Focus-Forming Assay to Assess Zika Virus Vector Competence in Mosquitoes

Take clarified lysates of mosquito bodies and legs, and saliva collected from distinct mosquito populations exposed to the Zika virus.

Incubate the samples with mammalian host cells to facilitate virus entry.

Overlay with methylcellulose to limit viral spread. The virus replicates and infects adjacent cells, forming infection foci.

Remove the overlay and wash.

Add a solvent mix to fix and permeabilize the cells, then air-dry.

Wash under agitation. Incubate with a blocking solution under agitation to prevent non-specific binding.

Incubate with virus-specific primary antibodies under agitation. Wash, then incubate with peroxidase-conjugated secondary antibodies under agitation. Wash again.

Introduce a substrate and incubate. The substrate reacts with peroxidase to produce a colored product, labeling infection foci. Rinse and air-dry.

Detect infection foci to identify Zika virus-positive mosquito samples. Calculate the percentage of positive mosquitoes and plot this data for body, legs, and saliva to assess viral infection, dissemination, and transmission rates, respectively.

The population showing the virus in saliva indicates higher transmission competence.

Carefully aliquot 100 microliters o