Bunyaviruses have a tripartite negative-sense RNA genome. Due to the segmented nature of these viruses, if two closely related viruses co-infect the same host or vector cell, it is possible that RNA segments from either of the two parental viruses are incorporated into progeny virions to give reassortant viruses. Little is known about the ability of tick-borne phleboviruses to reassort. The present study describes the development of minigenome assays for the tick-borne viruses Uukuniemi phlebovirus (UUKV) and Heartland phlebovirus (HRTV). We used these minigenome assays in conjunction with the existing minigenome system of SFTS phlebovirus (SFTSV) to assess the ability of viral N and L proteins to recognize, transcribe and replicate the M segment-based minigenome of a heterologous virus. The highest minigenome activity was detected with the M segment-based minigenome of cognate viruses. However, our findings indicate that several combinations utilizing N and L proteins of heterologous viruses resulted in M segment minigenome activity. This suggests that the M segment untranslated regions (UTRs) are recognised as a functional promoter of transcription and replication by the N and L proteins of related viruses. Further, virus-like particle assays demonstrated that HRTV glycoproteins can package UUKV and SFTSV S and L segment-based minigenomes. Taken together, these results suggest that co-infection of these viruses could lead to the generation of viable reassortant progeny. Thus, the tools developed herein could aid in understanding the role of genome reassortment in the evolution of these emerging pathogens under an experimental setting.
