Highlights • There are no approved antiviral drugs or recommended vaccines for feline coronavirus infection. • Plate-based image cytometry is used for high-throughput viral microneutralization assays. • Image cytometry is faster and more sensitive than traditional plaque reduction neutralization tests. • Cell seeding density, plate surface coating, virus concentration and incubation time, fluorescent labeling, and buffers were optimized. • Cross-neutralization between FCoV type I and II viruses was not observed. 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 and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, 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. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2–3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the 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.
【저자키워드】 high-throughput screening (HTS), viral titer, Feline coronavirus (FCoV), Plaque-Reduction neutralization tests (PRNT), Image cytometry, Celigo, 【초록키워드】 antibodies, Coronavirus infection, Vaccine, coronavirus, immune response, Vaccines, Variation, Infection, Antiviral compounds, virus, antiviral drug, Protein, sensitivity, Asymptomatic, therapeutic, death, vaccine candidate, plasma, viral neutralization, antigenicity, quantification, PRNT, microneutralization, Concentration, Image, Cytometry, serotype, type I, treat, clinical conditions, fluorescent, viral burden, viral infectious disease, plaques, fatal disease, buffer, labeling, plaque reduction neutralization, Effects, populations, Prevent, Cell, cytometer, robust, performed, was used, assays, addition, generate, approved, characterized, provide, majority, absence, faster, reducing, virus-infected cell, mutate, accelerate, time-consuming, automatically, cytolytic, immune correlate, wash buffer, 【제목키워드】 coronavirus, microneutralization, Cytometry,