Ribonucleic acid of barley yellow dwarf virus

Virions of the isolates of barley yellow dwarf virus transmitted by Macrosiphum avenae (MAV) and by Rhopalosiphum padi (RPV) contain a single component of single-stranded RNA of molecular weight 2.0 × 10⁶, estimated by sedimentation and gel electrophoretic mobility of formalinized RNA. The untreated ribonucleic acid of the RPV isolate appears to have a slightly more compact configuration than that of the MAV isolate, as shown by a slightly faster sedimentation coefficient (33.8 S vs 32.6 S) and a slightly faster migration rate in polyacrylamide gel electrophoresis. Virions of the MAV isolate had a $\text{280}\text{260}$ absorbance ratio of 0.52 and a $\text{230}\text{260}$ ratio of 0.82, whereas corresponding figures for RPV virions were 0.54 and 0.91. Both virions apparently have a high content of RNA.     

Protein component of two isolates of barley yellow dwarf virus

A single major protein was identified in preparations of the MAV and RPV isolates of barley yellow dwarf virus in tests based on SDS gel electrophoresis. The molecular weight of each protein was measured in experiments using a series of gels of different acrylamide concentrations. Although the precision of these determinations was affected by some anomalous migration of the proteins, we estimate the molecular weight of the protein of the MAV isolate to be 23,500, and that of RPV to be 24,450.     

Purification and antigenicity of three isolates of barley yellow dwarf virus

An isolate (RPV) of barley yellow dwarf virus transmitted specifically by Rhopalosiphum padi and an isolate (PAV) transmitted nonspecifically by both R. padi and Macrosiphum avenae were purified by procedures previously found satisfactory for another isolate (MAV) transmitted specifically by M. avenae. As with MAV, infectivity of RPV and PAV samples removed from sucrose gradient columns was associated with a dense polyhedral particle about 30 nm in diameter in shadowed preparations. No differences in sedimentation rate (sedimentation coefficient 115–118 S) among the 3 virus isolates were detected in parallel sucrose gradient centrifugation tests.     

Differences in cellular ultrastructural alterations between variants of barley yellow dwarf virus

Ultrastructural alterations in cells of tissues from oats (Avena sativa L.) infected with barley yellow dwarf virus (BYDV) differed markedly between vector-nonspecific and Rhopalosiphum padi-specific isolates of the virus. With the vector-nonspecific isolates, single-membraned vesicles containing fibrils appeared in the cytoplasm after penetration of the cell by the infectious agent. Densely staining filaments with amorphous material accumulated in clumps at isolated areas in the cytoplasm and later in small clusters in the nucleopores. Subsequent progressive stages involved distortion of the nuclear outline, massive clumping of heterochromatin, and the appearance of masses of filaments in the nucleoplasm and subsequently outside the nucleus. Presumed progeny virus particles were then first observed outside the nucleus among the filaments. Later, masses of virus particles and filaments accumulated throughout the cytoplasm. Ultrastructural alterations in early stages of infection with R. padi-specific isolates differed from those described above in the appearance of fibril-containing vesicles enveloped in a second membrane, the absence of filament clusters in the cytoplasm away from pitfields or in nuclear pores, and the presence of tubular aggregates in the cytoplasm. Later, presumed progeny virus particles were first observed in the nucleus (which otherwise remained normal in appearance) first around the nucleolus, and ultimately also in the cytoplasm. Filaments became numerous among the virus particles in the cytoplasm only at a late stage. Alterations with the R. padi-specific isolates also occurred in mitochondria and in phloem parenchyma walls. Similarities and differences in ultrastructural changes between cells infected with vector-nonspecific, R. padi-specific, and Macrosiphum avenae-specific isolates of BYDV, and the beet western yellows virus, are discussed.     

Optimal conditions for the use of cDNA probes to measure the concentration of barley yellow dwarf virus in barley (Hordeum vulgare)

Experiments which optimise the conditions for the measurement of the relative concentration of BYDV in barley (Hordeum vulgare) tissues using cDNA probes are described herein. These studies have shown that both the pH of the buffer and the ratio of buffer to tissue used to homogenise plant material greatly affects the amount of cDNA probe which hybridises to leaf extracts immobilised on nitrocellulose. These studies also showed that the measurement of this virus was greatly facilitated by using a dot-blot apparatus which allows samples contact with a piece of nitrocellulose 10 mm in diameter rather than a 3 mm (approx) diameter piece of nitrocellulose as is the case with most commercial dot-blot apparatuses. Further experiments using this technique showed that there was a large difference in the rate of replication of the PAV, BYDV serotype between BYDV-resistant and BYDV-susceptible cultivars of barley. These data suggest that a BYDV-resistant cultivar can easily be distinguished from a BYDV-susceptible one if the BYDV content of leaves is measured between 7 and 14 days after inoculation.     

Genetic structure in natural populations of barley/cereal yellow dwarf virus isolates from Alaska

Summary. The genetic structure of natural populations of Alaskan barley yellow dwarf virus (BYDV)-PAV, BYDV-PAS, and cereal yellow dwarf virus (CYDV)-RPV from barley (Hordeum vulgare L.) and oats (Avena sativa L.) in Alaska were analyzed between 2002 and 2004. PCR products spanning the viral coat protein gene of 187 isolates were cloned and sequenced. The majority (78%) were similar to BYDV-PAS, 19% were similar to CYDV-RPV, and only about 3% resembled BYDV-PAV. The CYDV-RPV isolates clustered in three groups: 44, 17, and 39% resembled RPS-like CP from Mexico, resembled RPV-like CP from New York, or formed a unique clade that was RPV/RPS recombinant CP, respectively. The patterns of genetic variation of PAS and RPV varied little over time or with respect to host plant. The difference in spatial and temporal population genetic structures of the PAS and RPV isolates suggests that these two viruses are influenced by different agroecological factors. Sequence of PCR products spanning the carboxyl terminus of the polymerase gene, the intergenic region, and most of the coat protein gene of RPV revealed two probable ancestral recombination events for some isolates.     

Role of accessory salivary glands in aphid transmission of barley yellow dwarf virus

Barley yellow dwarf virus (BYDV) was consistently observed in the basal lamina and in plasmalemma invaginations only of accessory salivary glands in each of 20 aphids (Sitobion avenae) reared on oats infected with the MAV or RPV isolates of BYDV. Virus particles were not found in any of 13 aphids reared in parallel on healthy oats. The MAV isolate was identified in aphids by indirect labeling with ferritin-conjugated antibody. Virus particles were also observed within cytoplasmic vesicles and intracellular canals that drain the accessory gland in all aphids reared on oats infected with MAV, which is transmitted by S. avenae, but in none of the aphids reared in parallel on oats infected with RPV, which S. avenae does not transmit. Visualization of only transmissible virus within accessory gland cytoplasmic structures indicates that the plasmalemma may be a site regulating virus uptake by the salivary gland. Presence of virus in intracellular canals and in coated pits and vesicles adjacent, to canals suggests possible transport mechanisms out of the accessory gland. The results are compatible with a virus-cell receptor mechanism for transmission specificity between luteoviruses and aphid vectors, and they suggest a route through aphids for viruses transmitted in the circulative manner.     

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene.