Impedance-Based Cellular Assay to Measure Multiplicity of Infection of Influenza Virus
DEŞİFRE METNİ
During influenza virus infection, the virus enters the cell through endocytosis and hijacks the host cell machinery, producing progeny virions. These get released, infecting neighboring cells, causing a cascade of infection.
To measure infectious influenza virus to cell ratio in culture — multiplicity of infection — add an appropriate density of freshly-obtained mammalian cell suspension into a microtiter plate.
The plate contains impedance-measuring gold microelectrodes embedded in the well bottom. Incubate, allowing cells to adhere to the well bottom and reach the exponential growth phase prior to infection.
Apply a small electrical potential between the microelectrodes. Cells adhered on the microelectrodes act as insulators, restricting current flow and increasing electrical resistance — impedance — which is measured.
Remove the media. Pipette the influenza virus suspension into the wells. Supplement with a suitable serine endoprotease.
During incubation, the serine endoprotease cleaves and activates the viral glycoprotein hemagglutinin, facilitating its binding to specific host cell receptors and subsequent virus cell entry.
Using host cell machinery, new viral particles are produced and released, infecting neighboring cells. As a result, the virus-infected cells undergo morphological changes and display cytopathic effects, leading to cell death — cells detach from the microelectrode surface, decreasing the impedance over time.
Determine the time taken for the impedance to reduce to half the initial impedance before virus addition. A shorter time frame suggests a higher multiplicity of viral infection.
