An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection
Transcription
Take a microplate with serial dilutions of a serum sample containing antibodies against the Zika virus.
Add Dengue reporter virus particles or RVPs, and incubate.
The engineered RVPs comprise an envelope with structural proteins and a nucleocapsid containing genomic RNA, encoding a fluorescent protein.
The structural homology of Dengue and Zika viruses' envelope proteins causes the antibodies to cross-react with the RVPs, forming a complex.
Add human leukemia cells impermissible to infection by RVPs.
The cells possess Fc receptors that recognize the RVP-bound antibodies, facilitating RVPs' entry inside endosomes — termed antibody-dependent enhancement or ADE.
Pellet the cells and discard the supernatant containing non-internalized RVPs. Add a growth media and incubate.
The pH-dependent fusion of RVP and endosomal membranes releases the nucleocapsid, which disassembles to release genomic RNA, enabling fluorescent protein expression.
Fix the cells and analyze them using flow cytometry.
A higher fluorescence at a suitable antibody concentration represents increased RVP internalization, indicating enhanced infection.
