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Sugar beet can be infected by many different viruses that can reduce yield; beet necrotic yellow vein virus (BNYVV) is one of the most economically important viruses of this crop plant. This report describes a new reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for identification of BNYVV. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection (without RNA extraction) of BNYVV was developed here and compared with DAS-ELISA and RT-LAMP assays. Our results show that the IC-RT-LAMP assay is a highly reliable alternative assay for identification of BNYVV.

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26 K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25 K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

The beet necrotic yellow vein virus (BNYVV) RNA-5-encoded p26 protein is involved in the accentuation of symptoms expression of infected Chenopodium quinoa plants and is capable of transcription activation (TA) in yeast. TA was previously localized within the first 55 residues of the p26 protein. Interestingly, TA did not occur when C-terminally deleted forms of p26 were used. We used a genetic screen in the yeast one-hybrid system to select restored TA from randomly generated mutants. The TA domain was found to be located within the first 17 residues. Alanine replacement of aspartic acids 11, 16, and 17 within the full-length p26 prevented TA but did not impair subcellular localization and the symptom expression.

The distribution of various Beet necrotic yellow vein virus (BNYVV) genotypes was studied using beet samples received from Germany and neighbouring countries. Almost exclusively B type BNYVV was detected in Germany, whereas in neighbouring countries BNYVV A types with different compositions of the amino acid tetrad in positions 67-70 of the RNA-3-encoded P25 are widely distributed. Neither A types nor the P type have been able to become established in Germany in the past decades, although there must have been many opportunities for their introduction from neighbouring countries. In one field, however, an RNA-5-containing BNYVV genotype closely resembling the Chinese isolate Har4 was found.

An isolate of Beet black scorch virus (BBSV) was obtained from Iranian sugar beet roots. Its genome organization closely resembles that of the previously described Chinese and North American isolates, but the nucleotide sequences of the three isolates differ considerably. Most of the nucleotide exchanges, however, are silent, and the Iranian and the Chinese isolates were serologically indistinguishable. Beets infected by the Iranian BBSV did not show black scorch symptoms, but severe root beardedness. This might have been caused by BBSV or the simultaneously present beet necrotic yellow vein virus, or both together.

The complete nucleotide sequences of RNAs 1 to 4 of Beet soilborne mosaic virus (BSBMV) were determined. The genomic organization of BSBMV is identical to Beet necrotic yellow vein virus (BNYVV), the type species of the genus Benyvirus. BSBMV RNA1 encodes a single large open reading frame (ORF) with similar replicase-associated motifs identified for BNYVV. BSBMV RNA2 has six potential ORFs with an organization resembling BNYVV RNA2. RNA3 and RNA4 resemble the analogous BNYVV RNAs, which encode proteins associated with symptom development and fungal transmission, respectively. The predicted ORFs on BNYVV and BSBMV reveal 23% to 83% amino acid identity and the overall nucleotide sequences are 35% to 77% identical. Based on sequence analyses, BSBMV is a new benyvirus that can be distinguished from BNYVV.

Computer analysis of published sequence data has consistently identified two complementary transmembrane domains in the coat protein readthrough domains of benyviruses, furoviruses and pomoviruses and in the P2 proteins of bymoviruses. These viruses differ in genome organisation but are all transmitted by plasmodiophorid fungi. The second domain is absent or disrupted in naturally-occurring deletion mutants that cannot be fungally-transmitted. In a non-transmissible substitution mutant of Beet necrotic yellow vein virus [Tamada et al. (1996) J Gen Virol 77: 1359-1367], the alignment of the helices is disrupted. From conserved patterns detected in transmembrane helix sequences and calculated relative helix tilts, structural arrangements consistent with tight packing of transmembrane helices were identified. These included ridge/groove arrangements between the two helices and strong electrostatic associations at the interfacial regions of the membrane. The data strongly suggest that these transmembrane helices facilitate the movement of virus particles across the fungal membrane.

PCR products representing c. 550 3' terminal bases of Beet soil-borne pomovirus (BSBV) RNA 3 were compared for sources of this virus from all major sugarbeet-growing areas in Germany. In none of these areas conspicious symptoms could be attributed to the presence of BSBV. Single strand conformation polymorphism analyses suggested that the BSBV genome may be very variable. This was confirmed by nucleotide sequence analysis. Each PCR product which was analysed showed sequence differences to others. Even the PCR products obtained from plants grown in the same soil sample were different. The highly variable nature of the BSBV genome is in contrast to the much more conserved nature of the Beet necrotic yellow vein virus genome. By means of the STAR programme a secondary structure was predicted for the 3' end of BSBV RNA 3, in which some areas are highly conserved, whereas others are characterized by a clustering of nucleotide exchanges.

About half of Japanese isolates of beet necrotic yellow vein virus (BNYVV) were found to contain RNA 5 molecules, which were also detected in virus isolates from China and France. Sequence comparisons of RNA 5 (nucleotides 327 to 1171) in 25 isolates showed that there are up to 8% sequence differences, and that RNA 5 variants fall into three groups: group I contains most of the Japanese and Chinese isolates, group II two Japanese isolates, and group III four French isolates. The group I isolates fall into three small clusters. In the 26 kDa coding region of RNA 5, there was a maximum of 1.5% nucleotide sequence differences (6 amino acid changes) within the group and 8.4% nucleotide sequence differences (17 amino acid changes) between the groups. Comparisons of the coat protein gene of RNA 2 revealed that most of the Japanese and Chinese isolates belonged to the A type strain, but some isolates were of the B type. The French isolates (P type) were closely related to those of the A type. Mixed infections of the two types of virus and the two groups of RNA 5 were detected in a small area of Hokkaido. BNYVV might have been introduced into Japan and China by a similar route from at least two origins. These results, together with other evidence, suggest that the three groups of RNA 5 variants separated from an original population a long time ago and, thereafter, the group I population diverged further into three clusters, which may have been associated with the A type strain rather than the B type.

Transgenic plants of Nicotiana benthamiana expressing single chain antibody fragments (scFv) specific for the coat protein of beet necrotic yellow vein virus (BNYVV) and non-expressing control plants were inoculated with BNYVV mechanically and by means of the vector Polymyxa betae. The scFv were presumably expressed in the endoplasmic reticulum (ER). The average time needed for infections to become detectable was longer in the scFv-expressing plants than in the non-expressing control plants. In addition, the scFv-expressing plants were partially protected against the pathogenic effects exerted by the virus on N. benthamiana plants in the late stages of infection.

The complete nucleotide sequences of beet necrotic yellow vein virus RNA-1 to RNA-4 of the Japanese isolate S (BNYVV-S) were determined and compared with those of French isolate (BNYVV-F2). The nucleotide sequences of the two isolates were very similar, differing by only 1.7% (RNA-1), 4.1% (RNA-2), 2.9% (RNA-3) and 3.6% (RNA-4), respectively. The differences of the amino acid sequences of the two isolates depended upon the open reading frames (ORF) as follows: P237, 1.4%; P22 (coat protein), 2.1%; 54k ORF, 3.4%; P42, 0.5%; P13, 1.7%; P15, 3.0%; P14, 7.0% P25, 6.4%; P31, 3.5%. Comparison of the coat protein and triple gene block (P42, P13 and P15) regions of RNA-2 with other isolates revealed that BNYVV-S was much more similar to the Yugoslavian isolate (BNYVV-Yu2) than to BNYVV-F2. The nucleotide differences between BNYVV-S and BNYVV-Yu2 were less than 1%. Based upon the grouping of BNYVV variants reported by Kruse et al. [10], BNYVV-S is thus considered to belong to the A type along with BNYVV-Yu2, whereas BNYVV-F2 is classified in the B type. Our data suggest that the Japanese isolate S may have been derived from European countries other than France or Germany.

Mechanisms of resistance to beet necrotic yellow vein virus (BNYVV) were studied by comparing the multiplication and distribution of BNYVV in root tissue of some beet accessions. Seedlings were infected either by soil containing resting spores of Polymyxa betae with BNYVV, or by a viruliferous zoospore suspension. With both inoculation methods high virus concentrations were obtained in rootlets of the susceptible cultivar 'Regina'. Using infested soil, low virus concentrations were found in the partially resistant cultivar 'Rima' and in the resistant accessions Holly and WB42. When a zoospore suspension was used, similar virus concentrations occurred in 'Rima' and Holly as in 'Regina', while a low virus concentration was found in WB42. By in situ localisation studies, using immunogold-silver labelling, virus was detected in 'Regina' after infection by soil or a zoospore suspension, but it could only be detected in the resistant accessions after infection by a zoospore suspension. In rootlets of 'Regina', 'Rima' and Holly, virus was found in the epidermis, cortex parenchyma, endodermis, and interstitial parenchyma, but in general not inside the vascular tissue. In WB42 the virus, occurring in small aggregates, seemed to be restricted to the epidermis and some cortex parenchyma cells. Comparing both the multiplication and distribution of BNYVV in rootlets of the accessions studied, it is concluded that the virus resistance mechanism in 'Rima' and Holly is different from that in WB42.

RNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5'-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.

An uneven distribution of the coat protein antigens of beet necrotic yellow vein (BNYVV) and beet soil-borne (BSBV) viruses in tap roots of naturally infected sugarbeets and of BNYVV coat protein antigen in leaves and petioles of mechanically inoculated sugarbeet seedlings was detected by means of tissue print-immunoblotting. BNYVV antigen-containing areas in the tap roots were usually found underneath a root beard. Occasionally BNYVV antigen was detected predominantly, but not exclusively in the xylem vessels.

Beet necrotic yellow vein virus (BNYVV) was detected in 288 of the 392 samples collected in Iran. A-type BNYVV was detected most frequently. The p25 coding region on BNYVV RNA-3 was amplified by RT-PCR and sequenced. Nine different variants of the highly variable amino acid tetrad at positions 67-70 of p25 were identified, i.e. ACHG, AHHG, AYHG, ALHG, AFHR, AFHG, AHYG, VLHG and VHHG. These are more different tetrad variants than have been reported from any other country. The first three variants were found most commonly. In 23 out of the 288 BNYVV-positive samples, we detected P-type BNYVV that had previously been identified only in France, Kazakhstan and recently in the UK. Surprisingly, none of these samples contained the fifth RNA species usually associated with P-type BNYVV in other countries. As in other BNYVV P-type sources, the p25 amino acid tetrad in positions 67-70 of the Iranian P-type consists of SYHG.

We investigated the genetic stability of recombinant potato virus X vectors presenting beet necrotic yellow vein virus (BNYVV) epitopes. Following N-terminal PVX coat protein (CP) fusion of the BNYVV epitopes, we inoculated Nicotiana benthamiana plants with recombinant (r)PVX and carried out five serial passages through systemically-infected plants. RT-PCR investigation of the BNYVV epitope sequences revealed the accumulation of several point mutations and deletions, predominantly affecting positively-charged residues. A comparison of the isoelectric point (pI) values and charges of the wild type and rCPs showed that the initial high rCP pI values had changed to values closer to that of the wild-type CP.

Two types of RNA-5-containing beet necrotic yellow vein virus (BNYVV) have been detected in the UK at different sites in Norfolk. On the basis of nucleotide (nt) sequence comparisons, one virus source (UK-MH) was clearly identified as P type BNYVV, a virus type that had previously only been detected in two widely separated parts of the world, France and Kazakhstan. The other virus source (UK-FF) has a complex genome composition. The analysed portions of its RNAs 2 and 4 are closely related to the corresponding portions in the RNAs of the East Asian A type isolate S, whereas those of its RNAs 1 and 3 resemble P type RNA 1 from Kazakhstan and European A type RNA 3, respectively. Interestingly, the P25 encoded on its RNA 3 has an unique TYHG tetrad in the highly variable amino acid positions 67-70. RNA 5 of the UK-FF BNYVV source shares properties with P type RNA 5, but also with East Asian types of RNA 5. The possible origin and epidemiology of BNYVV types is discussed.

We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yellow vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N-terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX particles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection.

Nucleotide sequence analyses revealed that the genomes of the various European types of Beet necrotic yellow vein virus (BNYVV), i.e. the A, B and P types, are strongly conserved. Almost identical sequences were found, for instance, for A types originating from The Netherlands, Italy and former Yugoslavia; these sequences were also almost identical to those determined c. 15 years ago by Bouzoubaa et al. (1985; 1986 and 1987). This sequence stability of BNYVV types is in contrast to a pronounced sequence variability observed with Beet soil-borne pomovirus, another Polymyxa-transmitted sugar beet virus with rod-shaped particles. Sequences of RNA 1, 2 and 4 of BNYVV sources from Kazakhstan were almost identical to those of the P type of BNYVV which so far had been found only in a small area around the French town of Pithiviers. RNA 5, which in Europe is found also only in the Pithiviers area, was detected in the bait plants grown in two out of three soil samples from different fields in Kazakhstan. It closely resembled RNA 5 from the Pithiviers area, but was very different from RNA 5 from various East Asian BNYVV sources.

One A and one B type isolate of Beet necrotic yellow vein benyvirus which had been passaged for more than 15 years on manually inoculated Chenopodium quinoa in our laboratory were found to have small deletions in the KTER-encoding domain on RNA 2 which is necessary for Polymyxa transmission. There were no indications that these isolates and a third one, in which intact RNA 2 was detected, contained mixtures of deleted and intact RNAs or that populations of RNAs 2 with different deletions including very large ones were present.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.