A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: A useful tool for inferring transmission pathways

The small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can be adapted for similar studies on other mosquito borne zoonoses.

A micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using two mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralising antibodies using the micro-PRNT.

There was significant agreement of the plaque neutralisation resulting from the micro-PRNT and standard PRNT techniques (R2 = 0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies, and on blood from RRV seropositive humans neutralised the virus by ≥50% until 48 hr post feeding. The vertebrate origin of the blood meal was also ascertained for the same samples, in parallel, using established molecular techniques. Conclusions: The small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can be adapted for similar studies on other mosquito borne zoonoses.