The role of accessory proteins in the replication of feline infectious peritonitis virus in peripheral blood monocytes

Abstract

The ability to productively infect monocytes/macrophages is the most important difference between the low virulent feline enteric coronavirus (FECV) and the lethal feline infectious peritonitis virus (FIPV). In vitro, the replication of FECV in peripheral blood monocytes always drops after 12h post inoculation, while FIPV sustains its replication in the monocytes from 45% of the cats. The accessory proteins of feline coronaviruses have been speculated to play a prominent role in virulence as deletions were found to be associated with attenuated viruses. Still, no functions have been ascribed to them. In order to investigate if the accessory proteins of FIPV are important for sustaining its replication in monocytes, replication kinetics were determined for FIPV 79-1146 and its deletion mutants, lacking either accessory protein open reading frame 3abc (FIPV-Δ3), 7ab (FIPV-Δ7) or both (FIPV-Δ3Δ7). Results showed that the deletion mutants FIPV-Δ7 and FIPV-Δ3Δ7 could not maintain their replication, which was in sharp contrast to wt-FIPV. FIPV-Δ3 could still sustain its replication, but the percentage of infected monocytes was always lower compared to wt-FIPV. In conclusion, this study showed that ORF7 is crucial for FIPV replication in monocytes/macrophages, giving an explanation for its importance in vivo, its role in the development of FIP and its conservation in field strains. The effect of an ORF3 deletion was less pronounced, indicating only a supportive role of ORF3 encoded proteins during the infection of the in vivo target cell by FIPVs.

Fig. 1

Kinetics of percentage of infected cells in FCoV-inoculated fcwf cells. Cells were inoculated with FIPV 79-1146, FIPV-Δ3, FIPV-Δ7 and FIPV-Δ3Δ7 at a m.o.i. of 0.02. At different time points post inoculation, cells were fixed and cytoplasmic nucleocapsid protein was visualized with an immunofluorescence staining. The data represent means ± SD of five replicate assays. Significant difference with FIPV 79-1146 is indicated with *p < 0.1; **p < 0.05; or ***p < 0.01.

Fig. 2

Confocal microscopy images of cytoplasmic expression of the nucleocapsid proteins (green) in fcwf cells 24 h after inoculation with FIPV 79-1146, FIPV-Δ3, FIPV-Δ7 and FIPV-Δ3Δ7 at a m.o.i. of 0.02. Nuclei were visualized with Hoechst 33342 (blue). Bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Kinetics of FCoV replication in fcwf cells. Cells were inoculated with FIPV 79-1146, FIPV-Δ3, FIPV-Δ7 and FIPV-Δ3Δ7 at a m.o.i. of 0.02. At different time points post inoculation, the intracellular and extracellular virus titers were determined. The data represent means ± SD of five replicate assays. Significant difference with FIPV 79-1146 is indicated with *p < 0.1; **p < 0.05; or ***p < 0.01.

Fig. 4

Kinetics of percentage of infected cells in FCoV inoculated monocytes isolated from the blood of three independent cats. Cells were inoculated with FIPV 79-1146, FIPV-Δ3, FIPV-Δ7 and FIPV-Δ3Δ7 at a m.o.i. of 0.02. At different time points post inoculation, cells were fixed and cytoplasmic nucleocapsid protein was visualized with an immunofluorescence staining.

Fig. 5

Kinetics of FCoV replication in feline monocytes of three cats. Cells were inoculated with FIPV 79-1146, FIPV-Δ3, FIPV-Δ7 and FIPV-Δ3Δ7 at a m.o.i. of 0.02. At different time points post inoculation, intracellular and extracellular virus titers were determined. The data represent means ± SD of triplicate assays.