Molecular characterization of the Tick-borne encephalitis virus Environments and replication

Abstract: The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains.In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden.The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/?PIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells.We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication.We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks.