Synthetic recovery of Yada Yada virus expands insect-specific alphavirus knowledge and facilitates production of chimeric viruses

In comparison to other arbovirus genera, few insect-specific alphaviruses have been discovered to date, with even fewer culturable to facilitate full characterisation. Using circular polymerase extension reaction (CPER), we report the recovery of an infectious clone of Yada Yada virus (YYV), which was initially detected by metagenomic sequencing of mosquito homogenates and is the first insect-specific alphavirus (ISA) discovered in the Asia-Pacific region. Using the infectious clone, we demonstrated that YYV did not replicate in a range of vertebrate cells and displayed mosquito genus-specific tropism by only replicating in Aedes -derived cell lines. The YYV infectious clone, in addition to one of Eilat virus (EILV; another ISA), generated by CPER and synthetic dsDNA fragments, facilitated the generation of the first monoclonal antibodies (mAbs) to ISAs. Through successful replacement of the structural proteins of YYV with those of three other ISAs, Agua Salud alphavirus (ASALV), Taï Forest alphavirus (TALV) and Mwinilunga alphavirus (MWAV), we established that a replication block for in vitro culture of TALV and MWAV in mosquito cells does not exist at virus entry. We also established that the majority of the anti-ISA mAbs were highly specific to YYV or EILV. Unexpectedly, ASALV structural proteins were recognized by cross-reactive mAbs made to chikungunya virus (CHIKV) and Ross River virus (RRV), suggesting a potential antigenic link between ASALV and pathogenic alphaviruses. To investigate YYV as a potential platform for producing vaccine and diagnostic antigens, YYV chimeras of pathogenic vertebrate-infecting alphaviruses (VIA) were made for CHIKV, RRV, Barmah Forest virus, Sindbis virus and Mayaro virus. These chimeras retained the antigenic properties of the parental VIAs and did not replicate in vertebrate cells.