Tomato spotted wilt Tospovirus genome reassortment and genome segment-specific adaptation

A system to associate specific genome segments with viral phenotypes and to study factors influencing genome reassortment was developed for tomato spotted wilt Tospovirus (TSWV). Reassortant isolates were generated by co-inoculating a TSWV isolate, TSWV-D, with TSWV-10 or TSWV-MD. The parental origin of each genome segment in putative reassortant isolates was determined by segment-specific restriction fragment length polymorphisms. The TSWV isolates readily exchanged genome segments in a nonrandom fashion. The S RNA from TSWV-D was dominant over the S RNA from TSWV-10. The intergenic region (IGR) of the S RNA was correlated with competitiveness of this genome segment in reassortant isolates. The less competitive S RNA contained a net increase of 62 nt, including a 33-nt duplication in the IGR. This duplicate sequence was highly conserved among isolates from the southeastern United States and an isolate from Bulgaria. The evidence supports the hypothesis that the IGR of the S RNA with an ambisense coding strategy serves a regulatory function which influences the occurrence of this segment in the viral population. In addition, it was demonstrated that stable parental phenotypes can be mapped to specific genome segments as well as generating novel phenotypes not associated with either parent.