due to environmental changes , the global incidence of tick - borne 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 .
ticks are widespread throughout europe and are the primary arthropod vector of both human and domestic animal disease agents . in terms of public health , the most important european tick is ixodes ricinus , the vector of the lyme borreliosis bacteria , .
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 tick - borne disease transmitted by i.  ricinus is lyme borreliosis , with over 85  000 new european cases annually , .
however , patients bitten by ticks can also be infected by many other zoonotic pathogens , including parasites , viruses and other bacteria , .
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 , , , .
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 , , , , , whereas novel tick - borne pathogens are continually being discovered , , , .
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 , , , , . for viruses
for instance , even though several tick - borne encephalitis cases have been reported in france , , 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 broad - range 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 .
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 .
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 , 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  l 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 .
newborn ( 72  h old ) type i interferon receptor knock - out mice ( ifnar , genetic background : a129svevbrd ) ) kindly provided by dr damien vitour ( virology unit , animal health laboratory , anses ) were used for viral isolation from tick extracts .
females ( 45 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 ) . crushed ticks ( individual adults or pooled nymphs )
one half was directly frozen at  80c 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 .
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 .
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 ( alsace sample ) , 131    10 ( ardennes sample ) and 62    10 ( 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 .
finally , de novo assembly was performed on all remaining reads ( 5.2    10 and 7.8    10 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 . for all viruses , primers and probes
eyach virus rna was detected in ticks by quantitative rt - pcr targeted to the vp2 gene ( primer f : 5-tggctgacaacatgacggata-3 ; primer r : 5-ggcctcacgatactttcgatt-3 ) ; probe ( 5-fam - acgggctcggtacttcggttgagat - bhq1 - 3 ) .
the quantitative rt - pcrs were performed in a final volume of 20  l using lightcycler 480 rna master hydrolysis probes ( roche applied science , penzberg , germany ) , with primers at 0.5  m , the probe at 0.25  m and 2  l of rna ( quantity range from 200 to 400  ng ) .
thermal cycling conditions were 63c for 3  min , 95c for 30  sec , 45 cycles at 95c for 10  sec , and 60c for 30  sec and one final cooling cycle at 40c 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 . ,
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 .
newborn ifnar mice ( 48 to 72  h old ) were directly inoculated with 20  l 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 .
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 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 ku133666ku133676 ,
ku133678 , ku133679 ) were compared to seven available genbank sequences of eyach virus using maximum likelihood trees ( genbank reference sequences af343052 , eu789374eu789377 , af282467 and af343053 ) .
the colorado tick fever virus was selected as an outgroup ( genbank reference sequence nc_004181 ) .
eyach virus inoculum obtained from the brain homogenates of infected newborn ifnar mice ( 2.4    10  copy/l ) was intraperitoneally injected into of1 mice : five females ( 100  l ) and nine newborn mice ( 40  l ) . for controls , adult and newborn of1 mice
were respectively intraperitoneally inoculated with 100  l and 40  l 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  l ) and adult ( 100  l ) mice at days 7 , 14 and 21 .
brains and spleens were individually crushed in 500  l 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 . 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  l of reaction mixture containing 0.4 l of sample loading reagent ( biomark ) , 1.6 l of roche rt mix ( roche ) , 0.4 l of rt superscript iii ( invitrogen ) , 0.16l of rox reference dye ( biomark ) , 0.4 l of primer stock ( mixture of primers and probe at a final concentration of 18 m for each primer and 4 m for probe ) , and 3.1 l rna ( quantity range from 620  ng to 1240  ng ) , or water for negative controls .
half of the 8 l reaction mix was loaded onto the chip with the integrated fluidic circuit controller , effectively partitioning 0.65  l ( equivalent to 0.25 l 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 25c for 10  min , one step of reverse transcription at 50c for 50  min , followed by a 5-min denaturation step at 95c , and lastly 45 cycles of 10 sec at 95c and 30 sec at 60c .
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 .
numbers of viral rna copies in the blood of females at days 7 , 14 and 21 were compared using the online kruskal 
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 .
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 .
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 .
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 contigs corresponded to the nairovirus and phlebovirus genera in both the ardennes and alsace samples , ( 68/395 and 39/395 contigs , respectively , for the alsace sample , and 25/150 and 16/150 contigs , respectively , for the ardennes sample ) .
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 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 .
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 .
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 ) .
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 attempt isolation of this virus .
the absence of all three phlebovirus segments in tick extracts may reflect low viral loads , or differences in pcr efficacies . to isolate the viruses ,
ifnar mouse brains were inoculated with tick extracts positive for all three segments of either the new nairovirus or the eyach virus .
eyach virus was detected by quantitative rt - pcr in rna extracted from mouse brains infected with eyach - positive tick extracts , whereas none of the new nairovirus segments were detected by rt - pcr in the brains of mice infected with new nairovirus - positive tick extracts .
sequencing one eyach virus - positive rna brain sample generated 17.9    10 reads with 109 contigs covering the 12 segments of the eyach viral genome ( table  4 ) .
each eyach virus consensus segment was submitted to genbank ( accession numbers : ku163321ku163332 ) .
contig identities comprised between 86% and 100% compared with the available eyach virus genome ( af282467.1af282478.1af282467.1af282468.1af282469.1af282470.1af282471.1af282472.1af282473.1af282474.1af282475.1af282476.1af282477.1af282478.1 ) .
hts resulted in 93.8% coverage of the entire eyach genome , with nucleotide identity ranging between 83.3% and 99.2% , depending on the analysed segment ( see supplementary material , fig . 
phylogenetic analysis of eyach virus segment 1 contigs ( accession numbers ku133666ku133676 , 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 ) .
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 . 
viral rna persisted in the blood of the four females until day 21 , with no statistical difference in copy numbers between days 7 , 14 and 21 ( p  0.1637 ) , but was not detected in either the spleen or brain of the five adult females at day 21 .
viral rna was present in the blood of all newborn mice at each time - point , with the highest observed copy number / ml at day 14 ( p  0.0344 ) .
viral rna was present in the spleens of 6/9 newborn mice and in the brains of 3/9 newborn mice at day 21 .
eyach viral copies were higher in newborn blood samples than in adult female blood samples ( day 7 , p  0.042 ; day 14 , p  0.001 ; day 21 , p  0.004 ) .
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 .
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 can not 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 , phlebovirus , orthobunyavirus ) , reoviridae ( coltivirus ) , rhabdoviridae ( vesiculovirus , sigmavirus ) and picornaviridae - like ( iflavirus , drosophila virus a ) with nucleotide identity to known viruses ranging from 20% to 100% . among these contigs ,
predominant sequences were for viral families , which include arboviruses , suggesting that they are unlikely to represent background noise .
most sequences shared < 80% identity with known viruses , potentially indicating new viral species . in both hts data groups ( obtained from alsace and ardennes samples ) , the greatest number of contigs with relatively low percentage identity to known viruses , belonged to viruses from the nairovirus and phlebovirus genera .
the nairovirus contigs demonstrated the closest taxonomies to pathogenic tick - transmitted nairoviruses , such as nairobi sheep disease virus , hazara virus , crimean 
congo haemorrhagic fever virus , or dugbe virus . concerning the phlebovirus - related contigs ,
most were related to non - pathogenic tick - associated viruses , such as blacklegged tick virus or uukuniemi virus .
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 tick - borne viruses , as tbev prevalence in european ticks is usually between 0.1% and 1.22% ,   and powassan virus prevalence in american ticks is often lower than 2%
. however , high nairovirus and phlebovirus tick infection rates have previously been reported ( from 5.7% to 23.5% ) , , .
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 .
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 .
subsequently , it was isolated in i.  ventalloi and i.  ricinus from france in 1981 . 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 .
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 , .
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 , .
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 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 , the heartland virus , and the bourbon virus in the usa .
but many other tick - borne viruses have been described without related human illness , , 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 .
 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
Introduction
Methods
Results
Discussion