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

Abstract

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.

Keywords: Celigo; Feline coronavirus (FCoV); High-throughput screening (HTS); Image cytometry; Plaque-Reduction neutralization tests (PRNT); Viral titer.

Fig. 1

Bright field and fluorescent images, and fluorescence intensity gating of positive and negative infection of CRFK cells using optimized scanning and analysis settings from image cytometry.

Fig. 2

Host cell seeding density and incubation time optimization results. (a) Bright field and confluence measurement overlay (pseudo-color green) for seeding densities 3000 – 6000 cells/well at 48 and 72 h showing coverage of CRFK cells on the well surface. (b) Time- and seeding density-dependent confluence percentages measured using image cytometry (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3

Fluorescent labeling optimization results. (a) Fluorescent images of AF488-labeled infected CRFK cells showing green fluorescence for labeling with primary antibody at 1 and 2 μg/mL and secondary antibody at 2 and 4 μg/mL. (b) Mean AF488 fluorescence intensities measured using image cytometry (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4

Optimal assay buffer and surface coating selection results. Comparison of the CRFK (a) confluence percentages and (b) infection rates between DMEM and DPBS, as well as between Advanced TC, collagen type I, poly-d-lysine, and poly-l-lysine surface coating.

Fig. 5

Viral concentration optimization results. (a) Viral concentration-dependent fluorescent images and (b) infection rates measurement showing the reduction of AF488-positive infected CRFK cells as viral concentration decreased. Both media and supernatant control did not show viral infection.

Fig. 6

Viral neutralization results showing dose-dependent infection rates measurements for 5 feline plasma samples with a titration from 1:10 to 1:1280. The virus and plasma only conditions showed high and low infection rates as expected. No noticeable neutralization effects were observed.