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