Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses

Abstract

Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.

FIG. 1.

CCHFV virus titers in cell culture supernatant of 10 tick cell lines. At biosafety level 4, tick cells were infected with 4,000,000 genome equivalents (GEQ) equaling 4000 plaque-forming units (PFU) of CCHFV strain IbAr 10200 (kindly provided by Dr. Michael Holbrook UTMB, Galveston, TX) in 500 μL of L-15 (Leibovitz) medium. IbAr 10200 had a titer of 6.2×10⁸ GEQ per mL (4×10⁵ PFU/mL). After incubation for 60 min at 31°C, the cells were centrifuged, the virus inoculum was carefully removed, appropriate culture medium was added, and the cells were incubated at 31°C for 21 days. Supernatant was collected from three replicate tubes on days 2, 5, and 7 postinfection (pi) and 140 μL aliquots were mixed with 560 μL of AVL buffer (QIAamp Viral RNA mini kit; Qiagen). A CCHFV-specific quantitative real-time reverse transcription–polymerase chain reaction using a recombinant RNA standard was performed (Wolfel et al. 2007). Assays were run on StepOnePlus (Applied Biosystems) and analyzed with StepOne Software v2.1. Virus titers (n=3 for each time point; mean+standard deviation) are reported as GEQ. Virus levels in supernatant were below the detection limit of the assay (dashed line; 3×10² GEQ/mL) in cell lines OME/CTVM21, OME/CTVM22, and RAE/CTVM1. CCHFV, Crimean-Congo hemorrhagic fever virus.

FIG. 2.

Tick cell lines infected with constructs of the mosquito-borne alphavirus Semliki Forest virus (SFV) (Tamberg et al. 2007) incorporating enhanced green fluorescent protein (eGFP), which facilitates identification of infected cells. Left panel: Rhipicephalus (Boophilus) decoloratus cell line BDE/CTVM14 at 24 h after infection with SFV4(3F)-eGFP, in which the eGFP is inserted into the nonstructural protein ORF and therefore localizes to virus replication complexes. Right panel: Ixodes scapularis cell line IDE8 at 24 h after infection with SFV4st-eGFP, in which the eGFP is inserted into the structural protein ORF and therefore is produced extensively in the cytoplasm of infected cells. Photomicrographs taken on an Axio Observer inverted microscope (Zeiss) with concurrent bright-field and UV illumination. Scale bar=50 μm. (Color images available at www.liebertonline.com/vbz)