Populations of genomic RNAs devoted to the replication or spread of a bipartite plant virus differ in genetic structure

Abstract

RNA viruses within a host exist as dynamic distributions of closely related mutants and recombinant genomes. These closely related mutants and recombinant genomes, which are subjected to a continuous process of genetic variation, competition, and selection, act as a unit of selection, termed viral quasispecies. Characterization of mutant spectra within hosts is essential for understanding viral evolution and pathogenesis resulting from the cooperative behavior of viral mutants within viral quasispecies. Furthermore, a detailed analysis of viral variability within hosts is needed to design control strategies, because viral quasispecies are reservoirs of viral variants that potentially can emerge with increased virulence or altered tropism. In this work, we report a detailed analysis of within-host viral populations in 13 field isolates of the bipartite Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). The intraisolate genetic structure was analyzed based on sequencing data for 755 molecular clones distributed in four genomic regions within the RNA-dependent RNA polymerase (RNA1) and Hsp70h, CP, and CPm (RNA2) open reading frames. Our results showed that populations of ToCV within a host plant have a heterogeneous and complex genetic structure similar to that described for animal and plant RNA viral quasispecies. Moreover, the structures of these populations clearly differ depending on the RNA segment considered, being more complex for RNA1 (encoding replication-associated proteins) than for RNA2 (encoding encapsidation-, systemic-movement-, and insect transmission-relevant proteins). These results support the idea that, in multicomponent RNA viruses, function can generate profound differences in the genetic structures of the different genomic segments.

FIG. 1.

Schematic representation of the ToCV genome in which analyzed regions are represented as dark shaded boxes. Mutations, identified by comparing each individual sequence with its corresponding consensus sequence are indicated with bars in the enlarged fragments. Mutations detected in more than one sequence are indicated by the number of times they have been identified. ▵, deletion; +, insertion; •, stop codon.

FIG. 2.

Acceptabilities of nucleotide substitutions identified in each genomic region, determined according to the SG system (17), and their relative frequencies. The values range from 0 (most drastic change) to 6 (synonymous change).

FIG. 3.

Phylogenetic relationships among sequences of RdRp, Hsp70h, CP, and CPm regions of the ToCV genome, inferred by the neighbor-joining method from multiple-sequence alignments. Each individual sequence is represented by a circle with a different color for each isolate. The circle sizes are proportional to the number of identical sequences in each node and are indicated with a number. Sequences belonging to each isolate are identified with a color code, as indicated at the bottom, and a letter: A for the isolates from Almería, M for the isolates from Málaga, and U for the isolates from Murcia. I, RNA1 type I sequences; II, RNA1 type II sequences. The bar below each tree indicates 0.0005 nucleotide substitutions per site. The asterisk indicates the type II sequence found in sample AT198/00. The arrow indicates the recombinant sequence found in sample AT80/99.