While most flaviviruses are defined as arboviruses based on their requirement to infect both the arthropod vector and the vertebrate host, a number of studies on several continents have reported the presence of insect-specific flaviviruses (ISFs), which replicate in mosquito cells but do not grow in vertebrate cells. The recent discovery of Palm Creek virus (PCV) in Darwin (Northern Territory) provided initial evidence that insect-specific flaviviruses also circulate within Australia (Hobson-Peters et al., 2013).
The focus of this thesis was to investigate the presence and diversity of insect-specific flaviviruses in mosquito populations in other locations of northern Australia, and their relationship with other flaviviruses. Novel broad-spectrum viral detection reagents (monoclonal antibodies) were utilized to detect viral isolates in inoculated cell cultures and compared to optimized RT-PCR protocols to detect flaviviral RNA directly in mosquito homogenates to determine the most efficient approach. The former was subsequently undertaken as a screening assay to identify viral-infected pools and the latter used to identify those cultures infected with novel ISFs. The study was conducted on three archival collections of mosquitoes from the Gulf in 2001 and from Kimberley in 2010. One hundred samples (pools) from each collection were chosen with a focus on Cq. xanthogaster (in which PCV was discovered), Ae. normanesis (a species in which novel ISF-like sequences were previously detected but no isolate obtained) and Cx. annulirostris, the most common mosquito species of Northern Australia, and a genus well known for harboring ISFs on other continents.
PCV-like viruses with up to 5% nucleotide sequence difference from the prototype PCV isolate were detected in 2 pools of Cq. xanthogaster collected from Kununurra (Kimberley) in 2010. This result was consistent with previous studies conducted in Darwin, where several isolates of PCV were obtained from this species, collected contemporaneously. While the infection rate in Cq. xanthogaster was 6% (2/33 pools) for Kununurra, a much higher rate was observed for this species in Darwin (33% - 6/18 pools). The isolation of a single PCV isolate from a pool of Cx. annulirostris mosquitoes from Kununurra was less expected. However a failure to re-isolate the virus from this species suggested it may have been due to a trace contamination of virus from mosquito parts (wings, legs etc) from infected Cq. xanthogaster mosquitoes collected in the same trap.
From the Gulf samples, five isolates of a novel ISF were obtained from Anopheles meraukensis mosquitoes collected near Karumba. This is the first isolation of an ISF from the genus Anopheles. When partial NS5 sequences of these new isolates were analysed, they were identical to one another and formed a separate cluster of ISFs, most closely related to PCV, with 71% nucleotide sequence identity, 63% amino acid sequence identity. The five isolates of the novel ISF from the Gulf were obtained from only 8 pools of An. meraukensis tested, suggesting a very high prevalence (>50%) in this species.
The isolation of two species of ISFs, at two different time points (ie. 2001 and 2010), from three different locations in northern Australia (Darwin, Kununurra and Karumba), with infection rates of over 50% in some mosquito species suggest that these viruses have a high prevalence in some populations of mosquitoes. Considering only one hundred pools of mosquitoes were tested from each location in this study, testing a larger sample size from a wider range of mosquito species in additional locations in Australia will likely reveal a high degree of genetic diversity and geographical range amongst ISFs on the continent.