The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.
The genome of Grapevine leafroll-associated virus-5 (GLRaV-5) was cloned, and the sequence of 4766 nt was determined. Degenerate oligonucleotide primers designed from the conserved closterovirus heat shock 70 protein (HSP 70) homologue were used to obtain viral-specific sequences to anchor the cloning of the viral RNA with a genomic walking approach. The partial nucleotide (nt) sequence of GLRaV-5 showed the presence of four open reading frames (ORF A through D), potentially coding for the HSP 70 homologue (ORF A); a 51-kDa protein of unknown function with similarity to GLRaV-3 p55 (ORF B); the viral capsid protein (ORF C); and a diverged viral duplicate capsid protein (ORF D). The ORF C was identified as GLRaV-5 viral capsid protein based on sequence analyses and the reactivity of the recombinant protein to GLRaV-5 specific antibodies by western blot analyses. The antiserum produced with the in vitro-expressed GLRaV-5 ORF C protein product specifically reacted with a 36-kDa polypeptide from GLRaV-5 infected vines but did not react with protein extracts from vines infected with other GLRaVs or uninfected vines. Furthermore, specific primers were designed for the sensitive detection of GLRaV-1 and GLRaV-5 by polymerase chain reaction.
Western blot immunoassay and enzyme-linked immunosorbent assay using different monoclonal antibodies (MAb) and polyclonal antisera (PA) revealed mixed infections of serologically related and unrelated grapevine leafroll associated viruses (GLRaVs) and grapevine corky bark associated virus (GCBaV) in symptomatic grapevines. A PA designated rootstock-scion incompatibility (RSI)-24 kDa, grapevine corky bark PA, and GLRaV-2b MAb reacted to polypeptides of approximately 24 kDa isolated from grapevines exhibiting rootstock-scion incompatibility, leafroll, and corky bark disease symptoms, suggesting that these isolates are infected with closely related viruses. A PA designated GLRaV-2 US detected virus specific polypeptides of 38, 37, 36, and 24 kDa, while a polyclonal antiserum designated GLRaV-2 FR detected a single virus-specific polypeptide of approximately 24 kDa. The reactivity of GLRaV-2 US to various polypeptides suggests that the immunogen used to produce this antiserum was a mixture of viruses. Apical meristems were excised and cultured to eliminate the infection of viruses in the grapevines showing RSI symptoms and in the cultivar French Colombard infected with GLRaV-1. The elimination of these viruses was confirmed by Western blot assay. These studies show that the Western blot assay can be used to detect and differentiate grapevine disease-associated closteroviruses.
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