Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

RNA viruses exist within a host as a population of mutant sequences, often referred to as quasispecies. Within a host, sequences of RNA viruses constitute several distinct but interconnected pools, such as RNA packed in viral particles, double-stranded RNA, and virus-derived small interfering RNAs.We aimed to test if the same representation of within-host viral population structure could be obtained by sequencing different viral sequence pools. Using ultradeep Illumina sequencing, the diversity of two coex-isting Potato virus Y sequence pools present within a plant was investigated: RNA isolated from viral particles and virus-derived small interfering RNAs (the derivatives of a plant RNA silencing mechanism). The mutational landscape of the within-host virus population was highly similar between both pools, with no notable hotspots across the viral genome. Notably, all of the single-nucleotide polymorphisms with a frequency of higher than 1.6%were found in both pools. Some unique single-nucleotide poly-morphisms (SNPs) with very low frequencies were found in each of the pools, with more of them occurring in the small RNA (sRNA) pool, possibly arising through genetic drift in localized virus populations within a plant and the errors introduced dur-ing the amplification of silencing signal. Sequencing of the viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Nonhomologous recombinations were commonly detected in the viral particle pool, with a hot spot in the 3 = untranslated and coat protein regions of the genome.We stress that they present an important but often overlooked aspect of virus population diversity