Virus-Derived DNA Forms Mediate the Persistent Infection of Tick Cells by Hazara Virus and Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF virus (CCHFV), a biosafety level (BSL)-4 pathogen. Ticks of the genus Hyalomma are the viral reservoir, and they represent the main vector transmitting the virus to its hosts during blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma-derived tick cell lines. However, the mechanism allowing the establishment of persistent viral infections in ticks is still unknown. Hazara virus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study virus/vector interactions. To investigate the mechanism behind the establishment of a persistent infection, we developed an in vitro model with Hyalomma-derived tick cell lines and HAZV. As expected, HAZV, like CCHFV, persistently infects tick cells without any sign of cytopathic effect, and the infected cells can be cultured for more than 3 years. Most interestingly, we demonstrated the presence of short viral-derived DNA forms (vDNAs) after HAZV infection. Furthermore, we demonstrated that the antiretroviral drug azidothymine triphosphate could inhibit the production of vDNAs, suggesting that vDNAs are produced by an endogenous retrotranscriptase activity in tick cells. Moreover, we collected evidence that vDNAs are continuously synthesized, thereby downregulating viral replication to promote cell survival. Finally, vDNAs were also detected in CCHFV-infected tick cells. In conclusion, vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks allowing their persistent infection. IMPORTANCE Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by CCHF virus (CCHFV). Ticks of the genus Hyalomma can be persistently infected with CCHFV representing the viral reservoir, and the main vector for viral transmission. Here we showed that tick cells infected with Hazara virus, a nonpathogenic model virus closely related to CCHFV, contained short viral-derived DNA forms (vDNAs) produced by endogenous retrotranscriptase activity. vDNAs are transitory molecules requiring viral RNA replication for their continuous synthesis. Interestingly, vDNA synthesis seemed to be correlated with downregulation of viral replication and promotion of tick cell viability. We also detected vDNAs in CCHFV-infected tick cells suggesting that they could represent a key element in the cell response to nairovirus infection and might represent a more general mechanism of innate immunity against RNA viral infection.

N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells.

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.

Evidence of the role of tick subolesin in gene expression.

BACKGROUND: Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression. RESULTS: Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites. CONCLUSION: Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks.

Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks.

We undertook a comparative study of the susceptibility of different tick cell lines to infection with the European subtype of tick-borne encephalitis virus (TBEV), prototype strain Neudoerfl. The growth of TBEV was investigated in lines derived from vector Ixodes ricinus L. ticks (IRE/CTVM18, 19, and 20), as well as non-vector ticks, namely Ixodes scapularis Say (IDE2), Boophilus microplus Canestrini (BME/CTVM2), Hyalomma anatolicum anatolicum Koch (HAE/CTVM9), Rhipicephalus appendiculatus Neumann (RA-257) and recently established and herein described lines from the argasid tick Ornithodoros moubata Murray (OME/CTVM21 and 22). All the tick cell lines tested were susceptible to infection by TBEV and the virus caused productive infection without any cytopathic effect. However, there was a clear difference between the TBEV growth in vector and non-vector cell lines, since I. ricinus cell lines produced 100-1000-fold higher virus yield than the non-vector cell lines. The lowest virus production was observed in O. moubata and R. appendiculatus cell lines.

The Tick Cell Biobank: A global resource for in vitro research on ticks, other arthropods and the pathogens they transmit.

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.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines.

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

Vaccinomics Approach to Tick Vaccine Development.

Ticks are blood-feeding arthropod ectoparasites that transmit disease-causing pathogens to humans and animals worldwide. Vaccines using tick antigens have proven to be cost-effective and environmental friendly for the control of vector infestations and pathogen infection and transmission. However, new strategies are needed to identify tick protective antigens for development of improved vaccines. These strategies will be greatly enhanced by vaccinomics approaches starting from the study of tick-host-pathogen molecular interactions and ending in the characterization and validation of vaccine formulations. The discovery of tick antigens that affect both tick infestations and pathogen infection/transmission could be used for vaccines targeting human and animal populations at risk and reservoir species to reduce host exposure to ticks while reducing the number of infected ticks and their vector capacity for pathogens that affect human and animal health. In this chapter, we describe methods of the vaccinomics platform using transcriptomics and proteomics for the identification of candidate protective antigens in Ixodes scapularis, the vector for human and animal granulocytic anaplasmosis, tick-borne encephalitis, and Lyme disease.

Transcriptional analysis of the major antigenic protein 1 multigene family of Cowdria ruminantium.

The major antigenic protein 1 (MAP1) of the tick-borne rickettsial pathogen Cowdria ruminantium is encoded by a multigene family containing conserved and variable genes. The part of a locus containing the map1 multigene family that was characterized contained three homologous, but non-identical map1 genes, designated map1-2, map1-1, and map1. Reverse transcriptase-polymerase chain reaction was used to study the transcriptional activity of these genes in isolates of C. ruminantium grown in bovine endothelial cells, in two different tick cell lines, and in Amblyomma variegatum ticks. The map1 gene was always transcribed, whereas transcription of map1-2 was not detected under any of the tested conditions. The map1-1 gene transcript was detected in A. variegatum ticks, but was not found in virulent C. ruminantium Senegal grown in bovine endothelial cells at 30 or 37 degrees C. Interestingly, transcripts of map1-1 were also found in different passages of the in vitro attenuated Senegal isolate grown in bovine endothelial cells, as well as in the Gardel isolate grown in two tick cell lines. When transcribed, map1-1 was present on a polycistronic messenger together with map1.

Cell membrane receptors and regulation of cell function in ticks and blood-sucking insects.

Immunoglobulins cross the midgut epithelium and enter the haemolymph of many blood-feeding arthropods without losing their immunological properties. Antigens essential to the survival of the blood-sucking arthropods which may be affected by the small amounts of specific antibody that cross the gut epithelium include membrane receptors or other factors which regulate cell function. Membrane receptors implicated in transmembrane signalling in response to specific neural and endocrine factors fall into three major classes: (1) gated ion channels, (2) agonist-stimulated tyrosine kinases and (3) receptors that interact with GTP-binding (G) proteins. Examples of all three types have been found in insects and ticks. A dopamine receptor interacts with a G-protein essential for controlling fluid secretion by the salivary glands of ixodid ticks. Another receptor in the ixodid tick salivary gland binds a neuropeptide from the tick synganglion and stimulates turnover of plasma membrane phosphoinositides, but its mechanisms of transmembrane signalling and function remain elusive. Another large class of membrane receptors are those concerned with endocytosis. Examples of receptor-mediated endocytosis include incorporation of vitellogenin by developing oocytes in mosquitoes and ticks and uptake of lysed blood-meal components by digest cells of the tick gut. Many cell membrane receptors and possibly hormones could serve as targets for vaccines in blood-feeding insects and ticks. The major challenge is to identify and characterize essential internal receptors and cellular components that are accessible to and affected by specific antibodies that are introduced into the body of blood-feeding arthropods.

Antigens identified by monoclonal antibodies in tissue sections of Boophilus microplus.

A panel of monoclonal antibodies (mAbs) raised against midgut antigens of Boophilus microplus were used to probe various stages and organs of the tick. One of the monoclonal antibodies in this panel (QU13) has previously been shown to recognize protective antigens. Of the 18 mAbs tested, all except two (QU5 and QU12) reacted with sections of adult midgut and Malpighian tubules using an avidin-biotin alkaline phosphatase method for immunostaining. MAbs QU1, QU2, QU3, QU4, QU12, QU13, and QU18 reacted specifically with the lumenal surfaces of type III acini of the salivary gland. These seven mAbs also stained the midgut in larval sections indicating that the antigens recognized were not stage specific. However, none of the seven mAbs tested recognised antigens in either the adult ovary or the developing egg. Antigens which were immunogold labelled by mAbs QU1, QU4, QU11, QU13, and QU15 in electron microscopy were located either on or near the surface of the microvilli of digestive cells from the midgut of the adult tick. We conclude that common antigens are present on the lumenal surfaces of the adult midgut, type III acini of the salivary gland, and the Malpighian tubules and that these antigens are also located in the larval gut.

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells.

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.

Rickettsia rickettsii growth and temperature-inducible protein expression in embryonic tick cell lines.

Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.

An immunocytochemical marker for the complex granules of tick salivary glands which traces e-granule shedding to interstitial labyrinthine spaces.

An antigenic protein (12C), previously isolated from salivary glands of Rhipicephalus appendiculatus, as a possible factor in host resistance to repeated tick infestation, is labeled by protein A-gold immunocytochemistry in adult feeding ticks. It is a component of the gland's complex a-, d- and e-granules and also appears within the chitinous walls of intercalated ducts. In females, between days 4 and 7 of feeding, labeled e-granules appear also within the labyrinthine spaces of acinus type III, apparently released from e-cells as the abluminal interstitial cells initiate formation of a basolateral labyrinth. Granules thus shed are fragmented by interstitial cell processes, some fragments being phagocytized, others disintegrating to single point label scattered throughout the labyrinth. The latter possibly may pass into the acinar lumen. By the eighth day the label is gone from the labyrinth.

Continuous cell lines from the tick Hyalomma anatolicum anatolicum.

The establishment of 5 continuous cell lines from embryonic tissues of the tick Hyalomma anatolicum anatolicum is reported. Each line comprises 2 or more cell types; they are maintained at 28 C and 32 C in L-15/H-Lac medium with 20% fetal calf serum, and have been cryopreserved successfully. Sustained and consistent growth was achieved only after 12-41 mo in culture.

Establishment and characterization of a diploid cell line from the tick, Dermacentor parumapertus Neumann (Acarina: Ixodidae).

Establishment of a continuous cell line (RML-14) from embryonic tissues of the tick Dermacentor parumapertus Neumann is reported. The culture medium employed consisted of a combination (2:1) of Eagle's and L-15 (Leibovitz) media supplemented with 20% fetal bovine serum, 10% tryptose phosphate broth, and 0.1% bovine plasma albumin. At the 8th passage, 99% of dividing cells had the female chromosome complement, among which more than 70% had a diploid chromosome number of 22. At the 13th passage, cell population showed approximately a 3-fold increase during the first 8 days of culture. As of December 1976, had been subcultured 40 times.

Effect of medium supplements on tick cells in culture.

The growth of tick cells in Leibovitz's L--15 medium supplemented with various concentrations of fetal bovine serum (FBS), tryptose phosphate broth (TPB), and tick egg extract (TEE) was evaluated using a protein assay. A continuous cell line from Rhipicephalus appendiculatus (RA 243) was compared with young lines of cells isolated from embryos of R. appendiculatus (RAE 25) and Rhipicephalus sanguineus (RSE 8). We found fetal bovine serum and tryptose phosphate broth both to be essential supplements. The addition of tick egg extract further stimulated growth. The yield of cellular protein in both young and continuous lines of tick cells increased as a function of the concentration of tryptose phosphate broth from 0 to 10%, and fetal bovine serum from 2.5 to 20%. The growth of the RA 243 line correlated negatively with the size of the inoculum and positively with the concentration of fetal bovine serum, as the greatest increase in cell protein was obtained when cells were seeded at a low density into a medium containing 20% fetal bovine serum. The addition of an extract prepared from eggs of R. sanguineus or Hyalomma excavatum improved yields of cultures and promoted cell growth at low population densities. The protein yield increased as a function of tick egg extract concentration, but 0.8% inhibited growth of the RA 243 line. The RA 243 line could be propagated in a medium supplemented with 10% tryptose phosphate broth, 5% fetal bovine serum, and 0.5% tick egg extract.

A new observation on spermateleosis in ticks.

The first phase of spermiogenesis in argasid and ixodid ticks is described. The inner core of the spermatid passes through the outer sheath so that speriogenesis is permitted to follow its course. The outer sheath is not ruptured in the process, but a well-defined operculum opens to let the inner core through. The opening of the operculum is a clear-cut indication of the beginning of spermiogenesis. In the examined species speriogenesis begins in Argasidae when the male transfers the spermatophore to the female genital opening, whereas in Ixodidae it starts only after the endospermatophore has reached the female genital tract.

Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.

Interest in tick-borne pathogens has been enhanced by the emergence of Lyme disease and, more recently, human and animal ehrlichioses. In order to facilitate investigations of the vector phase of tick-borne disease agents in vitro, several new cell lines derived from embryonated eggs of northern (IDE lines) and southern (ISE lines) populations of the tick Ixodes scapularis were developed. The establishment and characteristics of 4 IDE (IDE1, 2, 8, and 12) and 2 ISE (ISE5 and 18) lines were described. Primary cultures were initiated in L-15B medium at 31 C from a single egg mass each and established lines developed a morphologically distinct phenotype. Myoblasts were present during the first year after isolation in several lines as isolated clusters or sheets covering the whole flask. Cell line extracts resolved by isoelectric focusing were characterized for 3 isozymes (lactate dehydrogenase, malate dehydrogenase, and malic enzyme). The combined banding patterns allowed discrimination between Ixodes cell lines and a Rhipicephalus appendiculatus cell line. Two lines, i.e., ISE5 and ISE18, had unique isozyme bands. Chromosome numbers and morphology conformed to those described from tissue squashes of I. scapularis.

Spermatogenesis in the Ixodid tick Haemaphysalis (Herpetobia) sulcata (Acarina:Ixodidae).

Haemaphysalis sulcata (Acarina, Ixodidae) is a common ectoparasite of wild artiodactyls (mouflon and wild goat) in Sierra Nevada (Granada, southern Spain). A study of the spermatogonial meiosis of 15 H. sulcata males was carried out. The diploid complement is 2n = 21 and its sex determination is XX:XO. The behavior of the chromosomes in the different stages of meiosis was also investigated, and the possible presence of a secondary nucleolar organizer region in H. sulcata is discussed.