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# Virus Discovery Using Tick Cell Lines

Lesley Bell-Sakyi, Houssam Attoui

► To cite this version:

Lesley Bell-Sakyi, Houssam Attoui. Virus Discovery Using Tick Cell Lines. Evolutionary Bioinformatics, 2016, 12, pp.31-34. 10.4137/EBO.S39675. hal-01604255

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Distributed under a Creative Commons Attribution 4.0 International License
# Virus Discovery Using Tick Cell Lines

Lesley Bell-Sakyi¹ and Houssam Attoui²

¹Head of The Tick Cell Biobank, The Pirbright Institute, Pirbright, Surrey, UK. ²Group leader, National Institute for Agricultural Research (INRA), Department of Animal Health, UMR1161 Virology, INRA-ANSES-ENVA, Maisons Alfort, France.

Libertas Academica
FREEDOM TO RESEARCH

## Supplementary Issue: Evolution of the Insect Virome

**ABSTRACT:** While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks' genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area.

**KEYWORDS:** tick cell line, arbovirus, endogenous virus, endogenous viral element

**SUPPLEMENT:** Evolution of the Insect Virome

**CITATION:** Bell-Sakyi and Attoui. Virus Discovery Using Tick Cell Lines. *Evolutionary Bioinformatics* 2016:12(S2) 31–34 doi: 10.4137/EBO.S39675.

**TYPE:** Short Commentary

**RECEIVED:** May 13, 2016. **RESUBMITTED:** July 07, 2016. **ACCEPTED FOR PUBLICATION:** July 11, 2016.

**ACADEMIC EDITOR:** Liuyang Wang, Associate Editor

**PEER REVIEW:** Three peer reviewers contributed to the peer review report. Reviewers' reports totaled 331 words, excluding any confidential comments to the academic editor.

**FUNDING:** LB-S is supported by the United Kingdom Biotechnology and Biological Sciences Research Council (grant BBS/E/1/00001741). HA is supported by Institut National de la Recherche Agronomique core funding. The authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal.

**COMPETING INTERESTS:** Authors disclose no potential conflicts of interest.

**CORRESPONDENCE:** lesley.sakyi@pirbright.ac.uk

**COPYRIGHT:** © the authors, publisher and licensee Libertas Academica Limited. This is an open-access article distributed under the terms of the Creative Commons CC-BY 4.0 License.

Paper subject to independent expert blind peer review. All editorial decisions made by independent academic editor. Upon submission manuscript was subject to anti-plagiarism scanning. Prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. This journal is a member of the Committee on Publication Ethics (COPE).

Published by Libertas Academica. Learn more about this journal.

Ticks have been recognized as vectors of pathogenic protozoa and bacteria for over 120 years.¹⁻³ The first confirmed tick-borne viruses were identified in the early 1930s causing disease in sheep: the flavivirus louping ill virus, transmitted by *Ixodes ricinus* in Scotland,⁴ and the nairovirus Nairobi sheep disease virus, transmitted by *Rhipicephalus appendiculatus* in Kenya.⁵ Since then, ticks have been incriminated as vectors and/or reservoirs of numerous viruses of medical and/or veterinary importance,⁶,⁷ including the emerging pathogens Alkhumra hemorrhagic fever virus, severe fever with thromocytopenia virus, Heartland virus, and Bourbon virus.⁸⁻¹¹ In addition, many apparently nonpathogenic arboviruses have been isolated from ticks.⁶,⁷

Research on tick-borne viruses has benefited greatly from the availability of tick cell and tissue cultures. Almost as soon as techniques were developed for repeatedly producing primary tick cell cultures,¹²,¹³ they were put to use for experiments on arthropod-borne and other viruses, confirming the ability to replicate in tick cells and helping to clarify their host range.¹⁴⁻¹⁷ Continuous cell lines, first derived from ticks in the 1970s,¹⁸,¹⁹ have since been applied to many aspects of arbovirus research,¹¹,²⁰⁻²⁵ including clarification of actual and potential vector capacity.¹¹,¹⁴,¹⁵,²¹,²²,²⁶⁻²⁸ Replication was demonstrated in tick cells of viruses carried by ixodid and argasid ticks, mosquitoes, sand flies and midges as well as some viruses with

no known vector.¹⁵,²⁰,²⁹⁻³¹ Surprisingly, there are few reports of tick cells being used for the isolation of tick-borne viruses from field samples; primary tick cell cultures were found to be at least as sensitive as chick embryo cell cultures for the isolation of tick-borne encephalitis virus (TBEV) from vertebrate blood and brain suspension and tick suspension.³²,³³ An uncharacterized RNA virus, Cascade virus, was isolated from frozen triturated *Dermacentor occidentalis* ticks into a *Dermacentor variabilis* cell line (RML-15); Cascade virus also replicated well in amphibian (XTC-2) cells but poorly or not at all in mosquito cells, most vertebrate cells, and suckling mice.³⁴ The *Rhipicephalus microplus* cell line BME26 was used recently alongside Vero and BHK-21 cells for the isolation of a putative novel flavivirus, Mogiana tick virus from *R. microplus* ticks in Brazil.³⁵

Until recently, all the viruses detected in ticks had one characteristic in common—they could all replicate in vertebrate cells and indeed were initially isolated into vertebrate cells. However, it is now recognized that ticks themselves harbor a diverse microflora of vertically transmitted endosymbiotic bacteria and endogenous viruses³⁶⁻⁴⁰ that may not cycle between ticks and their vertebrate hosts. While endosymbiotic bacteria have been reported to occur in various different tick species for nearly a century,³,⁴¹⁻⁴⁴ very little is known about endogenous viruses of ticks.⁴⁵ An
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electron microscope study revealed the presence of virus-like particles in the salivary glands of adult female *R. microplus* ticks feeding on cattle<sup>46</sup>; the presence of large numbers of the particles was associated with possible abnormal tick-feeding behavior. A single apparently tick-only virus has been comprehensively described, sequenced, and characterized: the orbivirus St Croix River virus (SCRV)<sup>47</sup> was detected in, and replicates in, tick cell lines but has so far failed to infect or replicate in any of the vertebrate or insect cell lines tested.<sup>43</sup> Originally, SCRV was detected in the IDE2 cell line derived from the tick *Ixodes scapularis*,<sup>47,48</sup> suggesting that this prostriate tick was its natural host; subsequent detection of SCRV in two cell lines (RA243 and RA257) derived from developing adult *R. appendiculatus* ticks established nearly 20 years previously in a laboratory on a different continent<sup>18,49</sup> suggests that the virus may have originated from the latter metastriate tick species (Table 1). Electron microscopy revealed the presence of many more putative and potentially tick-only viruses in the majority of over 50 cell lines derived from 14 ixodid and two argasid tick species (Table 1).<sup>45,49,50</sup> These tick cell lines represent an abundant, easily accessible resource for virus discovery, as well as contributing to studies on virus ancestry and evolution. Studies on the evolution of SCRV and other tick-borne orbiviruses<sup>51-53</sup> suggested that SCRV is the oldest orbivirus identified to date and provided evidence of coevolution of orbiviruses and their respective arthropod host/vector. More recently, the *Amblyomma americanum* cell line AAE12<sup>54</sup> was found to contain novel orbivirus sequences (H. Attoui, P. Alberdi, C. Sharp, and L. Bell-Sakyi, unpublished data, 2012) (Table 1); if these sequences belong to

a replication-competent virus, it will be interesting to determine its relationship to SCRV and other orbiviruses.

In the past few years, high-throughput molecular screening and deep sequencing approaches applied to various tick species<sup>36,38,40,55,56</sup> have revealed tantalizing hints of many more potentially tick-only viruses belonging to diverse families, but for such putative viruses, if they cannot infect and replicate in vertebrate cells, tick cell lines will be needed to establish whether the sequences belong to intact, replication-competent viruses or to endogenous viral elements (partial or full-length DNA copies of the RNA genomes integrated into the genome of the vector/host; EVE) incorporated into the tick genomes. A proteomics informed by transcriptomics approach applied to RNAseq data from uninfected and TBEV-infected tick cells<sup>24</sup> revealed RNA sequences with similarity to picorna-like viruses and rhabdoviruses in the *I. scapularis* cell line IDE8 and rhabdoviruses and the iflavirus deformed wing virus in the *I. ricinus* cell line IRE/CTVM19 (D. Matthews, S. Weisheit, J. de la Fuente, and L. Bell-Sakyi, unpublished data, 2013) (Table 1). Rhabdovirus-like (weakly grouped with lyssaviruses), orthomyxovirus-like (related to quarjaviruses), and bunyavirus-like (related to Hazara and Uukuniemi viruses) endogenous virus elements were identified in the genome of *I. scapularis*,<sup>57</sup> the only tick species with a completely sequenced genome (https://www vectorbase.org/).<sup>58</sup> RNA sequences with similarity to genes of insect densoviruses, hepatitis E virus and the tick-borne pathogen African swine fever virus were detected in transcriptomes of *R. microplus* larvae and/or “tick gut” (unspecified life cycle stage) available on the CattleTickBase website (http://cattletickbase.ccgapps.com.au/). These data are not published,

<table><caption>**Table 1.** Viruses and viral RNA sequences detected in tick cell lines. The tick species and instar from which each cell line was derived have been presented previously (49).</caption><thead><tr><th>VIRUS OR VIRAL RNA SEQUENCES</th><th>TICK CELL LINE</th><th>REFERENCE</th></tr></thead><tbody><tr><td rowspan="2">St Croix River virus</td><td>IDE2</td><td>(47)</td></tr><tr><td>IDE8, RA243, RA257</td><td>(49)</td></tr><tr><td rowspan="4">Virus-like particles seen by electron microscopy</td><td>RA243</td><td>(50)</td></tr><tr><td>AAE2, AAE12, ANE58, AVL/CTVM13, AVL/CTVM17, BDE/CTVM14, BME/CTVM5, BME/CTVM23, BME/CTVM30, DALBE3, DVE1, HAE/CTVM9, IDE2, IDE8, IRE11, IRE/CTVM19, IRE/CTVM20, ISE6, ISE18, RAE/CTVM1, RAN/CTVM3, REE/CTVM28, REE/CTVM29, REE/CTVM31, RSE8</td><td>(49)</td></tr><tr><td>CCE1, CCE2, OME/CTVM21, OME/CTVM22, OME/CTVM24, OME/CTVM25, OME/CTVM26, OME/CTVM27</td><td>(45)</td></tr><tr><td>AAE12</td><td>H. Attoui, P. Alberdi, C. Sharp and L. Bell-Sakyi, unpublished data, 2012</td></tr><tr><td>RNA sequences with similarity to picorna-like viruses and rhabdoviruses</td><td>IDE8</td><td>D. Matthews, S. Weisheit, J. de la Fuente and L. Bell-Sakyi, unpublished data, 2013</td></tr><tr><td>RNA sequences with similarity to rhabdoviruses and the iflavirus deformed wing virus</td><td>IRE/CTVM19</td><td>D. Matthews, S. Weisheit, J. de la Fuente and L. Bell-Sakyi, unpublished data, 2013</td></tr></tbody></table>
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and it is unclear whether they represent authentic viral transcripts or EVE transcripts. It is noteworthy that reverse transcriptase activity, as well as retrotransposons, was detected in three of the *I. scapularis* embryo-derived cell lines (IDE2, IDE8, and ISE6) (H. Attoui and F. Mohd Jaafar, unpublished data. 2006). Reverse transcriptase activity may explain why DNA copies of RNA virus genomes occur in the *I. scapularis* genome. Interestingly, it has been shown that TBEV infection of Hep2 human cells resulted in the production of DNA copies of the viral genome and the stable integration of this DNA into the cellular genome backbone⁵⁹ and that TBEV DNA was found integrated into brain cells during progressive infection.⁶⁰ Whether or not a similar process occurs in TBEV-infected tick cells remains to be determined.

In mammalian genomes, endogenous retroviruses have been shown to exert essential regulatory gene expression functions,⁶¹ when integrated in specific positions of the mammalian genome. A decade ago, we identified a DNA form of a virus related to cell fusing agent virus in the *Aedes albopictus* mosquito cell line C6/36, including a 1557 amino acid open reading frame containing the NS1 to NS4B.⁶² Antibodies raised against recombinant NS1 and NS3 of this virus helped to identify expression of its NS3 in C6/36 cells (H. Attoui and F. Mohd Jaafar, unpublished data, 2006). The Dicer-2 of C6/36 cells is nonfunctional as a result of mutations,⁶³ making them highly permissive to a range of arboviruses; expression of viral protein such as NS3 (viral protease) from EVE may also enhance permissivity of cells to viral infections. Similar situations may exist in tick cells in which EVE are expressed.

In conclusion, the present portfolio of cell lines derived from different ixodid and argasid tick species, and new cell lines under development from additional species, constitutes a huge and hitherto underexplored and underutilized resource for the discovery, detection, isolation, and propagation of novel arthropod-borne viruses. It is likely that the application of molecular technologies such as those used in the studies mentioned previously,³⁵,³⁶,³⁸,⁴⁰,⁵⁵,⁵⁶ and novel pipelines for metagenomic analysis,⁶⁴ will very soon start to reveal the secrets of tick-borne viruses and endogenous viral elements harbored by tick cell lines and, by inference, the ticks from which they were derived.

## Acknowledgments

The authors would like to thank Ulrike Munderloh and Timothy Kurtti of the University of Minnesota for providing tick cell lines.

## Author Contributions

Composed, reviewed, and approved the final article: LB-S and HA.

## REFERENCES

1. Smith T, Kilbourne FL. *Investigations into the Nature, Causation and Prevention of Texas or Southern Cattle Fever*. Vol 1. Washington, DC: U.S. Dept. of Agriculture, Bureau of Animal Industry; 1893:7–269.

2. Lounsbury CP. Ticks and African Coast fever. *Agr J Cape Good Hope*. 1906;28:634–54.

3. Cowdry EV. Studies on the etiology of heartwater. II. *Rickettsia ruminantium* (N. Sp.), in the tissues of ticks transmitting the disease. *J Exp Med*. 1925;42:253–74.

4. Grieg JR, Brownlee A, Wilson DR, Gordon WS. The nature of louping ill. *Vet Rec*. 1931;11:325–33.

5. Daubney R, Hudson JR. Nairobi sheep disease. *Parasitology*. 1931;23:507–24.

6. Labuda M, Nuttall PA. Tick-borne viruses. *Parasitology*. 2004;129:5221–45.

7. Nuttall PA. Molecular characterization of tick-virus interactions. *Front Biosci*. 2009;14:2466–83.

8. Madani TA, Abuelzein EME, Bell-Sakyi L, et al. Susceptibility of tick cell lines to infection with Alkhumra haemorrhagic fever virus. *Trans R Soc Trop Med Hyg*. 2013;107:806–11.

9. Luo L-M, Zhao L, Wen H-L, et al. *Haemaphysalis longicornis* ticks as reservoir and vector of severe fever with thrombocytopenia syndrome virus in China. *Emerg Infect Dis*. 2015;21:1770–6.

10. Savage HM, Godsey MS Jr, Lambert A, et al. First detection of Heartland virus (Bunyaviridae: *Phlebovirus*) from field collected arthropods. *Am J Trop Med Hyg*. 2013;89:445–52.

11. Lambert AJ, Velez JO, Brault AC, et al. Molecular, serological and *in vitro* culture-based characterization of Bourbon virus, a newly described human pathogen of the genus *Thogotovirus*. *J Clin Virol*. 2015;73:127–32.

12. Rehacek J. Preparation of tissue cultures from the tick *Hyalomma dromedarii* Koch. *J Med Entomol*. 1965;2:161–4.

13. Yunker CE, Cory J. Effectiveness of refrigerated nymphs in tick tissue culture experiments. *J Parasitol*. 1965;51:686.

14. Rehacek J, Pesek J. Propagation of Eastern equine encephalomyelitis (EEE) virus in surviving tick tissues. *Acta Virol*. 1960;4:241–5.

15. Rehacek J. Cultivation of different viruses in tick tissue cultures. *Acta Virol*. 1965;9:332–7.

16. Yunker CE, Cory J. Growth of Colorado tick fever virus in primary tissue cultures of its vector, *Dermacentor andersoni* Stiles (Acarina: Ixodidae), with notes on tick tissue culture. *Exp Parasitol*. 1967;20:267–77.

17. Rehacek J, Rajcani J, Gresikova M. Relationship of Tribec virus to tick cells and tissues. *Acta Virol*. 1969;13:439–42.

18. Varma MGR, Pudney M, Leake CJ. The establishment of three cell lines from the tick *Rhipicephalus appendiculatus* (Acari: Ixodidae) and their infection with some arboviruses. *J Med Entomol*. 1975;11:698–706.

19. Bhat UKM, Yunker CE. Establishment and characterization of a diploid cell line from the tick, *Dermacentor parumapertus* Neumann (Acarina: Ixodidae). *J Parasitol*. 1977;63:1092–8.

20. Bell-Sakyi L, Kohl A, Bente DA, Fazakerley JF. Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses. *Vector Borne Zoonotic Dis*. 2012;12:769–81.

21. Ciota AT, Payne AF, Ngo KA, Kramer LD. Consequences of *in vitro* host shift for St. Louis encephalitis virus. *J Gen Virol*. 2014;95:1281–8.

22. Ciota AT, Payne AF, Kramer LD. West Nile virus adaptation to ixodid tick cells is associated with phenotypic trade-offs in primary hosts. *Virology*. 2015;482:128–32.

23. Tuppurainen ES, Venter EH, Coetzer JA, Bell-Sakyi L. Lumpy skin disease: attempted propagation in tick cell lines and presence of viral DNA in field ticks collected from naturally-infected cattle. *Ticks Tick Borne Dis*. 2015;6:134–40.

24. Weisheit S, Villar M, Tykalová H, et al. *Ixodes scapularis* and *Ixodes ricinus* tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis. *Parasit Vectors*. 2015;8:599.

25. Cerny J, Selinger M, Palus M, et al. Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells. *Virus Genes*. 2016;52:309–16.

26. Bhat UKM, Yunker CE. Susceptibility of a tick cell line (*Dermacentor parumapertus* Neumann) to infection with arboviruses. In: Kurstak E, ed. *Arctic and Tropical Arboviruses*. New York: Academic Press; 1979:263–75.

27. Lawrie CH, Uzcategui NY, Armesto M, Bell-Sakyi L, Gould EA. Susceptibility of mosquito and tick cell lines to infection with various flaviviruses. *Med Vet Entomol*. 2004;18:268–74.

28. Ruzek D, Bell-Sakyi L, Kopecky J, Grubhoffer L. Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks. *Virus Res*. 2008;137:142–6.

29. Hess WR. The growth of African swine fever virus in arthropod cells. *African Swine Fever*. Wilkinson PJ, ed. Luxembourg: Commission of the European Communities; 1983:124–31.

30. Munz E, Reimann M, Munderloh U, Settele U. The susceptibility of the tick cell line TTC 243 for Nairobi sheep disease virus and some other important species of mammalian RNA and DNA viruses. In: Kurstak E, Maramorosch K, Dubendorfer A, eds. *Invertebrate Systems In Vitro*. Amsterdam: Elsevier/North Holland Biomedical Press; 1980:338–40.
<figure><img src="image_5.png" /></figure>

31. Hepojoki J, Kipar A, Korzyukov Y, Bell-Sakyi L, Vapalahti O, Hetzel U. Replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells. *J Virol.* 2015;89:1119–28.

32. Rehacek J, Kozuch O. Comparison of the susceptibility of primary tick and chick embryo cell cultures to small amounts of tick-borne encephalitis virus. *Acta Virol.* 1964;8:470–1.

33. Rehacek J, Kozuch O. The use of *Hyalomma dromedarii* tick tissue cultures for the isolation of tick-borne encephalitis virus from its natural foci. *Acta Virol.* 1969;13:253.

34. Yunker CE, Cory J, Gresbrink RA, Thomas LA, Clifford CM. Tickborne viruses in western North America III. Viruses from man-biting ticks (Acari: Ixodidae) in Oregon. *J Med Entomol.* 1981;18:457–63.

35. Marayuma SG, Castro-Jorge LA, Ribeiro JMC, et al. Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in *Rhipicephalus microplus* ticks from Brazil. *Mem Inst Oswaldo Cruz.* 2014;109:38–50.

36. Nakao R, Abe T, Nijhof AM, et al. A novel approach, based on BLSOMs (batch learning self-organizing maps), to the microbiome analysis of ticks. *ISME J.* 2013;7:1003–15.

37. Tokarz R, Sameroff S, Leon MS, Jain K, Lipkin WI. Genome characterization of Long Island tick rhabdovirus, a new virus identified in *Amblyomma americanum* ticks. *Virol J.* 2014;11:26.

38. Tokarz R, Williams SH, Sameroff S, Leon MS, Jain K, Lipkin WI. Virome analysis of *Amblyomma americanum*, *Dermacentor variabilis*, and *Ixodes scapularis* ticks reveals novel highly divergent vertebrate and invertebrate viruses. *J Virol.* 2014;88:11480–92.

39. Narasimhan S, Fikrig E. Tick microbiome: the force within. *Trends Parasitol.* 2015;31:315–23.

40. Xia H, Hu C, Zhang D, et al. Metagenomic profile of the viral communities in *Rhipicephalus* spp. ticks from Yunnan, China. *PLoS ONE*. 2015;10: e0121609.

41. Scoles G. Phylogenetic analysis of the *Francisella*-like endosymbionts of *Dermacentor* ticks. *J Med Entomol.* 2004;41:277–86.

42. Parola P, Paddock C, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. *Clin Microbiol Rev.* 2005;18: 719–56.

43. Zhong J. *Coxiella*-like endosymbionts. In: Toman R, Heinzen RA, Samuel JE, Mege JL, eds. *Coxiella burnetii: Recent Advances and New Perspectives in Research of the Q Fever Bacterium*. Vol 984. Advances in Experimental Medicine and Biology; 2012:365–79. New York: Springer.

44. Pilloux L, Aeby S, Gaumann R, Burri C, Beuret C, Greub G. The high prevalence and diversity of *Chlamydiales* DNA within *Ixodes ricinus* ticks suggest a role for ticks as reservoirs and vectors of *Chlamydia*-related bacteria. *Appl Environ Microbiol.* 2015;81:8177–82.

45. Bell-Sakyi L, Attoui H. Endogenous tick viruses and modulation of tick-borne pathogen growth. *Front Cell Infect Microbiol.* 2013;3:25.

46. Megaw MWJ. Virus-like particles pathogenic to salivary glands of the tick *Boophilus microplus*. *Nature*. 1978;271:483–4.

47. Attoui H, Stirling JM, Munderloh UG, et al. Complete sequence characterization of the genome of the St Croix River virus, a new orbivirus isolated from cells of *Ixodes scapularis*. *J Gen Virol.* 2001;82:795–804.

48. Munderloh UG, Liu Y, Wang M, Chen C, Kurtti TJ. Establishment, maintenance and description of cell lines from the tick *Ixodes scapularis*. *J Parasitol.* 1994;80:533–43.

49. Alberdi MP, Dalby MJ, Rodriguez-Andres J, Fazakerley JK, Kohl A, Bell-Sakyi L. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines. *Ticks Tick Borne Dis.* 2012;3:137–46.

50. Munz E, Reimann M, Mahnel H. Nairobi sheep disease virus and *Reovirus*-like particles in the tick cell line TTC-243 from *Rhipicephalus appendiculatus*: experiences with the handling of the tick cells, immunoperoxidase and ultrahistological studies. In: Yunker CE, ed. *Arboviruses in Arthropod Cells In Vitro*. Vol 1. Boca Raton: CRC Press; 1987:133–47.

51. Belhouchet M, Mohd Jaafar F, Tesh R, et al. Complete sequence of Great Island virus and comparison with the T2 and outer-capsid proteins of Kemerovo, Lipovnik and Tribec viruses (genus *Orbivirus*, family *Reoviridae*). *J Gen Virol.* 2010;91:2985–93.

52. Mohd Jaafar F, Belhouchet M, Belaganahalli M, Tesh RB, Mertens PP, Attoui H. Full-genome characterisation of Orungo, Lebombo and Changuinola viruses provides evidence for co-evolution of orbiviruses with their arthropod vectors. *PLoS One*. 2014;9:e86392.

53. Belaganahalli MN, Maan S, Maan NS, et al. Genetic characterization of the tick-borne orbiviruses. *Viruses*. 2015;7:2185–209.

54. Singu V, Peddireddi L, Sirigireddy KR, Cheng C, Munderloh U, Ganta RR. Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in *Ehrlichia chaffeensis* and *Ehrlichia canis*. *Cell Microbiol.* 2006;8:1475–87.

55. Li C-X, Shi M, Tian J-H, et al. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. *Elife.* 2015;4:e05378.

56. Shi M, Lin XD, Vasilakis N, et al. Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the *Flaviviridae* and related viruses. *J Virol.* 2015;90:659–69.

57. Katzourakis A, Gifford RJ. Endogenous viral elements in animal genomes. *PLoS Genet.* 2010;6:e1001191.

58. Gulia-Nuss M, Nuss AB, Meyer JM, et al. Genomic insights into the *Ixodes scapularis* tick vector of Lyme disease. *Nat Commun.* 2016;7:10507.

59. Zhdanov VM, Bogomolova NN, Gavrilov VI, Andzhaparidze OG, Deryabin PG, Astakhova AN. Infectious DNA of tick-borne encephalitis virus. *Archiv Gesamte Virusforsch.* 1974;45:215–24.

60. Zhdanov VM, Terent'ev VF, Fateeva AP, Kitaeva LK, Gaidamovich SI. Integration of virus genome with the genome of brain cells during tick-borne encephalitis with a progressive course. *Vestn Akad Med Nauk SSSR.* 1977;7:13–5.

61. Chuong EB, Elde NC, Feschotte C. Regulatory evolution of innate immunity through co-option of endogenous retroviruses. *Science.* 2016;351:1083–7.

62. Crochu S, Cook S, Attoui H, et al. Sequences of flavivirus-related RNA viruses persist in DNA form integrated in the genome of *Aedes* spp. mosquitoes. *J Gen Virol.* 2004;85:1971–80.

63. Brackney DE, Scott JC, Sagawa F, et al. C6/36 *Aedes albopictus* cells have a dysfunctional antiviral RNA interference response. *PLoS Negl Trop Dis.* 2010;4:e856.

64. Verneau J, Levasseur A, Raoult D, La Scola B, Colson P. MG-digger: an automated pipeline to search for giant virus-related sequences in metagenomes. *Front Microbiol.* 2016;7:428.