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 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.

Mucosal Immune Response to Feline Enteric Coronavirus Infection.

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into naïve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFNγ responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.