sulphate (1.27 g/cc) compared to that of phenol-extracted dsRNAs (1.42 g/cc) and by their resistance to hydrolysis by ribonuclease at low ionic strength. Using a variety of conditions during purification, no virus-like particles were found to be associated with the dsRNAs. In Northern analysis, L-, M-, and S-RNAs failed to cross-hybridize with the genomic dsRNAs of La France isometric virus. We report here the first description of non-encapsidated, vesicle-associated, dsRNA genetic elements in the common cultivated mushroom.

Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus.

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.

Interactions and nuclear import of the N and P proteins of sonchus yellow net virus, a plant nucleorhabdovirus.

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.