Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.

The main areas for field-grown melon (Cucumis melo) production in Spain were surveyed for the occurrence and relative incidence of cucumber mosaic virus (CMV), papaya ringspot virus-watermelon strain (PRSV-W), watermelon mosaic virus-2 (WMV-2), and zucchini yellow mosaic virus (ZYMV) during the growing seasons of 1995 and 1996. Samples from 1,152 plants showing symptoms of virus infection were collected from commercial melon fields and analyzed by enzyme-linked immunosorbent assay (ELISA). CMV and WMV-2 were the most frequently found viruses, both by the number of locations and by their incidence in each location. In contrast, PRSV-W and ZYMV were detected in fewer sites and at lower incidences. PRSV-W was not found in 1996. In 79% of the samples, only one virus was detected; 15% of the samples were doubly infected. Both the incidence of plants showing symptoms of viral infection and the relative incidence of each of the four viruses varied according to the region, while the main trends of virus distribution were similar for 1995 and 1996.

We have studied the biological and molecular characteristics of a MNSV isolate collected in Spain (MNSV-Malpha5) and generated a full-length cDNA clone from which infectious RNA transcripts can be produced. The host range of MNSV-Malpha5 appeared to be limited to cucurbits and did not differ from that of MNSV-Dutch [4, 21]. However, differences were observed in the type of symptoms that both isolates could induce. A full-length cDNA of MNSV-Malpha5 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a 5'-end primer anchoring a T7 RNA promoter sequence and a 3'-end primer, and cloned. Uncapped RNAs transcribed from this cDNA clone were infectious and caused symptoms indistinguishable from those caused by viral RNA when mechanically inoculated onto melon, cucumber or watermelon plants. The complete genome sequence of MNSV-Malpha5 was deduced from the full length cDNA clone. It is 4271 nt long and, similarly to MNSV-Dutch, consists of 5' and 3' untranslated regions (UTRs) and five open reading frames (ORFs) coding for 29, 89, 42 and two small 7 kDa proteins. One notable difference between MNSV-Malpha5 and other sequenced MNSV isolates was found, as for MNSV-Malpha5 the first of the two small ORFs, which are contiguous in the genome, terminates with a genuine stop codon, whereas for MNSV-Dutch and other sequenced MNSV isolates it terminates with an amber codon. This suggested that the putative p14 readthrough protein that could be expressed from the MNSV-Dutch and other MNSV genomes could not be expressed from the MNSV-Malpha5 genome. Also, the nucleotide and amino acid sequences comparisons showed a distant relationship of MNSV-Malpha5 with other known MNSV isolates.

RNA 1 of the V strain of tomato aspermy virus (TAV) consists of 3410 nucleotides and contains one open reading frame (ORF) of 2982 nucleotides, resembling RNA 1 of cucumber mosaic virus (CMV) strains Q and Fny (68% and 66% identical, respectively) and of brome mosaic virus (BMV) (41% identical). In comparisons between amino acid sequences, three conserved regions (N-terminal, C-terminal and central) between TAV and each CMV were found. The N-and C-terminal regions were also conserved with BMV, and contained, respectively, consensus motifs for methyltransferases and for nucleic acid helicases. The 5' and 3' non-coding sequences were highly similar to those of TAV RNA 2. When the sequences for the genomic RNAs of the V and C strains of TAV, and of their encoded products, are compared with those reported for CMV strains representing either subgroup I (Fny-CMV) or subgroup II (Q-CMV) of CMV, it was found that the different virus-encoded proteins are conserved differently between these three viruses. Also, the divergence between TAV and both CMV subgroups has proceeded at different rates for the different ORFs. On the whole, the divergence between TAV and CMV is of the same order as that found between CMV subgroups I and II, which suggests that TAV, Q-CMV and Fny-CMV could be considered as representing three equivalent subgroups of a taxonomic entity.