Melbourne Veterinary School - Research Publications
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ItemDetection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines(ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2012)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.
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ItemPhenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus(PUBLIC LIBRARY SCIENCE, 2012-11)Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.
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ItemPKR acts early in infection to suppress Semliki Forest virus production and strongly enhances the type I interferon response(MICROBIOLOGY SOC, 2009-06)The double-stranded RNA-activated protein kinase (PKR) is a key regulator of protein translation, interferon (IFN) expression and cell survival. Upon infection of vertebrate cells in continuous culture, the alphavirus Semliki Forest virus (SFV) initiates apoptosis and IFN synthesis. To determine the effect of PKR on SFV infection, we studied the course of infection in wild-type (wt) mice, mice with a genetic deletion of PKR (PKR-/-) and mouse embryo fibroblasts (MEFs) derived from these mice. In MEFs, PKR delayed virus protein synthesis, production of infectious virus and caspase-3-activated cell death and reduced the yield of infectious virus by 90%. Small interfering RNA suppression of PKR levels in NIH-3T3 cells also reduced virus production and apoptosis. In MEFs, PKR was not required for initiation of IFN-beta gene transcription, but contributed strongly to the magnitude of this response. Levels of IFN-beta transcripts in PKR-/- MEFs at 8 h were 80% lower than those in wt MEFs and levels of functional IFN at 24 h were 95% lower. Following infection of wt and PKR-/- mice, SFV4 and SFV A7(74) were avirulent. PKR increased levels of serum IFN and the rate of clearance of infectious virus from the brain. In summary, in response to SFV, PKR exerts an early antiviral effect that delays virus protein production and release of infectious virus and, whilst PKR is not required for induction of apoptosis or activation of the type I IFN response, it strongly augments the type I IFN response and contributes to clearance of infectious virus from the mouse brain.