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

Abstract

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

Perception of Ticks and Tick-Borne Diseases Worldwide

“…in Bothriocroton undatum and within introduced and native rodents; a whole suite of Anaplasmatacae, Francisellaceae, Midichloriaceae, Coxiellaeceae, Bartonellaceae, Mycoplasmatcceae, and Rickettsiaceae species, including Neoehrlichia australis and Neoehrlichia arcana, Midichloria mitochondrii, Coxiella massiliensis, hemotropic mycoplasmas and novel species of Anaplasma, Ehrlichia, Rickettsia, Francisella; rhabdoviruses, chuviruses, coltivurses, flavivurses, and jingmenviruses; lastly, hemoprotozoa have also been recently discovered, including Thelieria spp., Babesia spp., Trypanosoma spp., and Hepatozoon spp. [229][230][231][232][233][234][235][236][237][238][239][240]. The genetic diversity of these TOIs mirrors the co-evolution of ticks and native wildlife.…”

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia

“…scapularis) or vertebrate (BSR-baby hamster kidney) cells lines are inoculated with cells retrieved from primary cultures of human tissue biopsies and buffy coat (see "Microbial isolation" section), and are incubated at the appropriate time and temperature. The supernatants are then harvested, and the cells fixed are in a solution of 4% formaldehyde with the addition of 0.5% Triton-X100 for 10 min at 4 • C [62,63]. A fixed-cell ELISA is then performed on plates using anti-dsRNA mAbs to detect long (>30 bp) double-stranded RNA (dsRNA), a molecule which is often present in RNA virusinfected cells as either the genomic form (e.g., Reoviruses) or as a replicative intermediate (e.g., flaviviruses) [64].…”

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade

“…Additionally, no virus was recoverable in intracerebral injection into neonatal mice with MPFV and NGOV. This lack of replication in vertebrate or invertebrate cells is unusual, given that tick orthoflaviviruses have historically been propagated and cultured using these methods [ 12 13 ]. The failure to isolate MPFV and NGOV through these techniques might suggest a unique ecological niche within the genus Orthoflavivirus , perhaps as arthropod-specific viruses with a limited capacity to infect vertebrates.…”