Analysis of the relative frequencies of the multipartite BNYVV genomic RNAs in different plants and tissues

Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil- borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive- sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two- step reverse- transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.