Australian isolates of cell fusing agent virus and Culex flavivirus provide new tools to study insect-specific flaviviruses

Cell fusing agent virus (CFAV) and Culex Flavivirus virus (CxFV) represent the prototype insect-specific flaviviruses (ISFs) found in Aedes and Culex species, respectively. While these viruses have been isolated from mosquitoes on several different continents, no isolates have been reported from Australian mosquitoes. In this study we successfully isolated CFAV from Aedes aegypti originating from Cairns in North Queensland, Australia and CxFV from Culex quinquefasciatus collected from Brisbane in South-East Queensland, representing the first field isolates for both viruses from Australia. Phylogenetic analysis of these isolates revealed that the new CFAV isolates were closely related to CFAV sequences previously detected by deep sequencing of pools of Aedes aegypti collected in North Queensland, while the genome sequences of the Brisbane CxFV isolates were most closely related to isolates of CxFV from Culex quinquefasciatus collected from Mexico in 2008. Using these Australian isolates, we report the first infectious clone created for CFAV, and the first infectious clone of the Australian strain of CxFV. Growth kinetics revealed that infectious clones of CFAV and CxFV replicated more efficiently than their wild-type counterparts, reaching titres more than 10 8 TCID 50 /ml. Deep-sequencing of these isolates indicated that differences in replication were due to co-infections of mosquito-derived native viruses with other viruses absent in infectious clone stocks. Using a panel of novel monoclonal antibodies raised against the CFAV virion proteins, we demonstrated antigenic relatedness between CFAV and CxFV and other lineage I ISFs, particularly those carrying the conserved FKW motif at positions 106–108 in the fusion loop region of the viral envelope protein. All generated mAbs bound CFAV with one mAb additionally recognising the ISFs CxFV, Palm Creek virus (PCV), Parramatta River virus (PaRV) and Niénokoué virus (NIEV). While these mAbs did not bind CxFV and CFAV proteins in mosquitoes injected with the infectious clones, the first immunofluorescence analysis of disseminated of CFAV in Aedes mosquitoes was visualised through anti-dsRNA staining, showing widespread infection across multiple mosquito tissues, including the midgut and ovaries. These advancements in the development of elementary tools provide opportunity to further our understanding of ISFs and pave the way for their potential application in laboratory research, as well as innovative mosquito-borne disease control strategies.