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Background A subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of  Ixodes  and  Amblyomma  ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks. Methods We performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry. Results Our sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the  Ixodes scapularis -derived ISE6 cell line supports replication of Australian members of the  Flaviviridae ,  Nairoviridae ,  Phenuiviridae  and  Reoviridae  families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick  Ornithodoros capensis . While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick  Ixodes holocyclus.  Conclusions Genetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the  Flaviviridae ,  Nairoviridae ,  Phenuiviridae  and  Reoviridae  families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines. Graphical Abstract

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks