Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines

Abstract

As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

Fig. 1

St Croix River virus segment 2 VP2 gene sequence alignment (ClustalW, using default parameters). The isolates from the 4 tick cell lines IDE2, IDE8, RA243, and RA257 show 98–99% similarity with the published sequence (GenBank accession number NC_005998).

Fig. 2

Alignment of sequences (ClustalW, using default parameters) from putative nairoviruses in tick cell lines BDE/CTVM12, 14 and 16 and HAE/CTVM8 and 9 with published sequences of the nairoviruses CCHF (CCHF BT958, EF123122), Dugbe (DUGV_NJT130, FJ422213), Ganjam (G619, AF504294), Hazara (JC280, M86624), Kupe (K611, EU257626), and Nairobi sheep disease (NSDV strain 62873, HQ286609).

Fig. 3

Transmission electron micrographs of 4 tick cell lines showing endogenous viruses (arrows). A (scale bar 1 μm) and B (scale bar 0.2 μm): Amblyomma variegatum cell line AVL/CTVM13; C (scale bar 5 μm) and D (scale bar 100 nm): Rhipicephalus (Boophilus) microplus cell line BDE/CTVM23; E (scale bar 2 μm) and F (scale bar 100 nm): Dermacentor albipictus cell line DALBE3; G (scale bar 1 μm) and H (scale bar 0.5 μm): Dermacentor variabilis cell line DVE1.