A single U/C nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus P25 protein promotes increased virus accumulation in roots of mechanically inoculated, partially resistant sugar beet seedlings.

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V(67)C(68)Y(129) P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A(67)H(68)H(129) P25. Mutants revealed that this was due only to the V(67) 'GUU' codon as opposed to the A(67) 'GCU' codon.

Beet soil-borne virus RNA 3--a further example of the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

The complete nucleotide sequence of RNA 3 of the Ahlum isolate of beet soil-borne virus (BSBV) was determined from cDNAs obtained with immunocaptured virus particles and denatured preparations of dsRNA. BSBV RNA 3 is unique among the plant virus RNAs studied so far in containing apparently only the coding sequences of a triple gene block (TGB). The derived amino acid sequences of the three putative TGB-encoded proteins showed the highest level of sequence similarities with those of the corresponding proteins of potato mop top furovirus (PMTV) followed by those of peanut clump furovirus and barley stripe mosaic hordeivirus. Progressively fewer similarities were found with the TGB-encoded proteins of beet necrotic yellow vein virus (uncertain classification), potato X potexvirus, and potato M carlavirus. The 3'-terminal 78 nucleotides of BSBV RNA 3 can be folded into a tRNA-like structure and a high degree of sequence similarity exists between the 122 3'-terminal nucleotides of BSBV RNA 3 and PMTV RNA 2. In other regions, however, no pronounced sequence similarities were found between the two RNAs, and PMTV RNA 2 contains an additional putative gene for a cysteine-rich protein downstream of the TGB. The two viruses are unrelated serologically. BSBV RNA 3 adds a further variant to the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

Detection of beet necrotic yellow vein virus strains, variants and mixed infections by examining single-strand conformation polymorphisms of immunocapture RT-PCR products.

Single-strand conformation polymorphism analysis was found to be a powerful tool for rapidly assigning large numbers of beet necrotic yellow vein virus (BNYVV) isolates to a known strain group as well as for detecting mixed infections, minor variants or new strain groups. The prevalence of the B-type in Germany and France and the A-type in most other countries was confirmed. Minor variants with a very restricted distribution were detected occasionally. New rhizomania outbreaks in Great Britain were caused either by the A- or B-type or mixtures of both suggesting introduction of BNYVV from several sites abroad. An entirely different BNYVV type (P-type) was identified in a small area in France. Evidence for further strain groups in China was also obtained.

Location, size, and complexity of epitopes on the coat protein of beet necrotic yellow vein virus studied by means of synthetic overlapping peptides.

Five regions on the coat protein of BNYVV which had been shown previously to be involved in the formation of continuous epitopes were further analyzed by means of synthetic overlapping peptides. It was found that at least some of these regions may encompass several overlapping epitopes (or parts thereof). Four monoclonal antibodies (MAbs) which were known to be specific for the C-terminus of BNYVV coat protein (amino acids 182-188 = RTSPPGQ) were found to react with different sets of peptides which had either the sequence RTS, RTSP, RTSPP, or PPGQ in common. Two other MAbs which also had been shown previously to be specific for the C-terminus of BNYVV coat protein failed to react with overlapping decapeptides. Two epitopes which were previously located in the areas of amino acids 115-125 and 125-140 could now be located more precisely on the sequences SANVRRD (amino acids 115-121) and AESSG (amino acids 128-132), respectively. Replacement studies with alanine showed that not all amino acids within these sequences are equally important for antibody binding. On the other hand, amino acids outside these sequences may strongly influence the reactivity of epitopes. The accessibility of amino acid sequences on the particles of BNYVV is discussed.

Single- and double-stranded RNAs associated with an isolate of beet soil-borne virus.

Ethidium bromide staining of electrophoretically separated ssRNAs and dsRNAs as well as northern blot analyses with cDNA clones suggested that the genome of the Ahlum serotype of beet soil-borne virus (BSBV) consists of two major ssRNA species of approximately 3.6 and 3.2 kb, respectively, and possibly a minor ssRNA of approximately 6.0 kb. A few of our clones hybridized with both the 3.6-kb and the 3.2-kb RNAs, the majority of the clones, however, hybridized only with the 3.2-kb RNA. The 3.2-kb RNA is, therefore, apparently not a degradation product or a partially deleted form of the 3.6-kb RNA. None of our clones hybridized with the faint band(s) of the 6.0-kb ssRNA(s) which was produced by RNA extracts of some of our virus preparations. A fourth ssRNA of approximately 3.0 kb, which hybridized with the same clones as the 3.2-kb RNA, was found at relatively high concentrations in RNA extracts from purified virus, but not in total RNA extracts from leaves. Its origin is unknown. It is apparently not derived from the 3.2-kb RNA by the loss of a VPg or a poly(A) tail. Hybridization tests with 32P-labelled poly(dT) suggested that none of the RNAs of BSBV is polyadenylated. With respect to size and the lack of a poly(A) tail the RNAs of BSBV are more similar to those of definitive furoviruses than to those of beet necrotic yellow vein virus which is only a possible member of the furovirus group and has RNAs which readily hybridized with poly(dT).

Effect of recombinant beet necrotic yellow vein virus with different RNA compositions on mechanically inoculated sugarbeets.

Beet necrotic yellow vein virus (BNYVV) inocula with different RNA compositions were prepared from infectious transcripts of RNAs 3 and 4 and the Rg 1 isolate, which has a genome consisting only of RNAs 1 and 2. The recombinant viruses were inoculated on 6- to 8-day-old sugarbeet seedlings by 'vortexing'. Inocula containing RNAs 1 and 2 or 1, 2 and 4 produced some growth reduction, but the most dramatic effects, with yield reductions of about 95% in a highly susceptible variety, were seen when RNA 3 was also present in the inoculum. Under these conditions the side roots were brown and brittle and often deteriorated, but 'root beardedness' was not observed. This might be due to the fact that our experiments were done in the absence of Polymyxa betae. Alternatively, the heavy inoculation at a very young age may either have weakened the plants to such an extent that extensive root proliferation was impaired or it may have led to rapid deterioration of the proliferating rootlets, which would therefore be lost prior to or during removal of the tap roots from the soil. In the presence of RNA 3 the virus concentrations in tap roots were markedly increased suggesting that this RNA facilitates the multiplication and/or spread of the virus in root tissues.