Alphavirus and flavivirus infection of Ixodes tick cell lines: an insight into tick antiviral immunity
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Arthropod-borne viruses, arboviruses, have the ability to replicate in both vertebrates and invertebrates and are transmitted to susceptible vertebrate hosts by vectors such as mosquitoes and ticks. Ticks are important vectors of many highly pathogenic arboviruses, including the flavivirus tick-borne encephalitis virus (TBEV) and the nairovirus Crimean-Congo haemorrhagic fever virus. In contrast, alphaviruses are principally mosquito-borne and have been isolated only rarely from ticks; ticks have not been implicated as their vectors. Nevertheless, the alphavirus Semliki Forest virus (SFV) replicates in cell lines derived from many different tick species, including those of the genus Ixodes, which includes vectors of TBEV and its lesspathogenic relative Langat virus (LGTV). In vertebrate cells, arboviruses generally cause cytopathic effects; however, arbovirus infection of arthropod cells usually results in a persistent low-level infection without cell death. While little is known about antiviral immunity in tick cells, the immune system of other arbovirus vectors such as mosquitoes has been studied extensively over the last decade. In insects, pathways such as RNA interference (RNAi), JAK/STAT, Toll, Imd and melanisation have been implicated in controlling arbovirus infection, with RNAi being considered the most important antiviral mechanism. In tick cells, RNAi has been shown to have an antiviral effect, but current knowledge of other immunity pathways is limited and none have been implicated in the antiviral response. In the present study, SFV and LGTV replication in selected Ixodes spp. tick cell lines was characterised and the Ixodes scapularis-derived cell line IDE8 was identified as a suitable cell line for this project. Potential antiviral innate immunity pathways were investigated; putative components of the tick JAK/STAT, Toll and Imd pathways were identified by BLAST search using available sequences from well-studied arthropods including the fruit fly Drosophila melanogaster. Using gene silencing, an attempt was made to determine whether these pathways play a role in controlling SFV and LGTV infection in tick cell lines. Selected genes were silenced in IDE8 cells using long target-specific dsRNA and cells were subsequently infected with either SFV or LGTV. Effects of gene silencing on virus replication were assessed by quantitative real time PCR (qPCR) or luciferase reporter assay. Effects on infectious virus production were measured by plaque assay. Replication of the orbivirus St Croix River virus (SCRV), which chronically infects IDE8 cells, was also quantified by qPCR after silencing of selected genes. Interestingly, SFV or LGTV infection of IDE8 cells resulted in a significant increase in SCRV replication, possibly as a result of interference with antiviral pathways by SFV and LGTV or possibly due to diversion of cellular responses from sole control of SCRV. No evidence for an antiviral role for the JAK/STAT or Toll pathways was found in IDE8 cells. However, an antiviral effect was observed for protein orthologues putatively involved in the RNAi response. Argonaute proteins play an important role in translation inhibition and target degradation mediated by RNAi, and silencing of selected Argonaute proteins resulted in a significant increase in SFV and SCRV replication. The carboxypeptidase CG4572 is essential for an efficient antiviral response in D. melanogaster, and supposedly involved in the systemic RNAi response. A putative tick orthologue of CG4572 was identified and this appeared to be involved in the antiviral response in IDE8 tick cells. When expression of CG4572 was silenced and cells subsequently infected with SFV or LGTV, replication of both viruses was significantly increased. In addition, it was shown that three mosquito orthologues of CG4572 also had an antiviral role against SFV in Aedes mosquito cells. In conclusion, of the tick cell lines investigated, IDE8 provided a suitable model system for investigating tick cell responses against arboviruses and new insight into the nature of the tick cell antiviral response was gained.