Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines

Abstract

Investigating type I feline coronaviruses (FCoVs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. This has been a perennial challenge as type I FCoV strains do not easily adapt to cell culture. Here we characterize replication kinetics and plaque formation of a model type I strain FIPV Black in Fcwf-4 cells established at Cornell University (Fcwf-4 CU). We determined that maximum virus titers (>10⁷ pfu/mL) were recoverable from infected Fcwf-4 CU cell-free supernatant at 20 h post-infection. Type I FIPV Black and both biotypes of type II FCoV formed uniform and enumerable plaques on Fcwf-4 CU cells. Therefore, these cells were employable in a standardized plaque assay. Finally, we determined that the Fcwf-4 CU cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type I interferon than were Fcwf-4 cells purchased from ATCC.

Keywords: AK-D cells; FIPV; Fcwf-4 cells; Feline coronavirus; Feline macrophage-like cell line; Plaque assay.

Fig. 1

Fcwf-4 Cornell University (CU) cells produce high titers of cell-free type I FIPV with rapid growth kinetics. A) Virus growth kinetics measured from cell-free supernatants of AK-D (black), Fcwf-4 ATCC (blue), and Fcwf-4 CU (red) cells infected with FIPV Black (MOI=0.1) over 96 h. Arrows indicate time of peak titer presented as plaque-forming units (pfu)/mL. Titer determined by plaque assay on AK-D cells in triplicate; error bars ± SD. B) Cell-associated and cell-free virus titers were determined following infection of Fcwf-4 CU, AK-D, and Fcwf-4 ATCC cells with FIPV Black (MOI=0.1). Cell-free titer was determined from cell-clarified supernatants; cell-associated titer was determined from suspended cell monolayers following three freeze-thaw cycles alternating between −80 °C and 37 ^oC. Samples were taken at hours post-infection (hpi) just prior to, at, and following the maximum (max) virus titer for each cell type. Titers determined by plaque assay on AK-D cells in triplicate; error bars ± SD.

Fig. 2

FIPV Black-induced cell cytopathic effect varies between infected cell types. A) Progression of cell cytopathic effects (CPE) induced by FIPV Black over time in Fcwf-4 and AK-D cells (MOI=0.1). Localized cell syncytia formation (black, closed arrows) and discrete cell death (white, open arrows) indicated on brightfield images taken at 20x magnification. B) Immunofluorescence staining of FIPV Black-infected Fcwf-4 CU and AK-D cells (MOI=0.1) at indicated hours post-infection (hpi). Nucleocapsid protein staining (Red; CCV2-2) indicates infected cells; nuclei stained in blue (Hoesch3342). Images taken at 60x magnification.

Fig. 3

Evaluating FIPV Black plaque development and titer over time in different feline cell lines. Tenfold serial dilutions (10^-3-10^-6) of virus inoculum, derived from 24 h cell-free supernatant of Fcwf-4 CU cells infected with FIPV Black (MOI=0.1), were applied to AK-D, Fcwf-4 ATCC, and Fcwf-4 CU indicator cells. Following Oxoid agar/media overlay, plates were incubated for 2 (top), 3 (middle), or 4 (bottom) days before cells were fixed with 3.7% formaldehyde and stained with 0.1% crystal violet. Titers were calculated by plaque counts and are presented as plaque forming units (pfu)/mL. ND indicates that titers were not determined.

Fig. 4

Fcwf-4 CU cells differ in morphology and IFN-responsiveness from Fcwf-4 ATCC. A) Single-cell morphology of feline bone marrow-derived macrophages (fBMDMs), Fcwf-4 CU, and Fcwf-4 ATCC cells determined by Wright-Giemsa stain following cytospin preparation. Images were taken at 100 × . B) Interferon stimulated gene (ISG) 54 transcript levels from Fcwf-4 ATCC (grey) and Fcwf-4 CU (black) cell lines stimulated with increasing concentrations of feline type I interferon (IFNα). After 6 h, total RNA was extracted and analyzed by qPCR for ISG54 and feline beta-actin. ISG54 mRNA expression was normalized to β-actin via the delta Ct method (2^-ΔCt[ΔC_t=C_{t(gene of interest)}-C_t(β-actin)]), then presented as relative expression over corresponding mock (basal) expression. Data are representative of three independent experiments performed in triplicate and presented as means ± SD. Values were analyzed by unpaired t-test. * * P < 0.01; * ** P < 0.001; * ** * P < 0.0001.

Fig. 5

Fcwf-4 CU cells and are permissive to both biotypes of type II FCoV. A) Cell cytopathic effect at 12 and 24 h post-infection (hpi) of Fcwf-4 CU cells infected with indicated FCoV strains (MOI=0.1). B) FCoV growth kinetics in Fcwf-4 CU cells (MOI=0.1) determined by C) plaque assay on Fcwf-4 CU indicator cells. Tenfold serial dilutions of cell-free virus inoculum were applied to cells. Following Oxoid agar/media overlay, cells were fixed in 3.7% formaldehyde and stained using 0.1% crystal violet after 24 h. FCoV used were the type I strain FIPV Black and the type II strains FIPV WSU 79–1146 and FECV WSU 79–1683. Plaque assay performed in triplicate; error bars ± SD.