Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells.

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

An Ixodes scapularis cell line with a predominantly neuron-like phenotype.

The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 ß-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection.

BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

Expression of heat shock proteins and subolesin affects stress responses, Anaplasma phagocytophilum infection and questing behaviour in the tick, Ixodes scapularis.

We characterized the effects of subolesin and heat shock protein (HSP) expression on Ixodes scapularis Say (Acari: Ixodidae) stress responses to heat shock and feeding, questing behaviour and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) infection. Ticks and cultured tick cells were analysed before and after subolesin, hsp20 and hsp70 gene knock-down by RNA interference. The results of these studies confirm that HSPs are involved in the tick cell response to heat stress and that subolesin and HSPs are both involved in the tick response to blood-feeding stress and A. phagocytophilum infection. Subolesin and hsp20 are involved in the tick protective response to A. phagocytophilum infection and hsp70 expression may be manipulated by the pathogen to increase infectivity. Importantly, these results demonstrate that subolesin, hsp20 and hsp70 expression also affect tick questing behaviour. Overall, this research demonstrates a relationship between hsp and subolesin expression and tick stress responses to heat shock and blood feeding, A. phagocytophilum infection and questing behaviour, thereby extending our understanding of the tick-host-pathogen interface.

An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat virus infection of a tick cell line.

Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection. ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene-specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay. IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV. Based on the results, GSTs are possibly utilized both by cells and the virus for mutual survival and proliferation.

Confirmation of occurrence of Ixodes (Pholeoixodes) rugicollis Schulze et Schlottke, 1929 (Acari: Ixodidae) in Poland, including the morphological description and diagnostic features of this species.

Research on feline and canine ticks was conducted in the years 2004-2005 in the vicinities of Przemysl, in the village of Torki (Medyka region) and in Przemysl. Ticks were collected by examining thoroughly the bodies of cats and dogs. Ticks were preserved in 70% ethyl alcohol and their taxonomic identity was determined. It was confirmed that dogs and cats in the study sites carried four species of ticks: Ixodes (Ph.) rugicollis, I. (Ph.) hexagonus, I. (Ph.) crenulatus and I. (I.) ricinus. Ixodes (Ph.) rugicollis was shown to be a common species among the ectoparasites on these hosts and therefore is considered now to be a regular component of the Polish fauna. Morphological characteristics of all the development stages of I. rugicollis are given in the paper.

Tick-Borne Encephalitis Virus Adaptation in Different Host Environments and Existence of Quasispecies.

A highly virulent strain (Hypr) of tick-borne encephalitis virus (TBEV) was serially subcultured in the mammalian porcine kidney stable (PS) and Ixodes ricinus tick (IRE/CTVM19) cell lines, producing three viral variants. These variants exhibited distinct plaque sizes and virulence in a mouse model. Comparing the full-genome sequences of all variants, several nucleotide changes were identified in different genomic regions. Furthermore, different sequential variants were revealed to co-exist within one sample as quasispecies. Interestingly, the above-mentioned nucleotide changes found within the whole genome sequences of the new variants were present alongside the nucleotide sequence of the parental strain, which was represented as a minority quasispecies. These observations further imply that TBEV exists as a heterogeneous population that contains virus variants pre-adapted to reproduction in different environments, probably enabling virus survival in ticks and mammals.

Babesia bigemina: in vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells.

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.

Characterization of an iron-inducible Haemaphysalis longicornis tick-derived promoter in an Ixodes scapularis-derived tick cell line (ISE6).

BACKGROUND: Ticks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick. RESULTS: Here, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter's activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate. CONCLUSION: In this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.

Tissue-specific signatures in tick cell line MS profiles.

BACKGROUND: The availability of tick in vitro cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions. RESULTS: Three cell lines obtained from a hard tick, Ixodes ricinus, and two from I. scapularis were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and I. ricinus tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines. CONCLUSIONS: Several organ-specific MS signals were revealed in the profiles of tick cell lines.

Mutational analysis of gene function in the Anaplasmataceae: Challenges and perspectives.

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.

Anaplasma phagocytophilum modifies tick cell microRNA expression and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 pathway.

The microRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms including innate and adaptive immune pathways to control bacterial, parasite and viral infections, and pathogens could modify host miRNA profile to facilitate infection and multiplication. Therefore, understanding the function of host miRNAs in response to pathogen infection is relevant to characterize host-pathogen molecular interactions and to provide new targets for effective new interventions for the control infectious diseases. The objective of this study was to characterize the dynamics and functional significance of the miRNA response of the tick vector Ixodes scapularis in response to Anaplasma phagocytophilum infection, the causative agent of human and animal granulocytic anaplasmosis. To address this objective, the composition of tick miRNAs, functional annotation, and expression profiling was characterized using high throughout RNA sequencing in uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cells, a model for tick hemocytes involved in pathogen infection. The results provided new evidences on the role of tick miRNA during pathogen infection, and showed that A. phagocytophilum modifies I. scapularis tick cell miRNA profile and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 (Robo2) pathway. Furthermore, these results suggested new targets for interventions to control pathogen infection in ticks.

Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line.

BACKGROUND: Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 µM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment. RESULTS: Cell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 µM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 µM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 µM (P < 0.01). CONCLUSIONS: The adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Anaplasma phagocytophilum p44 mRNA expression is differentially regulated in mammalian and tick host cells: involvement of the DNA binding protein ApxR.

The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37 degrees C than in A. phagocytophilum-infected HL-60 cells cultured at 28 degrees C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.

Isolation and establishment of the raccoon Ehrlichia-like agent in tick cell culture.

Feral animals are reservoirs of emerging human pathogens, as well as carriers of closely related wildlife diseases. The latter may interfere with epidemiologic studies by inducing cross-reactive antibodies, or by providing false positive signals in PCR based tests. We cultured a novel intracellular bacterium from the blood of two raccoons (Procyon lotor): RAC413 and RAC414. RAC413 had been experimentally inoculated with blood from a wild-caught raccoon, and provided the material for a blood passage into RAC414. The microbes grew in Ixodes scapularis (black-legged tick) cells, line ISE6, inoculated either with the leukocyte or erythrocyte fraction of anticoagulated blood. Giemsa-stained cells sampled two and three months after initial inoculation of the cultures revealed inclusions similar to those of Ehrlichia sp., except that individual bacteria commonly were elongated and clustered within endosomes. Electronmicroscopy confirmed the presence of irregularly shaped bacteria with evenly granular bacterioplasm bounded by a unit membrane. 16S rDNA sequencing identified the microbes as the raccoon Ehrlichia-like agent previously detected in feral raccoons from Georgia, United States. In conclusion, the availability of a culture isolate of this agent will facilitate future studies to determine its biology, epidemiologic significance, vector association, and host range. The Ehrlichia-like agent infecting raccoons joins a growing list of tick-borne agents cultivable in tick cells.

Host range specificity of flaviviruses: correlation with in vitro replication.

Vector-borne flaviviruses have been traditionally grouped into either mosquito-borne or tick-borne group. However, this vector range specificity has sometimes been questioned because of the puzzling records of occasional isolation of mosquito-borne viruses from ticks and of tick-borne viruses from mosquitoes. In this study, host range of the flaviviruses representing not only the two vector-borne groups but also insect flaviviruses and vertebrate viruses that are not arboviruses was comprehensively reexamined by a serial passage experiment in vitro by using cell cultures derived from mosquitoes, ticks, and vertebrates. The results showed that the host range specificity in the four groups of viruses, based on replication for five consecutive passages as a criterion to evaluate the ability of viruses to replicate in three different cell cultures, agreed with the conventional grouping as well as phylogenetic clustering. Thus, this assay provides useful, supplementary information regarding host range for those flaviviruses when their natural host range is unknown, ambiguous, or questionable.

Phagocytosis of the Lyme disease spirochete, Borrelia burgdorferi, by cells from the ticks, Ixodes scapularis and Dermacentor andersoni, infected with an endosymbiont, Rickettsia peacockii.

Tick cell lines were used to model the effects of endosymbiont infection on phagocytic immune responses. The lines tested for their ability to phagocytose the Lyme disease spirochete, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), were ISE6 and IDE12 from the black-legged tick, Ixodes scapularis Say (Acari: Ixodidae) and DAE15 from the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of D. andersoni, was used as a representative tick endosymbiont. 70-80% of uninfected or R. peacocciz-infected IDE12 and DAE15 cells phagocytosed heat-killed borreliae and 80-90% of IDE12 and DAE15 cells phagocytosed viable spirochetes. ISE6 cells were permissive of spirochetes; less than 1% of these cells phagocytosed borreliae, and spirochetes remained adherent to the cells seven days after inoculation. Cytochalasin B blocked phagocytosis of killed and viable borreliae by IDE12 cells, and prevented phagocytosis of killed spirochetes by DAE15 cells, whereas viable spirochetes successfully invaded cytochalasin-treated DAE15. IDE12 and DAE15 cells degraded borreliae within phagolysosome-like compartments. Time-lapse microscopy showed that DAE15 cells phagocytosed borreliae more rapidly than IDE12 cells. IDE12 and DAE15 cells eliminated most adherent spirochetes within 7 days of inoculation. Thus, endosymbiont infection does not significantly interfere with the phagocytic activity of immunocompetent tick cells.

Targeting the tick/pathogen interface for developing new anaplasmosis vaccine strategies.

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.

Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line.

Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated I. scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of I. scapularis-associated virus-1, which was reported in a recent metagenomic study of I. scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks.

In vitro cultivation of a south African isolate of an Anaplasma sp. in tick cell cultures.

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08' E, 25 degrees 38' S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatienne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).