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Diversity of viruses in Ixodes ricinus, and characterization of a neurotropic strain of Eyach virus

Ticks transmit more pathogens—including bacteria, parasites and viruses—than any other arthropod vector. Although the epidemiological status of many tick-borne bacteria is very well characterized, tick-borne viruses are still relatively under-studied. Recently, several novel tick-borne viruses have been isolated from human febrile illnesses following tick bites, indicating the existence of other potential new and unknown tick-borne viruses. We used high-throughput sequencing to analyse the virome of Ixodes ricinus, the main vector of tick-borne pathogens in Europe. The majority of collected viral sequences were assigned to two potentially novel Nairovirus and Phlebovirus viruses, with prevalence rates ranging from 3.95% to 23.88% in adults and estimated to be between 0.14% and 72.16% in nymphs. These viruses could not be isolated from the brains of inoculated immunocompromised mice, perhaps indicating that they are unable to infect vertebrates. Within the I. ricinus virome, we also identified contigs with >90% identity to the known Eyach virus. Initially isolated in the 1980s, this virus was indirectly associated with human disease, but had never been extensively studied. Eyach virus prevalence varied between 0.07% and 5.26% in ticks from the French Ardennes and Alsace regions. Eyach virus was successfully isolated following intracerebral inoculation of immunocompromised mice with Eyach virus-positive tick extracts. This virus was also able to multiply and persist in the blood of immunocompetent mice inoculated by intraperitoneal injection, and caused brain infections in three of nine juveniles, without any obvious deleterious effects.


Importance
Due to environmental changes, the global incidence of tickborne disease is increasing worldwide, thus tick-borne diseases have been listed alongside diseases with high emergence risk. Several tick-borne pathogens have already emerged in certain geographical regions, such as tick-borne encephalitis virus and Crimean-Congo haemorrhagic fever virus, and novel tick-borne pathogens are continually being discovered. A proportion of these viruses are zoonotic (severe fever with thrombocytopenia syndrome virus in China, the Heartland virus and the Bourbon virus in the USA) but the pathogenicity of many others is poorly documented. Consequently, it is extremely important to determine the pathogenicity of these viruses, and their potential involvement in undiagnosed animal or human febrile illness or encephalitis.

Introduction
Ticks are widespread throughout Europe and are the primary arthropod vector of both human and domestic animal disease agents [1]. In terms of public health, the most important European tick is Ixodes ricinus, the vector of the Lyme borreliosis bacteria [2,3]. Ixodes ricinus can transmit many varieties of pathogens, including bacteria, parasites and viruses, due to specific biological adaptations and its capacity to feed on numerous different animal species. The most prevalent tickborne disease transmitted by I. ricinus is Lyme borreliosis, with over 85 000 new European cases annually [2,3]. However, patients bitten by ticks can also be infected by many other zoonotic pathogens, including parasites, viruses and other bacteria [4,5]. Some of these pathogens were initially identified in ticks decades before their association with human disease (such as Borrelia miyamotoi), whereas others have only been discovered very recently (such as the Bourbon virus). The global incidence of tick-borne disease is increasing worldwide as the result of environmental changes, so tick-borne diseases are now highlighted as having significant emergence risk [6][7][8][9]. Several tick-borne pathogens have already emerged in specific geographical regions, such as the tick-borne encephalitis virus (TBEV), louping ill virus, Powassan virus, deer tick virus, severe fever with thrombocytopenia syndrome virus and Crimean-Congo haemorrhagic fever virus [10][11][12][13][14], whereas novel tickborne pathogens are continually being discovered [15][16][17][18]. These factors highlight the importance of studying viral epidemiology in tick populations.
Although ticks have the potential to transmit many different viruses, most studies surveying tick-borne pathogens in Europe have focused on bacterial and/or parasitic pathogens. Numerous reports detailing parasitic or bacterial prevalence in either European ticks or animal reservoirs are published every year [19][20][21][22][23]. For viruses however, the situation is completely different. For instance, even though several tick-borne encephalitis cases have been reported in France [24,25], recent data on TBEV prevalence in I. ricinus or animal reservoirs in France do not exist. Moreover, no data are available regarding the prevalence of other tick-borne viruses in Europe. This lack of knowledge and relevant data within ticks is surprising, but may be explained by the fact that it is far easier to detect bacterial and parasitic DNA, because they possess the conserved rRNA genes that are most often targeted by broadrange molecular testing. Subsequently, many laboratories, including ours, have focused their research on bacteria and parasites rather than viruses.
In this study, we aimed to describe the global picture of viruses carried by French I. ricinus, using RNA deep sequencing to identify and better characterize both DNA and RNA viruses that replicate in ticks. We identified 545 assembled contigs related to eukaryotic viruses. The vast majority of hits mapped to families known to include arboviruses, and the greatest number of contigs probably designated a novel Nairovirus and a new Phlebovirus. The only known virus identified in the ticks was the Eyach virus, first isolated in Europe in the 1980s, and which has since been forgotten. Here, we demonstrated its capacity to multiply and persist in the blood of OF1 mice, and its ability to colonize murine brains.

Methods
Tick collection and extract preparation Questing I. ricinus female ticks (268 in Ardennes), male ticks (228 in Ardennes) and nymphs (285 and 1455 in Ardennes and Alsace, respectively) were collected by flagging from northeastern France (Ardennes in 2012 49.574177, 4.802835; Alsace in 2010 47.918066, 7.146111). All collected ticks were washed as previously described [26], nymphs were pooled into groups of 15 individuals (116 pools in total) and adults were individually treated. Before RNA extraction, tick samples were crushed in 300 μL Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. A pathogen-free colony was obtained as follows; females from the field were engorged on rabbits and allowed to lay eggs. DNA and RNA samples were extracted from female ticks and PCRs were performed to test for the presence of Borrelia spp., Bartonella spp., Anaplasma spp., Rickettsia spp., Francisella spp. and Coxiella spp. Only larvae from 'pathogen-free' female ticks were conserved and maintained in our colony before use in high throughput sequencing (HTS) experiments.

Animal and ethical issues
Newborn (72 h old) type I interferon receptor knock-out mice (IFNAR −/− , genetic background: A129SvEvBrd) [27]) kindly provided by Dr Damien Vitour (Virology Unit, Animal Health Laboratory, ANSES) were used for viral isolation from tick extracts. Females (4-5 weeks old) and newborn OF1 mice (72 h old) (Charles River Company, Wilmington, MA, USA) were used for studying the course of the Eyach virus infection. Animal experiments were carried out in strict accordance with appropriate animal care practices as recommended by European guidelines. Protocols were approved by the ANSES-ENVA-UPEC Ethics Committee for Animal Experimentation (Agreement Number: 13-021).

RNA extraction
Crushed ticks (individual adults or pooled nymphs) were divided into two equal samples. One half was directly frozen at −80°C for subsequent viral isolation, the other half was used for total RNA extraction using the Nucleospin RNA II kit (Macherey-Nagel, Duren, Germany) following the manufacturer's instructions.
High throughput sequencing and bioinformatic analysis of sequences obtained from tick extracts RNA samples were pooled relating to their geographical area (one Ardennes pool and one Alsace pool). The pooled RNA samples were retro-transcribed to cDNA, and randomly amplified using the multiple displacement amplification (MDA) protocol with phi29 polymerase and random hexamers as described previously [26]. Library preparations and sequencing with an Illumina HiSeq2000 sequencer were outsourced to DNAVision (Charleroi, Belgium). Wild and pathogen-free samples were sequenced to a depth of 200 × 10 6 (Alsace sample), 131 × 10 6 (Ardennes sample) and 62 × 10 6 (pathogen-free sample) paired-end reads of 101 bp. Raw sequence reads were trimmed according to their quality score. At the time of analysis, there was no publicly available reference genome for I. ricinus, so tick sequences were removed from the analysis by subtracting sequences identical to those derived from a pathogen-free reference sample using the SOAP2 aligner tool [28]. Finally, de novo assembly was performed on all remaining reads (5.2 × 10 6 and 7.8 × 10 6 for the Ardennes and Alsace tick samples, respectively) using CLC ASSEMBLYCELL software (version 4.0), producing 16 094 and 174 841 contigs in the Ardennes and Alsace tick samples, respectively. Taxonomic assignation of these contigs was achieved by successive sequence alignment using nucleotide and protein databases and the BLAST algorithm. Contigs were assigned the closest known taxonomy according to their identity percentage, and distant alignments were not considered.

Virus prevalence identification in ticks
For all viruses, primers and probes were derived from high throughput contigs (HTS-contigs). Eyach virus RNA was detected in ticks by quantitative RT-PCR targeted to the vp2 gene (Primer F: 5 0 -TGGCTGACAACATGACGGATA-3 0 ; Primer R: 5 0 -GGCCTCACGATACTTTCGATT-3 0 ); probe (5 0 -FAM-ACGGGCTCGGTACTTCGGTTGAGAT-BHQ1-3 0 ). The quantitative RT-PCRs were performed in a final volume of 20 μL using Lightcycler 480 RNA Master Hydrolysis Probes (Roche Applied Science, Penzberg, Germany), with primers at 0.5 μM, the probe at 0.25 μM and 2 μL of RNA (quantity range from 200 to 400 ng). Thermal cycling conditions were 63°C for 3 min, 95°C for 30 sec, 45 cycles at 95°C for 10 sec, and 60°C for 30 sec and one final cooling cycle at 40°C for 30 sec. Each RNA sample was run in duplicate.
To detect the novel Nairovirus and Phlebovirus RNA, the cDNA synthesis step was performed with random hexamers (Superscript III RT; Invitrogen Inc., Carlsbad, CA, USA). Amplification of L, M and S segments from each new Nairovirus and Phlebovirus was performed using 22 specific primer pairs (Table 1) designed from HTS-contigs. All PCR amplifications were performed by using the Taq Core kit (MPBio, Illkirch, France) following the manufacturer's instructions.

Viral isolation from positive tick extracts
Newborn IFNAR -/mice (48 to 72 h old) were directly inoculated with 20 μL of tick extracts positive for either the novel Nairovirus or Eyach virus (pool of ticks numeros E48 and E134). Mice were observed daily, and if paralysis occurred, mice were euthanized, and their brains were harvested and homogenized in medium. RNA was extracted using the Nucleospin RNA II Macherey-Nagel kit following the manufacturer's instructions, viral RNA was detected by quantitative RT-PCR as described above.

Full genome sequence and phylogenetic analysis of Eyach virus
Purified RNA from mouse brains infected with Eyach-positive tick extracts was amplified as previously described for tick RNA and sequenced on a Life Technologies Proton sequencer using a single chip (around 80 × 10 6 reads). Genome sequences were derived from de novo assembled contigs. For phylogenetic analysis, HTS-contig sequences of Eyach virus strains from the RNA-dependent RNA polymerase gene (segment 1) from French ticks (deposited in GenBank, accession numbers KU133666-KU133676, KU133678, KU133679) were compared to seven available GenBank sequences of Eyach virus using maximum likelihood trees (GenBank reference sequences AF343052, EU789374-EU789377, AF282467 and AF343053). The Colorado tick fever virus was selected as an outgroup (GenBank reference sequence NC_004181).

Infection of OF1 mice with Eyach virus isolated from tick extracts
Eyach virus inoculum obtained from the brain homogenates of infected newborn IFNAR −/− mice (2.4 × 10 5 copy/μL) was intraperitoneally injected into OF1 mice: five females (100 μL) and nine newborn mice (40 μL). For controls, adult and newborn OF1 mice were respectively intraperitoneally inoculated with 100 μL and 40 μL of 1× PBS. Mice (infected and negative controls) were observed and weighed daily until day 21. Recorded clinical signs included significant weight loss, weakness, ruffled fur, hunched posture, ataxia, tremors and occasionally hind leg paralysis.
Blood samples (infected and negative controls) were collected from newborn (40 μL) and adult (100 μL) mice at days 7, 14 and 21. Mice were killed at day 21, and brains and spleens were harvested.

RNA preparation from mouse tissues and blood
Brains and spleens were individually crushed in 500 μL Dulbecco's modified Eagle's medium before RNA extraction. RNA was extracted from total blood, brain or spleen homogenates using the Nucleospin RNA II Macherey-Nagel kit following the manufacturer's instructions.

Quantification of Eyach virus in mouse tissues and blood
For each RNA sample (brain, spleen, blood), digital RT-PCR was performed to quantify Eyach vp2 segments. Digital RT-PCR amplifications were performed on a Fluidigm BioMark System using digital array microfluidic chips (Fluidigm Corporation, San Francisco, CA, USA).
Reactions were performed in 8 μL of reaction mixture containing 0.4 μL of sample loading reagent (BioMark), 1.6 μL of Roche RT Mix (Roche), 0.4 μL of RT superscript III (Invitrogen), 0.16μL of ROX reference dye (BioMark), 0.4 μL of Primer stock (mixture of primers and probe at a final concentration of 18 μM for each primer and 4 μM for probe), and 3.1 μL RNA (quantity range from 620 ng to 1240 ng), or water for negative controls. Half of the 8 μL reaction mix was loaded onto the chip with the Integrated Fluidic Circuit controller, effectively partitioning 0.65 μl (equivalent to 0.25 μL RNA; quantity range to 50 ng to 100 ng) into each of the 770 chambers per panel. The one-step digital RT-PCR programme involved a step of 25°C for 10 min, one step of reverse transcription at 50°C for 50 min, followed by a 5-min denaturation step at 95°C, and lastly 45 cycles of 10 sec at 95°C and 30 sec at 60°C. The Digital PCR Analysis software, part of the BioMark System (Fluidigm Corporation) was used to count the number of positive chambers out of the total number of chambers per panel. Poisson distribution was used to estimate the average number of template copies per chamber in a panel. All samples were characterized by a corresponding absolute quantity per microlitre. No positive chambers were observed in the negative controls.

Statistical analysis
Numbers of viral RNA copies in the blood of females at days 7, 14 and 21 were compared using the online Kruskal-Wallis test (Aus Vet Animal Health Service http://epitools.ausvet.com.au/ content.php?page=home). The same statistical analyses were performed for newborn mice. Numbers of viral RNA copies in the blood of females compared with newborn mice at day 7, day 14 and day 21 were done using the online Wilcoxon rank sum test. The differences were considered statistically significant if p <0.05.

Results
Ixodes ricinus viral metatranscriptomic analysis Metatranscriptome analysis was performed on two pooled I. ricinus RNA samples: subsequently termed the 'Alsace sample' and the 'Ardennes sample', and detected pathogens including replicating DNA and RNA viruses, including 174 841 and 16 094 assembled contigs were generated for the Alsace and Ardennes samples respectively. Of these contigs, 395 and 150 had best hits corresponding to eukaryote viruses belonging to viral genera comprising eukaryotic viruses of vertebrates and/or arthropods. We excluded plant or fungi viruses for the sake of clarity. Fig. 1(a,b) illustrates the viral family distribution of read counts used to assemble the 395 and 150 contigs. The vast majority of hits mapped to virus families known to replicate in arthropods. Most belonged to Bunyaviridae (Nairovirus, Phlebovirus, Orthobunyavirus), Reoviridae (Coltivirus), Rhabdoviridae (Vesiculovirus, Sigmavirus), Picornaviridae-like (Iflavirus, Drosophila virus A) families (see Table 2 for the Alsace sample and Table 3 for the Ardennes sample). Hits with high nucleotide identity (94% -100%) were all assigned to the identical Coltivirus virus, i.e. Eyach virus. We identified 19 contigs corresponding to the 12 segments normally present within Coltivirus genomes. All other hits were distant from known viruses (50% mean nucleotide identity, range 25%-72%), most belonged to families known to replicate in arthropods. The most frequently occurring These contigs corresponded to all three viral genome segments (L, M and S). None of the contigs corresponded to any of the three viral genome segments from other viral genera belonging to the Bunyaviridae family. We then focused NMNI on the little-known or new viruses for whom HTS analyses gave the most complete data. Subsequently, we evaluated the prevalence of the Eyach virus, and the likely novel Nairovirus and Phlebovirus members in ticks, by identifying infected ticks, and then used crushed tick extracts in further isolation and characterization studies.

RNA virus prevalence in I. ricinus
The prevalence of the Eyach virus, novel Nairovirus and novel Phlebovirus RNA viruses was calculated in adult ticks (female and male) from the Ardennes, and was estimated in pooled nymphs from both the Alsace and Ardennes regions.
The prevalence of Eyach virus RNA was 1.87% in female adult ticks (5/268) and 0.88% in adult males (2/228). In the Ardennes samples, 1/19 nymph pools (each comprising 15 nymphs) was positive, indicating that in nymphs, prevalence ranged between 0.35% (if only one nymph was positive per pool) and 5.26% (if all 15 nymphs were positive in each pool). In Alsace ticks, 1/97 nymph pools was positive, with an estimated nymph prevalence ranging between 0.07% and 1.03%.
Concerning the new Nairovirus, only the L segment was detected in adult ticks from Ardennes, with a prevalence of 23.88% in females (64/268) and 16.67% in males (38/228). In nymphs from Alsace, all three segments were detected with prevalences ranging between 4.81% and 72.16% (70/97) for the L contig, between 0.14% and 2.06% (2/97) for the M contig, and between 0.48% and 7.22% (7/97) for the S contig. All three segments were present in two nymph pools (E48 and E134). Interestingly, both of these pools were also positive for the Eyach virus. New Nairovirus members were not detected in the Ardennes nymphs.
For the new Phlebovirus member(s), only the L segment was detected in adult ticks from Ardennes with a prevalence of 5.60% in females (15/268), and 3.95% in males (9/228). In the nymph pools from Ardennes, only the L and M fragments were detected: 13/19 nymph pools were positive for the L segment, indicating that in nymphs, the L-segment prevalence ranged between 4.56% and 68.42%; the M contig was detected in 1/19 nymph pools, with a prevalence between 0.35% and 5.26%. No pools were simultaneously positive for both the L and M fragments. In Alsace nymphs, only the L segment was detected with a prevalence ranging between 2.82% and 42.27% (41/97).

Viral isolation from positive tick extracts
We attempted Eyach virus and new Nairovirus isolation from tick extracts positive for all three segments. As no tick extracts carried more than one new Phlebovirus segment, we did not  Fig. S1).
Phylogenetic analysis of Eyach virus segment 1 contigs (Accession numbers KU133666-KU133676, KU133678, KU133679) revealed that Eyach genotypes identified in the Alsace and Ardennes regions were more closely related to the German Eyach virus isolated from I. ricinus, than to other French Eyach viruses isolated from I. ricinus and Ixodes ventalloi collected in the west of France (Fig. 2).

Eyach viruses exhibit neurotropism in newborn OF1 mice
We then investigated whether the virus could replicate in immunocompetent mice (adults and juveniles) following intraperitoneal inoculation. Infected adults and newborn mice presented no obvious clinical signs of disease (such as weight loss, weakness, ruffled fur, hunched posture, ataxia, tremor) compared with controls.
To assess Eyach viral load in the blood, spleen and brain of each mouse, Eyach virus RNA was evaluated at days 7, 14 and 21 by digital RT-PCR (Fig. 3). Viral Eyach RNA was detected in

Discussion
In this study we analysed the virome of I. ricinus, the most relevant tick species to animal and human health in Europe. Our ultimate aim was to uncover new or unsuspected tick-borne animal or human viruses in I. ricinus. We identified a multitude of diverse eukaryotic viral sequences related to major arbovirus families. Interestingly, most of the eukaryotic viral sequences detected in I. ricinus belonged to probable new viruses, as only one previously known virus was identified, the Eyach virus. This had been identified in the 1980s as a potential human pathogen in ticks but has not been studied subsequently.
Here, we isolated an Eyach viral strain with neurotropism in mice, confirming that this virus has the capacity to infect vertebrates and traverse the blood-brain barrier. Although we cannot draw conclusions regarding viral pathogenicity from these data, our results lay the foundation for several studies regarding the pathogen itself as well as their possible hosts and reservoirs.
Following the analysis of the two sets of HTS sequences, we obtained contigs belonging to Bunyaviridae (Nairovirus, Ticks had relatively high infection rates for both genera in the two geographical areas studied: between 3.95% and 23.88% in adults, and between 0.14% and 72.16% in nymphs. These rates are higher than have typically been reported for other tickborne viruses, as TBEV prevalence in European ticks is usually between 0.1% and 1.22% [29,30] and Powassan virus prevalence in American ticks is often lower than 2% [31]. However, high Nairovirus and Phlebovirus tick infection rates have previously been reported (from 5.7% to 23.5%) [14,32,33]. Possible explanations for the high prevalence rates may be either due to extensive bioavailability of susceptible viraemic hosts, or because these viruses can be transovarially transmitted from females to offspring. In the latter case, these viruses could then be considered as endogenous tick viruses, where the ticks themselves play the role of long-term reservoirs [34]. Endogenous tick viruses are not necessarily able to infect vertebrates, and for many, almost nothing is known about their biology or their potential pathogenic effects on humans or animals. The inability to detect sequences corresponding to these new Nairovirus after inoculating the brains of immunocompromised mice with positive tick extracts suggests that these viruses are unable to multiply in this particular vertebrate host.
In addition to contigs corresponding to probable viruses, we identified contigs sharing more than 90% identity with another known virus. These contigs were all assigned to the same virus, Eyach virus. This virus, a member of the Coltivirus genus within the Reoviridae family, was first isolated in I. ricinus collected from Germany in 1972 [35]. Subsequently, it was isolated in I. ventalloi and I. ricinus from France in 1981 [36]. Since this time and despite the lack of surveillance, this virus is evidently still present in France. The Eyach virus was indirectly incriminated in cases of encephalitis and polyradiculoneuritis in the former Czechoslovakia, as antibodies to the virus were identified in sera from patients with a neurological syndrome, but no formal confirmatory viral identification occurred [37]. Nevertheless, solid demonstration of causality is still lacking.
An animal reservoir for the Eyach virus, if it does exist, remains unidentified, even though anti-Eyach virus antibodies have been identified in many animal species in France, including the European rabbit (Oryctolagus cuniculus), rodents, sheep, deer and mountain goats [38,39]. We demonstrated that Eyach virus prevalence (0.07%-5.26%) was similar in Ardennes and Alsace ticks and was similar to rates of TBEV, which is endemic in the studied regions [29,30]. So far, no viral RNA has been isolated from animals or humans; however, successful isolation of the virus following intracerebral inoculation of mice with infected tick extract, indicated that the virus could possibly multiply in vertebrate hosts. This result was reinforced by the demonstration that after intraperitoneal inoculation in immunocompetent mice, the virus was able to multiply and persist in the blood for up to 21 days after infection. Finally, we identified the virus in the NMNI brains of suckling mice as evidence of its neurotropism. More advanced analysis of the Eyach virus should be performed to determine the presence of lesions and/or to determine specific cellular tropism in the brain. The long-lasting viraemia of Eyach virus observed in OF1 mice might suggest that rodents could be the natural reservoir of this virus. However, a preliminary prevalence study performed on bank voles collected in the same area as the ticks (Ardennes) did not reveal the presence of the virus (Cosson et al., unpublished data). In summary, there is still no clear evidence that the Eyach virus infects humans or animals, so this will require additional investigation.
Currently, new tick-borne viruses are often isolated from humans with febrile illness or death following a tick bite, as occurred for severe fever with thrombocytopenia syndrome virus in China [18], the Heartland virus [16], and the Bourbon virus in the USA [15]. But many other tick-borne viruses have been described without related human illness [17,33], leading to new challenges and questions in the field of tick-borne disease research: are those new viruses potential pathogens for humans, and even more importantly, could these new viruses be linked to human disease.