Ixodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed. RNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV). Differential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway. This systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmenvirus (JMV) group holds a special place among flaviviruses and flavi-like viruses because they have a segmented ssRNA(+) genome. We detected Alongshan virus (ALSV), which is a representative of the JMV group, in ten pools of adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions. Three of the ten strains were isolated in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most ALSV virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream open reading frame in the glycoprotein-coding segment of ALSV and other members of the JMV group.

The genus Flavivirus includes related, unclassified segmented flavi-like viruses, two segments of which have homology with flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3. This group includes such viruses as Jingmen tick virus, Alongshan virus, Yanggou tick virus and others. We detected the Yanggou tick virus in Dermacentor nuttalli and Dermacentor marginatus ticks in two neighbouring regions of Russia. The virus prevalence ranged from 0.5% to 8.0%. We detected RNA of the Alongshan virus in 44 individuals or pools of various tick species in eight regions of Russia. The virus prevalence ranged from 0.6% to 7.8%. We demonstrated the successful replication of the Yanggou tick virus and Alongshan virus in IRE/CTVM19 and HAE/CTVM8 tick cell lines without a cytopathic effect. According to the phylogenetic analysis, we divided the Alongshan virus into two groups: an Ixodes persulcatus group and an Ixodes ricinus group. In addition, the I. persulcatus group can be divided into European and Asian subgroups. We found amino acid signatures specific to the I. ricinus and I. persulcatus groups and also distinguished between the European and Asian subgroups of the I. persulcatus group.

There has been an emergence and expansion of tick-borne diseases in Europe, Asia and North America in recent years, including Lyme disease, tick-borne encephalitis and human anaplasmosis. The primary vectors implicated are hard ticks of the genus Ixodes. Although much is known about the host response to these bacterial and viral pathogens, there is limited knowledge of the cellular responses to infection within the tick vector. The bacterium Anaplasma phagocytophilum is able to bypass apoptotic processes in ticks, enabling infection to proceed. However, the tick cellular responses to infection with the flaviviruses tick-borne encephalitis virus (TBEV) and louping ill virus (LIV), which cause tick-borne encephalitis and louping ill respectively, are less clear. Infection and transcriptional analysis of the Ixodes ricinus tick cell line IRE/CTVM20 with the viruses LIV and TBEV, and the bacterium A. phagocytophilum, identified activation of common and distinct cellular pathways. In particular, commonly-upregulated genes included those that modulate apoptotic pathways, putative anti-pathogen genes, and genes that influence the tick innate immune response, including selective activation of toll genes. These data provide an insight into potential key genes involved in the tick cellular response to viral or bacterial infection, which may promote cell survival and host resistance.

Tick cell lines are increasingly used in many fields of tick and tick-borne disease research. The Tick Cell Biobank was established in 2009 to facilitate the development and uptake of these unique and valuable resources. As well as serving as a repository for existing and new ixodid and argasid tick cell lines, the Tick Cell Biobank supplies cell lines and training in their maintenance to scientists worldwide and generates novel cultures from tick species not already represented in the collection. Now part of the Institute of Infection and Global Health at the University of Liverpool, the Tick Cell Biobank has embarked on a new phase of activity particularly targeted at research on problems caused by ticks, other arthropods and the diseases they transmit in less-developed, lower- and middle-income countries. We are carrying out genotypic and phenotypic characterisation of selected cell lines derived from tropical tick species. We continue to expand the culture collection, currently comprising 63 cell lines derived from 18 ixodid and argasid tick species and one each from the sand fly Lutzomyia longipalpis and the biting midge Culicoides sonorensis, and are actively engaging with collaborators to obtain starting material for primary cell cultures from other midge species, mites, tsetse flies and bees. Outposts of the Tick Cell Biobank will be set up in Malaysia, Kenya and Brazil to facilitate uptake and exploitation of cell lines and associated training by scientists in these and neighbouring countries. Thus the Tick Cell Biobank will continue to underpin many areas of global research into biology and control of ticks, other arthropods and vector-borne viral, bacterial and protozoan pathogens.