To perform comparative analysis of the S1/S2 cleavage site between FECVs and FIPVs, we identified cases of FIP that were confirmed postmortem by using immunohistochemistry, the standard for FIP diagnosis; archival immunohistochemistry-positive formalin-fixed tissues were used as the source of FIPV RNA.
Analysis of the S1/S2 cleavage site of FIPV sequences shows that it has much more variability, both within the narrow furin cleavage recognition motif (P4-P1) and in residues extending out of it (P8-P5 and P2'-P4') (Figure 2A).
To test whether the identified FIPV S1/S2 mutations have an effect on cleavability by furin, we performed an in vitro proteolytic assay.
FIPV UU3 was obtained from a lymph node of a cat infected with FECV UCD; the presence of feline infectious peritonitis in the cat was pathologically confirmed.
To identify key differences between FIPV and FECV, we analyzed their genomes and proteomes; for each nucleotide or amino acid position, we determined the rate at which FIPVs differed from all FECVs at that position.
This was done by counting, for every nucleotide position, the number of FIPV genomes for which the identify at that position differed from that in all FECV genomes.
When compared by phylogenetic analysis, the nucleotide sequences of FIPV and FECV M genes distributed into paraphyletic patterns rather than in monophyletic clusters (Figure, panel A).
FECV 586 and FIPVs 584 and 585 from cattery A; FECV 620 and FIPVs 615 and 622 from cattery G; FECV 10 and FIPV 8 from cattery F).
We developed a study of naturally occurring FECV and FIPV using molecular genetic tools by collecting samples from field cases of FIP (cases) and FECV-positive but asymptomatic cats (controls).
Formalin-fixed sections (3 [micro]m thick) were cut from paraffin blocks and placed on glass slides for immunohistochemical (IHC) testing, as previously described, with CoV p56, a cross-reacting antibody for the demonstration of feline coronavirus (FECV and FIPV biotypes) (9,10) (Washington Animal Disease Diagnostic Laboratory, Pullman, WA, USA) (Figure 2).
Antibody-dependent enhancement of FIPV has been demonstrated in vitro in feline macrophages as well as in stable human and mouse macrophage cell lines (29).
Inoculation of cats with a recombinant vaccinia virus expressing the S protein FIPV 79-1146 sensitized cats and led to accelerated disease after FIPV challenge (37), while inoculation with recombinant vaccinia viruses expressing the M or N proteins did not (38).