Restriction fragment length polymorphism analysis of reverse transcription-PCR products reveals the existence of two major strain groups of beet necrotic yellow vein virus

Abstract

Beet necrotic yellow vein virus (BNYVV)-infected sugarbeets were obtained from many parts of Europe and also from some sites in Asia and the U.S.A. Reverse transcription (RT)-PCR products of more than I kbp were obtained for four different regions of the viral genome which may be particularly important with respect to the pathogenic properties of the virus, i.e. for the coat protein and the 42K protein-encoding regions on RNA 2 and for major parts ofRNAs 3 and 4. Restriction fragment length polymorphism (RFLP) patterns obtained with these PCR products revealed the existence of two major strain groups of BNYVV, named type A and type B. The A type was detected in Greece, the former Yugoslavia, Slovakia, parts of Austria, Italy, Spain, parts of France, Belgium, The Netherlands and England as well as in Asia (Turkey, Kazachstan, China and Japan) and the U.S.A. The B type occurs in Germany and parts of France. Mixed infections were detected at the borderline regions between areas of the A and B types. Comparisons of published and newly determined nucleotide sequences of the respective parts of the BNYVV genome indicate that the percentage of nucleotide differences between the A and the B type is approximately 3 % for the respective regions of RNAs 2 and 3 and approximately 1.5 % for RNA 4. Nucleotide sequences appear to be remarkably stable within each of the two strain groups. The majority of the nucleotide differences between the A and B types occur in the third triplet position. The amino acid changes in the coat protein area are outside the four previously determined antigenic regions that are accessible on the surface of the virus particles and are involved in the formation of continuous and presumably also discontinuous epitopes. This may explain why serological differences between the two strain groups have not been found.

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

“…The restriction patterns obtained for the B-type corresponded to those which were to be expected from the nucleotide (nt) sequences published by Bouzoubaa et al (1985Bouzoubaa et al ( , 1986Bouzoubaa et al ( , 1987 for the French BNYVV isolates F2/F13. The restriction patterns obtained for the A-type, however, were in agreement with those to be expected from the nt sequences of RNAs 3 and 4 of isolate G1 (Bouzoubaa et al, 1985), the coat protein gene region on RNA 2 of isolates from Belgium, former Czechoslovakia and former Yugoslavia (Yu2) (Meulewater et al, 1989;Kruse et al, 1994) and the triple gene block region on RNA 2 of isolate Yu2 (Kruse et al, 1994). The G1 isolate studied by Bouzoubaa et al (1985) probably does not originate from Germany, but possibly from former Yugoslavia (Kruse et al, 1994).…”

“…The restriction patterns obtained for the A-type, however, were in agreement with those to be expected from the nt sequences of RNAs 3 and 4 of isolate G1 (Bouzoubaa et al, 1985), the coat protein gene region on RNA 2 of isolates from Belgium, former Czechoslovakia and former Yugoslavia (Yu2) (Meulewater et al, 1989;Kruse et al, 1994) and the triple gene block region on RNA 2 of isolate Yu2 (Kruse et al, 1994). The G1 isolate studied by Bouzoubaa et al (1985) probably does not originate from Germany, but possibly from former Yugoslavia (Kruse et al, 1994). The small number of amino acid exchanges that occur in the coat protein regions of the A-and the B-types and of a deviating isolate from China lie outside the four antigenic regions which we have identified previously (Commandeur et al, * Author for correspondence.…”

“…Fax +49 531 299 3006. e-mail r.koenig @ bba.d400.de 1994). This might explain the lack of serological differences between strain groups of BNYVV (Kruse et al, 1994).…”

“…By means of restriction fragment length polymorphism (RFLP) analyses of RT-PCR products we have recently demonstrated the existence of two major strain groups of beet necrotic yellow vein virus (BNYVV) which were named type A and type B (Kruse et al, 1994). The B-type was detected mainly in France and Germany, while the A-type was found in most other countries.…”

Characteristics of Beet Soilborne Mosaic Virus, a Furo-like Virus Infecting Sugar Beet

“…In addition, RNA isolated from purified virion preparations and fractionated using the PolyATtract System 1000 (Promega) according to the manufacturer's instructions was used as template for synthesis of radiolabeled viral cDNA probes by incorporation of [α-32 P]dCTP using an oligo dT primer and reverse transcriptase as described (34). The PolyATtract System was originally designed for use in separating mRNA from total RNA extractions, and a similar system has been used in isolating BNYVV RNA from infected root tissue (23). Northern blots were prepared by separating the viral RNAs on 1% agarose gels and blotting them by capillary transfer to ZetaProbe membranes according to the supplier's instructions (Bio-Rad).…”