The relative infectivities and genomic characterisation of three distinct mastreviruses from South Africa.

The genomic nucleotide sequences of the cloned agroinfectious genomes of three South African mastreviruses obtained from Zea mays, a Setaria sp., and Panicum maximum (designated MSV-Kom, MSV-Set, and PanSV-Kar respectively), were determined. Additionally, their relative infectivities and virulence were analysed in a range of differentially susceptible wheat, maize, and barley genotypes. MSV-Kom produced moderate to severe streak symptoms in all maize genotypes tested, but only moderate to very mild symptoms in the wheat and barley genotypes. MSV-Set infected only the susceptible to tolerant maize genotypes, but was generally more severe in the barley and wheat genotypes than MSV-Kom. PanSV-Kar was incapable of infecting any of the wheat and barley genotypes and only produced very mild symptoms on the three most sensitive maize genotypes. Genomic characteristics in common with related mastreviruses were identified. Phylogenetic analysis indicated that while MSV-Kom was closely related to previously sequenced MSV isolates, MSV-Set and PanSV-Kar represented distinctly novel strains of MSV and PanSV respectively. In the case of MSV-Set, this is the most distantly related MSV strain yet characterised.

Complete nucleotide sequence of sugarcane streak Monogeminivirus.

The complete nucleotide sequence of the genome of the Monogeminivirus sugarcane streak virus (SSV) was determined from cloned replicative form DNA. The genome is contained in one DNA circle of 2,758 nucleotides, and has four open reading frames with the potential to encode proteins of MW > 10 kDa: two in the viral (+) sense and two in the complementary (-) sense. Each open reading frame has a counterpart among the open reading frames reported for other Monogeminiviruses. A potential binding site for a DNA replication primer and potential transcriptional control sequences were identified on the (+) strand, and a possible intron on the (-) strand. Phylogenetic analysis of coat protein and replication-associated protein sequences of SSV and other grass-infecting geminiviruses indicate that SSV, although distinct from any other virus, is part of an "African streak virus subgroup" of Monogeminiviruses.

Genome typing of southern African subgroup 1 geminiviruses.

The relatedness of subgroup 1 geminiviruses from a variety of naturally infected southern African graminaceous hosts was compared by DNA cross-hybridization, restriction endonuclease mapping and partial sequencing. Cross-hybridization divided the viruses into three groups: those closely related to maize streak virus (MSVs), and separate groups comprising a Panicum sp. virus (PanSV) and two sugarcane viruses (SSVs). Restriction mapping and comparisons, and phylogeny reconstructions from map data, showed that mapped and sequenced maize viruses were all highly similar; that two viruses of grasses and wheat bore limited resemblance to each other and to MSV, and that a mapped local and a sequenced Kenyan PanSV were similar, but that these and the two SSVs were dissimilar to each other and to all other subgroup 1 geminiviruses. The conclusions were: that maize viruses and the two viruses of wheat and grasses are probably strains of MSV; that two SSVs are only distantly related and distinct from MSVs; that the PanSVs are closely related to one another, but also distinct from other viruses; that all of the viruses in this study are part of a 'MSV-related sub-subgroup' of geminiviruses. Partial sequencing of cloned genomes reinforced conclusions drawn from other data, and indicated a definite relationship between the mapped and sequenced Panicum viruses. The implications of the results for taxonomic and epidemiological purposes are discussed.

Characterization of the sugarcane streak agent as a distinct geminivirus.

The relationship between maize streak virus and the geminivirus causing streak in sugarcane was investigated. The DNA of sugarcane streak virus does not cross-hybridize detectably with that of maize streak virus and vice versa. Restriction mapping of native replicative form viral DNA (genome size 2.7 kb) and of cloned viral DNA, combined with limited sequencing and estimated DNA sequence divergence, showed that sugarcane streak virus is as unrelated to maize streak virus and digitaria streak virus as these are different from each other. The virus is only distantly related to wheat dwarf virus and chloris striate mosaic virus. Based on these results, we propose that the agent causing sugarcane streak is a distinct geminivirus.

Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence.

The application of the polymerase chain reaction DNA amplification technique to the detection and typing of isolates of maize streak virus (MSV) and other related geminiviruses of grasses is described. The oligonucleotide primers used for amplification were 17-mers which contained a number of degeneracies. An approximately 250 base pair fragment was amplified from all geminivirus-infected grass and cereal samples tested. The amplification reaction was specific, working down to a concentration of 50 fg/ml of MSV-specific plasmid-cloned DNA and with a 10(-9) dilution of MSV-infected maize DNA extract. DNA could also be amplified from distantly related geminiviruses, including two different sugarcane viruses, digitaria streak virus and another as yet uncharacterized virus of a Panicum sp. Amplified DNA from a Mauritian sugarcane isolate (SSV-M) was cloned and sequenced. Sequence comparison and phylogenetic analysis showed that this sequence differed sufficiently from the analogous region of other geminiviruses for SSV-M to be considered a distinct virus. The use of the polymerase chain reaction for the amplification of gemini- and other virus genomes or genomic fragments for typing, mapping, phylogenetic analysis and taxonomy is discussed.

Characterization of southern African isolates of maize streak virus: typing of three isolates by restriction mapping.

The genomic replicative form DNAs (RF-DNA) of three maize streak virus isolates (MSV-CT, MSV-PE, and MSV-SW) from widely separated locations in southern Africa were characterized by restriction endonuclease mapping in order to assess the feasibility of using the technique to determine genetic variability between isolates. The viruses were transmitted to and propagated in laboratory-grown maize by the leafhopper vector Cicadulina mbila (Naudé). MSV-PE produced more severe symptoms than MSV-CT and MSV-SW; the isolates were serologically identical in 'western' immunoblot tests, but distinct in 'sandwich' enzyme-linked immunosorbent assays. RF-DNA of all three isolates was prepared from infected maize; the RF-DNA of MSV-CT and MSV-PE was cloned in a plasmid vector in Escherichia coli. Restriction maps were generated from this cloned DNA and from the RF-DNA of MSV-SWA. The maps were similar in regions expected to be conserved, but there were also important differences between all isolates. The implications of these results, and of relationships amongst these and other sequenced isolates of MSV, are discussed.