Could Australian ticks harbour emerging viral pathogens?

Tick-borne viruses contribute significantly to the disease burden in Europe, Asia and the US. Historically, some of the most well-known viruses from this group include the human pathogens, tick-borne encephalitis virus and Crimean-Congo haemorrhagic fever virus. More recently multiple emerging tick-borne viruses have been associated with severe disease in humans with Bourbon virus and Heartland virus isolated from patients in the US and severe fever with thrombocytopenia syndrome virus reported from China, Japan, and South Korea. Such examples highlight the need for broader approaches to survey arthropod pathogens, to encompass not only known but novel pathogens circulating in Australian tick populations.

There are currently 70 recognised species of ticks in Australia, with 22 reported infesting on humans^1,2. Ixodes holocyclus is the most significant tick in terms of human and animal health in Australia, causing a myriad of problems from toxin-induced paralysis, mostly in domestic animals³, to allergic reactions in humans⁴. The feeding behaviour of I. holocyclus as a three-host tick, makes it a successful vector for Rickettsial pathogens that cause Queensland tick typhus, Flinders Island spotted fever, and Australian spotted fever⁵. Research into ‘Debilitating Symptom Complexes Attributed to Ticks’ has led to the discovery of several new bacterial species in I. holocyclus and other native Australian ticks from families containing known human pathogens^6,7. Despite this, little is known of the viruses carried by Australian terrestrial ticks, with most isolations from seabird ticks collected on offshore islands^8–10.

Viruses isolated from Australian seabird ticks show similarities with tick-borne pathogens in other parts of the world (Table 1). The flavivirus Gadgets Gully virus (GGYV), first isolated from Ixodes uriae ticks in 1985, clusters with the mammalian tick-borne flavivirus group including human pathogens Powassan virus and Tick-borne encephalitis virus⁹. While GGYV has not been associated with disease in humans, a serological survey demonstrated evidence for infection in inhabitants of a research station on Macquarie Island¹¹. Similarly, a second flavivirus, Saumarez Reef virus (SREV), was isolated from Ixodes eudyptidis and Ornithodoros capensis ticks taken from nests of sooty terns (Onychoprion fuscatus) and silver gulls (Chroicocephalus novaehollandiae) after reports of illness and tick bites by technicians servicing weather stations on Saumarez reef (~330 km off the Central Queensland coast)¹⁰. Although no serological evidence of SREV infection was gathered, it was noted that the closely related Tyuleniy virus showed a 6% seroconversion rate in inhabitants of the Commodore Islands¹².

Table 1. Virus isolates from ticks collected in Australia and New Zealand.

In 2002, a disease outbreak in shy albatross (Thalassarche cauta) on Albatross Island (129 km north-west of Burnie, Tasmania) led to the isolation of Hunter Island Group virus. Next-generation sequencing identified the virus as a phlebovirus related to human pathogens severe fever with thrombocytopenia syndrome virus and Heartland virus¹³. Following this, sequencing of Albatross Island virus (ABIV), an isolate from I. eudyptidis ticks from the same location in 1983, showed that the two viruses were the same species¹⁴.

More recently, a novel orbivirus and two bunyaviruses were reported from I. uriae collected during a survey of ticks on penguins at Macquarie Island¹⁵. The two bunyaviruses, tentatively named Catch-me-Cave virus and Finch Creek virus, show similarities to previously isolated Precarious Point and Taggert viruses^9,16. However, full genome sequencing is required to confirm whether these viruses are contemporary strains of known viruses or new species. During this study, GGYV was also re-isolated from I. uriae suggesting that it is probable that the viruses first described between 1975 and 1985 are still circulating in the tick and bird populations on Macquarie Island¹⁵.

In contrast to the panel of viruses isolated from Australian seabird ticks, attempts to screen terrestrial tick populations have yielded only three viruses, all from Argas robertsi, a tick also found in Asia. Lake Clarendon virus (CSIRO704) was isolated from A. robertsi ticks collected at the Lake Clarendon cattle egret colony in Gatton, Queensland in 1980¹⁷. The virus was not identified and showed no relatedness to known arboviruses by serum-neutralisation and complement-fixation tests. Neutralising antibodies in cattle egret sera suggested the virus was able to infect the birds with no apparent disease¹⁷. To our knowledge, this isolate remains unidentified. A second virus was isolated from A. robertsi ticks at the same colony following reports of death in nestling chicks. This virus, originally designated CSIRO1499, was shown to have no serological similarity to Lake Clarendon virus. Experimental infections subsequently showed that the isolate was able to infect and cause mortality in birds¹¹. More recently, the full genome sequence of this virus revealed it to be closely related to Dera Ghazi Khan virus (family Nairoviridae) and the name Vinegar Hill virus (VINHV) was proposed⁸. Serological surveys found antibodies to VINHV in 3.4% of avian samples and 1% of human serum samples tested¹¹. Finally, a virus isolated from A. robertsi in the Northern Territory (NT15470) was suggested to be a member of the Dera Ghazi Khan genogroup, thought to be either a strain of Kao Shuan virus (KSV) or a close relative¹⁸. As no genome sequence is available for this isolate, it remains to be seen whether it is in fact KSV or another isolate of VINHV.

To our knowledge, there have been no virus isolations from an Australian terrestrial hard tick species. Next generation sequencing performed on I. holocyclus ticks collected in New South Wales and Queensland have allowed some insights into their potential virome¹⁹. Recently we published the genome of a novel iflavirus assembled from the transcriptome of I. holocyclus salivary glands (Figure 1a)²⁰. Interestingly, related iflaviruses have also been identified in the Ixodes scapularis cell line (ISE6) and in ticks collected from China^21,22. Several bunyavirus nucleocapsid sequences have also been identified in I. holocyclus transcriptome sequence data but whether these sequences belong to a virus or an integration in the host genome is yet to be elucidated (Figure 1b). This data indicates that the virome of Australian terrestrial ticks may mirror that of terrestrial ticks found in the northern hemisphere.

Figure 1. (a) Evolutionary relationship of Ixodes holocyclus iflavirus within the family Iflaviridae. Mid-point rooted maximum likelihood phylogenetic tree was constructed based on an alignment of peptidase and RdRP proteins corresponding to position 2036–2944 of the IhIV polyprotein. (b) Summary of virus sequences identified in I. holocyclus by transcriptome sequencing¹⁹.

We have developed a broad-spectrum screening system that detects viral isolates in cell cultures inoculated with mosquito homogenates. The MAVRIC system (Monoclonal antibodies against viral RNA intermediates in cells) targets long (>30 bp) double-stranded RNA molecules, produced during replication of viruses, in a sequence-independent manner²³. MAVRIC led to the isolation of at least 9 new viral species from 7 different families allowing the identification of numerous viruses previously not known to exist in Australian mosquito populations²⁴. Based on the success of MAVRIC in mosquito screening, we aim to apply this system to screen Australian terrestrial ticks. One barrier is the lack of suitable cell lines derived from Australian tick species and their hosts. While the use of vertebrate cell cultures (generally BHK-21 and Vero cell lines) has proven successful for the isolation of mosquito and seabird tick viruses in Australia^9,25, alternative cell lines reflecting the common hosts of terrestrial ticks (i.e. marsupials), may need to be considered for tick virus isolation on the mainland. Furthermore, while mosquito cell culture has been well established, tick cell culture has proven more difficult requiring a complex mix of vitamins and minerals which must be formulated in-house²⁶.

Ixodes holocyclus iflavirus was unable to replicate in the I. scapularis cell line (ISE6), but appeared to replicate in the host tick raising the question of the suitability of cell lines derived from ticks of the northern hemisphere to isolate Australian tick-borne viruses^20,27. Phylogenetic analyses have demonstrated that I. holocyclus and I. uriae are divergent from ‘other’ Ixodes species²⁸. Preliminary analysis from our group has suggested that some of the vertebrate-infecting viruses of I. uriae and I. eudyptidis are able to replicate in the ISE6 cell line (Figure 2), however this remains to be demonstrated for viruses of terrestrial ticks. Bell-Sakyi and Attoui recently discussed the role of tick cell culture in virus discovery, particularly in relation to tick-specific viruses²⁹. In this instance, the development of cell lines from Australian native ticks may be necessary.

Figure 2. Anti-flavivirus E protein (green) staining in Ixodes scapularis (ISE6) cells infected with Australian tick-borne flaviviruses Gadgets Gully virus (GGYV) and Saumarez Reef virus (SREV).

Finally, the risk that Australian seabird-associated tick viruses pose to human health should be considered. A study undertaken to investigate the health risk posed to residents and tourists on the islands in the Great Barrier Reef and Coral Sea by seabird-associated arboviruses identified two isolates in O. capensis collected on Masthead Island, which appeared closely related to VINHV³⁰. This may demonstrate the potential for incursion of seabird-associated viruses to the mainland and vice versa. Serological surveys undertaken after the initial isolation of ABIV identified a black noddy (Anous minutus) from Heron Island off the coast of Queensland with neutralising antibodies to the virus³⁰. Finally, SREV was isolated from O. capensis ticks found on Saumarez reef off the coast of Queensland and 2000 km away in Tasmania¹⁰.

While our knowledge of the viruses harboured by Australian ticks is still limited, the data thus far suggests that our tick viromes may mirror those seen in the northern hemisphere. Next generation sequencing of terrestrial ticks performed by our group and others will greatly contribute to the characterisation of tick-viruses in Australia. In this context, a recent deep sequencing study of Australian ticks by Eddie Holmes’ group at the University of Sydney, has identified a plethora of novel viral sequences that will provide a useful reference for further studies (unpublished data available online: https://www.biorxiv.org/content/early/2018/08/07/386573). Complementary to next generation sequencing, a system for efficient isolation of newly discovered viruses will allow for complete characterisation. Finally, comprehensive characterisation of the current tick-borne virus isolates held in archive is required to avoid re-discovery of these viruses in future studies.

The authors declare no conflicts of interest.