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Binding('tagging') of a virus-specific oligonucleotide 'sticker' to RNA transcripts copied from PCR products caused retardation of transcript mobility in gel. This enables detection of specific sequences within the RNA transcripts, and a virus strain (PVY(NTN)) could thus be positively identified. We have demonstrated further that oligonucleotides that contained virus sequences originated from different genomic locations varied in their inhibitory effect on the rate of transcript migration in gel; thus, the most effective oligonucleotide could be chosen. Combinations of different strain-specific oligonucleotides had additive retarding effects on transcript migration. The conditions for annealing oligonucleotides to the RNA transcripts were studied, including concentrations of oligonucleotides and salt. A higher electrophoresis temperature (up to 45 degrees C) reduced the gel retardation phenomena, which indicated a conformation mechanism. The applicability of 'tagging' of RNA transcripts with a strain-specific oligonucleotide for virus strain differentiation is discussed.

A method for the differentiation of virus strains based on the shift in electrophoretic mobility of partially annealed RNA transcripts is described. Oppositely oriented RNA transcripts of the NTN- and N-strains of PVY, complementary at their 3'-end variable (strain-specific) region, were annealed to form a partial duplex which moved more slowly in gel than heterologous (NTN+N) unpaired transcripts. Thus, the two virus strains could be identified by annealing to a known reference transcript. The rate of duplex migration was correlated with transcript lengths and could be tightly controlled thereby. Thus, a higher degree of resolution was obtained than with transcript conformation polymorphism, which is empirical and unpredictable in nature.

An improved procedure for the resolution of RNA transcripts by electrophoretic gel retardation, mediated by annealing to specific homologous oligonucleotiedes is described. The N and NTN strains of PVY served as a model system. Non-polymorphic but sequence-diverse RNA transcripts were copied from PCR products of the two virus strains. The transcripts were resolved by gel electrophoresis, because of the differential retardation effect caused by the binding of strain-specific homologous oligonucleotides. The two PVY strains were thus differentiated. Applicability of this method to virus strain differentiation in general is discussed.

Mosaic symptoms were seen on the ornamental plant Bupleurum falcatum (Apiaceae) grown in a commercial nursery in Israel. Examination by electron microscopy (EM) of samples from diseased plants revealed elongated filamentous virus particles. Here we provide evidence that B. falcatum is an alternate host for Lettuce mosaic potyvirus (LMV). Several lines of evidence indicated that Bupleurum was infected by LMV, including double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunosorbent electron microscopy, and sap and aphid transmission of LMV to lettuce and other host plants. Additionally, general primers derived from a conserved region of the 3’-terminus of the potyvirus genome were used in a reverse transcription-polymerase chain reaction (RT-PCR) assay and gave an expected amplification product of 672 bp. A similar approach was carried out to amplify the corresponding fragment from LMV-infected lettuce. The PCR product was cloned and sequenced; it encompassed an open reading frame encoding 153 amino acids and a 3’ untranslated region (UTR) of 273 nucleotides, very similar to the coat protein (CP) and the 3‵UTR nucleotide sequence, respectively, of other LMV isolates. Analysis of the amino acid sequences of the putative CP of the Bupleurum isolate showed complete identity to the lettuce isolate of LMV. The Bupleurum isolate had no new nucleotide changes which have not been established in other published LMV strain sequences. It is concluded that LMV is the causal agent of the disease in B. falcatum in Israel. [ABSTRACT FROM AUTHOR]