Genetics and pathogenesis of feline infectious peritonitis virus

Abstract

Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.

Figure 1

Alternative phylogenetic predictions of the in vivo mutation hypothesis versus the dual circulating virulent/avirulent hypothesis. A) The in vivo mutation transition hypothesis predicts paraphyly of feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic feline coronavirus (FCoV) isolates). B) The circulating virulent/avirulent strain hypothesis predicts reciprocal monophyly of FIV-cases versus FECV asymptomatic. Numbers represent individual cat (or locale), which is either FIPV case (red) or FECV asymptomatic (blue). Evidence presented in this article supports the circulating dual virulent and avirulent strains.

Figure 2

A) Histopathologic and immunohistochemical (IHC) results from 23 necropsied cats positive for antibodies against feline coronavirus. Liver, lung, spleen, colon, jejunum, stomach, heart, kidney, lymph node were evaluated by IHC. Feline infectious peritonitis (FIP) cases are highlighted in gray. Pos, positive; Neg, negative; ND, not done. B) Representative tissues from cat no. FCA-4653, spleen (histopathologic) showing granuloma (arrow); magnification ×20. C) Representative tissues from cat no. FCA-4590, small intestine (IHC); magnification ×20. D) Red staining indicates binding of coronavirus antibody (CoV p56, arrow); magnification ×100.

Figure 3

A) Feline coronavirus genome indicating PCR products obtained (bars). Structural proteins are shaded in dark gray; nonstructural proteins are shaded in light gray. B) Forward and reverse primers used to amplify virus segments are listed in 5′ → 3′ orientation. The number of source cats and cloned sequences generated from feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic cats are presented. Pol, polymerase; NSP, nonstructural protein; FIPV, feline infectious peritonitis virus.

Figure 4

Maximum-likelihood (ML) phylogenetic tree of unique sequences from 3 feline coronavirus (FCoV) genes membrane, nonstructural protein 7b (NSP 7b), and spike-NPS3 (see Figure 3) gene sequences showing monophyly correlating to disease status. Cloned sequences from feline infectious peritonitis (FIP) cases are shown in red; feline enteric coronavirus (FECV) asymptomatic cats are shown in blue, and FCoV virulent strain from Aju-92 (cheetah) is in green. Number of cats and number of clones assessed are listed in Figure 3, panel B. A) membrane 575-bp sequences (ML –ln L = 3086.20787 best tree found by maximum parsimony [MP]: length = 493, confidence interval [CI] = 0.551724, retention index [RI] = 0.0926505); B) NSP 7b 736-bp sequences (ML –ln L = 4556.60497 best tree found by MP: length = 452, CI = 0.608, RI = 0.942; C) spike-NSP3 1017-bp sequences (ML –ln L = 2804.53198 best tree found by MP: length = 280, CI = 0.800, RI = 0.954). The number of FIP cases and FECV asymptomatic cats and number of cloned sequences is indicated in parenthesis in the key. Each sequence is labeled as follows: first letter indicates source farm (W, Weller Farm; F, Frederick Animal Shelter; S, Seymour Farm; M, Mount Airy Shelter; A, Ambrose Farm), 4-digit cat identification number, tissue source (fe, feces; af, ascites fluid; co, colon; li, liver; sp, spleen; in, intestine; je, jejunum; ln, lymph node), 2-digit year (e.g., 04 = 2004), and the number of clones for each sequence. Bootstrap values are shown (maximum parsimony/minimum evolution/maximum likelihood) above branches. Where maximum likelihood tree was congruent with maximum parsimony tree, branch lengths are indicated below branches; the number of homoplasies is in parenthesis after the branch length. Virus sequence obtained from cat no. 4590 in May 2004 and at the time of death due to FIP in December 2004 is indicated by box. Scale bars indicate substitutions/site.

Figure 5

Diagram of membrane protein containing 3 transmembrane helices, an external N terminus and an internal carboxy terminus. Approximate position of 5 variable diagnostic amino acid sites (see Table 2) as determined by sequence comparison to severe acute respiratory syndrome coronavirus (32). Amino acid residue, polarity, and hydrophobicity or hydropholicity is stated.