Abstract
Recombination is a major force driving virus evolution, especially in viruses that have high genome stability and persistently infect hosts with multiple strains. CTV, an RNA virus with a genome of approximately 20 kb, occurs frequently as a complex of multiple strains in its natural hosts, vegetatively propagated and long-lived citrus plants. To examine the CTV complex at the sequence level and to assess the extent of inter-strain recombination, we performed deep-sequencing analysis of a natural CTV complex, FS2-2, using the high-throughput, 454 pyrosequencing technique. Entire CTV genomes in FS2-2 were amplified with four sets of universal RT-PCR primers and subjected to the 454 sequencing analysis in a 1/16-region sequencing run in the Genome Sequencer FLX. Over 2.2 megabases of high quality sequences were obtained from 8722 sequencing reads with an average read length of 256 nucleotides. Three divergent, high coverage (27X-43X) genome contigs corresponding to the genomes of the three co-infecting CTV strains were subsequently assembled from the sequence reads. Additionally, a large percentage (4.7%) of the sequencing reads represented recombinants between the three strains. A genome-wide recombination map for each of the CTV strains in FS2-2 was constructed using these recombinant sequences, revealing a systematic and unprecedented scale of recombination activity throughout the CTV genome. Recombination was more active in the more highly conserved 3' halves of the CTV genomes. This unprecedented, genome-wide recombination provides a plausible explanation for rapid evolution and extreme diversity of an RNA virus whose genome is remarkably stable for years in hosts infected with a single pure strain.