Double-stranded RNA analysis of strawberry plants affected by June yellows

Using an improved method for extraction of dsRNA from strawberry leaf tissue, small quantities of several dsRNA species with mol. wt greater than 1.0 × 10⁶were detected in strawberry plants free from known strawberry viruses but affected by June yellows (JY). No such dsRNA species were detected in plants of Fragaria vesca or seven strawberry cultivars known to be free from JY. Neither JY symptoms nor these dsRNA species were detected in healthy strawberry and F. vesca plants graft-inoculated with tissue from JY-affected plants. It is not known whether the JY-associated dsRNA species are those of a causal agent of JY or are a consequence of the JY condition. Nevertheless, the detection of such dsRNA species in plants affected by JY may offer a possible objective method for detecting the incipient condition in symptomless strawberry plants. However, the concentrations of dsRNA in JY-affected plants are very low and dsRNA analysis is thought not to be sufficiently reliable for routine testing of plants. The occurrence of anomalous dsRNA species in extracts from some strawberry plants was caused by dsRNA from two-spotted spider mites (Tetranychus urticae) infesting the plants.     

Presence of double‐stranded RNAs and virus‐like particles in the entomopathogenic fungus Metarhizium anisopliae

Nucleic acids from 41 strains of Metarhizium anisopliae, obtained from different parts of the world were extracted and examined by electrophoresis. Strong bands of double‐stranded RNA (dsRNA) were detected in two isolates from Brazil, V215 and V291, which had, respectively, 13 and 9 distinct bands ranging in size from ca. 0.75 to 3.5 kb. Icosahedral virus‐like particles (VLPs) (ca. 33 nm in diameter) were observed by transmission electron microscopy in extracts of these isolates. The VLPs and dsRNA were both absent from a clone of the isolate V291 which had been subcultured successively on solid medium. Bioassays against the aphid Myzus persicae showed no detectable difference in virulence between the clone of V291 which contained dsRNA and the clone that did not.     

Use of Rotavirus Virus-Like Particles as Surrogates To Evaluate Virus Persistence in Shellfish

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log₁₀ of VLPs was depurated after 1 week in warm seawater (22°C). Phage MS2 was depurated more rapidly (about 2 log₁₀ in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10¹⁰ and 10⁹ particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10⁵ particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.     

Isolation and characterization of a new dsRNA virus fromWickerhamia fluorescens

Virus-like particles (VLPs) were isolated from the yeastWickerhamia fluorescens strain CCY61-1-1. The VLPs are approximately 42 nm in diameter and contain only one species of dsRNA molecule. The apparent length of the dsRNA determined by native agarose gel electrophoresis was 4.6 kbp. Analysis of protein content of the VLPs showed them to contain one major capsid protein with an apparent molar mass of 74.5 kDa.     

Probable mechanical transmission of a virus-like agent from rose rosette disease-infected multiflora rose to Nicotiana species

As part of efforts to identify the causal agent of the rose rosette disease (RRD) of multiflora rose (Rosa multiflora Thunb.), root tip extracts from both symptomatic and nonsymptomatic roses were used to mechanically inoculate leaves of Nicotiana glutinosa. Pale green spots were observed along the margins of the major leaf veins only on leaves inoculated with extracts prepared from symptomatic rose plants. Light microscopy revealed abnormal development of the palisade and spongy mesophyll cells in the symptomatic tissue, although no virus‐like particles (VLPs) were observed by electron microscopy. However, VLPs were observed in cells from tissue adjacent to the leaf veins and bordered by the pale green spots. Inoculation of N. benthamiana with extracts from symptomatic N. glutinosa initially did not result in visible symptoms on N. benthamiana inoculated leaves. However, approximately 4 wk post inoculation, splitting of leaf tissue across and along major leaf veins in expanding leaves occurred. In later stages of leaf expansion some leaves split in regions not associated with veins. Light microscopy of thick sections revealed separation between palisade cells and groups of small dead cells in the mesophyll tissue of expanding systemically infected leaf blades. Electron microscopy revealed crystalline arrays in the cytoplasm of mesophyll cells. No abnormal cellular changes were observed in plants inoculated with extracts prepared from nonsymptomatic rose plants.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

Characterization and localization of a virus-like particle in aDrechslera species

A virus-like particle (VLP) has been found in a species of the fungusDrechslera. Four double-stranded RNA species with sizes of 3.8, 2.8, 2.7, and 2.2 kbp were isolated using CF-11 cellulose. These dsRNAs are associated with a 35-nm particle at a density of 1.37 g/cm³ in CsCl. The particle contains a major protein of 117 kDa and a minor protein of 89 kDa. In cellular fractionations of 2-week-old cultures, the VLPs are found in the mitochondrial pellet, but do not band with mitochondria in sucrose step gradients. In 1-week-old cultures, however, the VLP dsRNA is found in the cytoplasmic fraction. VLPs have not been reported previously in this genus.     

Analysis of the 3-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3^′-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed “degradative PCR” and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3^′-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

Characterization of an Isolate of Zucchini Yellow Mosaic Virus form Cucumber in Singapore

Zucchini yellow mosaic virus (ZYMV) was first found in cucumber in Singapore in 1989. This virus was propagated in Cucurbita pepo cv. First Taste and mechanically transmitted to 12 species of six families. It induced milder symptoms than the Connecticut and Florida strains of ZYMV in infected leaves of C. pepo cv. Zucchini Elite. ZYMV-S is neither seed nor aphid transmissible. Immunoelectron microscopy revealed that ZYMV-S is distantly related to WMV-2, Moroccan WMV, and TelMV, but not, related to PRSV or ZYFV. Cytoplasmic pinwheels and scrolls were observed in ultrathin sections of infected leaf cells by light, confocal laser scanning, and transmission electron microscopy. The molecular weights of the viral coat protein and cytoplasmic inclusion protein, RNA and dsRNA were estimated to be 3.2 × 10^?1, 6.1 × 10⁴, 3.23 × 10⁶ and 6.53 × 10⁶ daltons, respectively.     

An Epstein-Barr Virus Mutant Produces Immunogenic Defective Particles Devoid of Viral DNA

Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4⁺ T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.     

Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco

Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.     

<Emphasis Type="Italic">Trichoplusia ni</Emphasis> cells (High Five<Superscript>TM</Superscript>) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines

Virus-like particles (VLPs) consisting of the influenza A virus proteins haemagglutinin (HA) and matrix protein (M1) represent a new alternative approach for vaccine design against influenza virus. Influenza VLPs can be fast and easily produced in sufficient amounts in insect cells using the baculovirus expression system. Up to now, influenza VLPs have been produced in the Spodoptera frugiperda cell line Sf9. We compared VLP production in terms of yield and quality in two insect cell lines, namely Sf9 and the Trichoplusia ni cell line BTI-TN5B1-4 (High Five^TM). Additionally we compared VLP production with three different HAs and two different M1s from influenza H1 and H3 strains including one swine-origin pandemic H1N1 strain. Comparison of the two cell lines showed dramatic differences in baculovirus background as well as in yield and particle density. Taken together, we consider the establishment of the BTI-TN5B1-4 cell line advantageous as production cell line for influenza VLPs.     

Characterization and mapping of cryptic alien introgression from <Emphasis Type="Italic">Aegilops geniculata</Emphasis> with new leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr57</Emphasis> and <Emphasis Type="Italic">Yr40</Emphasis> in wheat

Leaf rust and stripe rust are important foliar diseases of wheat worldwide. Leaf rust and stripe rust resistant introgression lines were developed by induced homoeologous chromosome pairing between wheat chromosome 5D and 5M^g of Aegilops geniculata (U^gM^g). Characterization of rust resistant BC₂F₅ and BC₃F₆ homozygous progenies using genomic in situ hybridization with Aegilops comosa (M) DNA as probe identified three different types of introgressions; two cytologically visible and one invisible (termed cryptic alien introgression). All three types of introgression lines showed similar and complete resistance to the most prevalent pathotypes of leaf rust and stripe rust in Kansas (USA) and Punjab (India). Diagnostic polymorphisms between the alien segment and recipient parent were identified using physically mapped RFLP probes. Molecular mapping revealed that cryptic alien introgression conferring resistance to leaf rust and stripe rust comprised less than 5% of the 5DS arm and was designated T5DL·5DS-5M^gS(0.95). Genetic mapping with an F₂ population of Wichita × T5DL·5DS-5M^gS(0.95) demonstrated the monogenic and dominant inheritance of resistance to both diseases. Two diagnostic RFLP markers, previously mapped on chromosome arm 5DS, co-segregated with the rust resistance in the F₂ population. The unique map location of the resistant introgression on chromosome T5DL·5DS-5M^gS(0.95) suggested that the leaf rust and stripe rust resistance genes were new and were designated Lr57 and Yr40. This is the first documentation of a successful transfer and characterization of cryptic alien introgression from Ae. geniculata conferring resistance to both leaf rust and stripe rust in wheat.     

Partial characterisation of a new virus in brusca from the Venezuelan lowlands

Flexuous thread‐like virus particles c. 650–700 nm in length were isolated from brusca (Senna pallida) plants showing stunting, mosaic, vein yellowing and leaf malformation. The virus was mechanically transmitted to healthy Senna pallida, Cassia obovata and Cassia emarginata L. plant species. Virus particles sedimented in sucrose density gradients as one component, with a bouyant density of 1.2 g cm^?3 in caesium chloride equilibrium gradients. Virions contained a molecule of ssRNA with an apparent size of 6.4 kb. The dsRNA pattern showed one main band of about 12 kb, and two subgenomic dsRNA of c. 10 and c. 5.4 kb. Analyses of purified virus preparations by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) resolved two coat protein subunits, with mol. wt of c. 28 000 and 26 000 daltons. In Western blotting the virus coat proteins reacted with an homologous polyclonal antiserum and with an antiserum to Lettuce infectious yellow virus. Electron microscopic observations of cells from infected plants showed the accumulation of cytoplasmic vesiculate inclusion bodies and crystalline aggregates of virus particles within phloem tissue. Some of the physicochemical and ultrastructural properties of this virus resemble those of a Closterovirus; however, differences show it to be sufficiently distinct from any previously reported viruses. We proposed the name of Senna chlorotic stunt (SeCSV) for this virus.     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.