Molecular characteristics of segment 5, a unique fragment encoding two partially overlapping ORFs in the genome of rice black-streaked dwarf virus

Rice black-streaked dwarf virus (RBSDV), a ds-RNA virus in Fijivirus genus with family Reoviridae, which is transmitted by the small brown planthopper, is responsible for incidence of maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD). To understand the variation and evolution of S5, a unique fragment in the genome of RBSDV which encodes two partially overlapping ORFs (ORF5-1 and ORF5-2), we analyzed 127 sequences from maize and rice exhibiting symptoms of dwarfism. The nucleotide diversity of both ORF5-1 (π = 0.039) and ORF5-2 (π = 0.027) was higher than that of the overlapping region (π = 0.011) (P < 0.05). ORF5-2 was under the greatest selection pressure based on codon bias analysis, and its activation was possibly influenced by the overlapping region. The recombinant fragments of three recombinant events (14NM23, 14BM20, and 14NM17) cross the overlapping region. Based on neighbor-joining tree analysis, the overlapping region could represent the evolutionary basis of the full-length S5, which was classified into three main groups. RBSDV populations were expanding and haplotype diversity resulted mainly from the overlapping region. The genetic differentiation of combinations (T127-B35, T127-J34, A58-B35, A58-J34, and B35-J34) reached significant or extremely significant levels. Gene flow was most frequent between subpopulations A58 and B35, with the smallest |Fst| (0.02930). We investigated interactions between 13 RBSDV proteins by two-hybrid screening assays and identified interactions between P5-1/P6, P6/P9-1, and P3/P6. We also observed self-interactive effects of P3, P6, P7-1, and P10. In short, we have proven that RBSDV populations were expanding and the overlapping region plays an important role in the genetic variation and evolution of RBSDV S5. Our results enable ongoing research into the evolutionary history of RBSDV-S5 with two partly overlapping ORFs.