We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.

The results obtained here have expanded our knowledge of mycoviral diversity, horizontal transfers, and putative cross-kingdom events. To date, this study presents the most extensive and wide diversity collection of mycoviruses infecting the necrotrophic fungus B. cinerea .

The long flexuous virions of the Closteroviridae have a unique bipolar architecture incorporating two coat proteins, with most of the helical nucleocapsid encapsidated by the major coat protein (CP) and a small portion of one end encapsidated by the minor coat protein (CPm). It is not known whether CPm encapsidates the genomic RNA and, if so, which end and what effects transition between the two coat proteins. Two other virus-encoded proteins, an HSP70 homolog (HSP70h) and an ≈61-kDa protein, are required to augment virion assembly. In this work, we examine the in vivo encapsidation of Citrus tristeza virus by its CPm in the absence of CP. In the absence of other assembly-related proteins, CPm protected a family of 5′ coterminal RNAs, apparently because of pausing at different locations along the genomic RNA. Most of the nucleocapsids formed by CPm were short, but a few were full-length and infectious. Mutations within the 5′ nontranslated region demonstrated that the CPm origin of assembly overlaps the previously described conserved stem-and-loop structures that function as a cis-acting element required for RNA synthesis. Thus, in the absence of CP, the CPm encapsidation is initiated from the 5′ end of the genomic RNA. Coexpression of HSP70h and the p61 protein with CPm in protoplasts restricted encapsidation to the 5′ ≈630 nucleotides, which is close to the normal boundary of the bipolar virion, whereas the presence of either HSP70h or the p61 protein alone did not limit encapsidation by CPm.