High-throughput viral microneutralization method for feline coronavirus using image cytometry

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations.

In general, to assess immune responses and screen for therapeutic and vaccine candidates, the plaque reduction neutralization test (PRNT) is conducted. However, this assay is time-consuming, low-throughput, and typically requires 2 – 3 days for the formation and manual counting of cytolytic plaques.  Operator variation can generate uncertainty in the results and digital records are not automatically created. Furthermore, the host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay.

To address these issues, we developed a novel high-throughput viral microneutralization assay using a plate-based image cytometer to quantify virus-infected cells. We utilized the developed microneutralization assay to screen for immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that image cytometry-based high-throughput viral microneutralization assay provides a robust and efficient method for rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.