Significance of Coronavirus Mutants in Feces and Diseased Tissues of Cats Suffering from Feline Infectious Peritonitis

1. Introduction

2. Results and Discussion

2.1. Structural and accessory genes of Feline coronavirus from the feces and diseased tissues of cats with FIP are virtually identical with the exception of truncating mutations in the 3c gene

Complete structural (S, E, M, N) and accessory (3a-c and 7 a, b) gene sequences were obtained from diseased omentum of the four related cats that died of FIP and the isolates designated were FIPV-UCD11, 12, 13 and 14 (Table 1). The numbers of nucleotides sequenced for isolates FIPV-UCD11 to UCD14 are shown in Table 1, while the relationship of the FIPVs isolated from the 4 related Scottish Fold cats is shown in Figure 1. The overall sequence identity for the nine structural and accessory genes was ≥ 99% with only a small number of mutations among the four highly related viruses (Table 1). Mutations consisted of minor SNP changes, and less commonly deletions that appeared to be randomly scattered among the genes that were sequenced; about one half of the mutations resulted in amino acid changes (Table 2). Among the 9 structural and accessory genes of the four related cats, the highest genetic variability was in the 3c gene, followed by the S and M genes (Table 2). The least variability was detected in the 3b, 7a, and 3a accessory genes. Among all of the genes sequenced, only the 3c genes of the FIPV isolates had SNPs that resulted in premature stop codons or deletions that caused frame shifts; both resulting in a variable truncation of the 3c protein (Figure 2).

Table 1. Name and biotype designation of coronavirus isolates (including variants) from diseased tissues of eight cats dying of FIP. The genes that were sequenced, their mutability, degree of relatedness, and the nature of the functional mutation in the 3c gene are given for each cat.

Table 1. Name and biotype designation of coronavirus isolates (including variants) from diseased tissues of eight cats dying of FIP. The genes that were sequenced, their mutability, degree of relatedness, and the nature of the functional mutation in the 3c gene are given for each cat.

Cat name	Isolate	Genes sequenced	# SNPs/nts sequenceda	% Sequence identityb	Status of 3c	GenBank Accession #
Red	FIPV-UCD11a	S,E,M,N,3a-c,7a,b	36/8959	99.59	Early stop	FJ917519
	FIPV-UCD11b		41/8959	99.54		FJ917520
Toby	FIPV-UCD12	S,E,M,N,3a-c,7a,b	51/8984	99.43	Deletion	FJ917521
	FECV-UCD5		50/8984	99.43	Intact	FJ917522
Tux	FIPV-UCD-13	S,E,M,N,3a-c,7a,b	35/8950	99.61	Deletion	FJ917523
Lucy	FIPV-UCD-14	S,E,M,N,3a-c,7a,b	25/8958	99.72	Early stop	FJ917524
388210	FIPV-UCD-15a	E,M,N,3a-c,7a,b	nac /4608	92	Deletions in 3b-c and early stop	FJ917525
388406	FIPV-UCD-16	E,M,N,3a-c,7a,b	na/4635	92	Early stop	FJ917526
Cat-T	FIPV-UCD-17	E,M,N,3a-c,7a,b	na/4660	92	Early stop	FJ917527
392312	FIPV-UCD-18a	E,M,N,3a-c,7a,b	na/4529	91	Deletions	FJ917528
	FIPV-UCD-18b					FJ917529

^a SNP differences between tissue virus and the consensus sequences of the FIPVs from the four related cats.

^b The percentage sequence identity was determined by comparison to the consensus sequences of the FIPVs of the four related Scottish Fold cats.

^c na - not applicable; sequence difference >6-12%).

Table 2. Mutational variations in nucleotide and resulting amino acid sequences among the structural and accessory genes of FIPV isolates from four closely related cats involved in the same FIP outbreak.

Table 2. Mutational variations in nucleotide and resulting amino acid sequences among the structural and accessory genes of FIPV isolates from four closely related cats involved in the same FIP outbreak.

Isolate	S	3a	3b	3c*	E	M	N	7a	7b
	4413nt/ 1471aa	213nt/ 71aa	221nt/ 74aa	714nt	249nt/ 83aa	796nt/ 265aa	1128nt/ 376aa	306nt/ 102aa	621nt/ 207aa
UCD11a	19/12	0/0	0/0	6nt	3/2	4/1	1/1	0/0	3/2
UCD12	24/15	1/1	0/0	7nt	1/0	8/5	7/4	1/1	2/1
UCD13	23/10	0/0	0/0	4nt	1/1	4/4	3/2	0/0	0/0
UCD14	12/5	0/0	0/0	3nt	0/0	2/1	2/2	0/0	2/1

* None of the 3c genes of these FIPV isolates encoded a functional protein.

Figure 1. Phylogenetic analysis based on the sequences encoding for structural (E, M and N) and accessory (3a-c and 7a,b) genes. FIPV-UCD11a, 12, 13, and 14, and FECV-UCD4, were isolated from 4 related kittens and a healthy housemate. FIPV-UCD15a, 16, 17, and 18a were isolated from unrelated and geographically disparate cats.

Figure 2. Cartoons show the 3a-c and E gene segments of FIPV-UCD11 to 18 and a cat-passaged variant of FIPV-UCD11b (FIPV11b.2). Deletions are indicated with triangles with number of nucleotides (nt) indicated; nonsense mutations are indicated with filled diamonds.

The omentum viruses from Red consisted of two distinct variants, as determined by sequences obtained from multiple overlapping PCR products (Table 1). These variants were designated FIPV-UCD11a and -UCD11b. There were only five SNPs scattered across the nine structural and accessory genes between the two variants. Two variants were also sequenced from the omentum of Toby, one with a non-functional 3c gene (FIPV-UCD12) and one with a functional 3c gene (FECV-UCD5). These two variants were identical in sequence except for a single-base deletion in the 3c of one of the variants (FIPV-UCD12).

Four additional unrelated cats (392312, 384062 and 388210) from Paradise, Menlo Park, and San Jose, CA, respectively, and Cat-T from Mountlake Terrace, WA were included in the study. The three cats seen at the VMTH suffered from the non-effusive form of FIP, while the Washington state cat died of effusive FIP. The E, M, N, 3a-c and 7a, b genes were amplified from the omentum or organ granulomas of all four animals. Viruses were readily detected in the diseased tissues of cats 388210, 388406, Cat-T, and 392312 and designated FIPV-UCD15 to UCD18, respectively (Table 1). FIPV-UCD15a possessed a two-nucleotide deletion near the end of the 3c gene and a second deletion of 48-nucleotide involving the terminus of 3b and beginning of 3c (Figure 2). Mutations of the 3c gene in FIPV-UCD16 and -UCD17 involved premature stop codons. Two variants with six scattered SNPs and an identical deletion in the 3c genes were identified in organ granulomas of cat 392312 and designated FIPV-UCD18a and -UCD18b (Table 1).

All of the structural and accessory genes that were sequenced for the eight different FIP cats appeared to be intact, except for the 3c genes. The 3c genes from all eight isolates contained deletions or SNPs that either produced truncating frame shifts or premature stop codons (Figure 2). The sequence relationship of the four unrelated FIPV isolates to each other and to the FIPV isolates from the four related Scottish Fold cats is shown in Figure 1. The overall genetic similarity for the E, M, N, and 3a-c, 7a, b genes ranged from 89-99% among the 8 FIPV isolates. The four FIPVs from unrelated cats showed sequence identity of 89-92% to each other and to the FIPVs from the four related cats.

Feces or colonic scrapings from the four related cats and a fifth unrelated housemate contained feline coronaviruses (Table 3). The amount of viral RNA in feces in cats with FIP was much lower than in diseased omentum and obtaining complete sequences of all 9 genes was not always possible. Therefore, the actual genes sequenced for each fecal coronavirus isolate are shown in Table 3. Coronaviruses isolated from the feces of two cats, Tux and Toby, were ≥99% identical and contained identical 3c gene mutations to the omental viruses from the same cats. The coronavirus isolated from Lucy’s feces (designated FECV-UCD3) had an intact (i.e., wild type or non-deliterious) 3c and its sequence was otherwise 99% identical to the sequence of FIPV-UCD14 found in her diseased omentum. The sequence obtained from the fecal virus of Simba, a housemate of Lucy, also had an intact 3c gene and was designated FECV-UCD4. FECV-UCD4, was most closely related to the FIPV isolated from Lucy and was 99.7% related to the consensus nucleotide sequences of coronaviruses obtained from the four related FIP cats (Figure 1,Table 2).

Table 3. Name and biotype designation of coronavirus isolates (including variants) from the feces of eight cats dying of FIP. The genes that were sequenced, their mutability, degree of relatedness to lesional isolates, and the nature of the functional mutation in the 3c gene are given for each cat.

Table 3. Name and biotype designation of coronavirus isolates (including variants) from the feces of eight cats dying of FIP. The genes that were sequenced, their mutability, degree of relatedness to lesional isolates, and the nature of the functional mutation in the 3c gene are given for each cat.

Cat Name	Isolate	Genes sequenced	#SNPs/nts sequenceda	% sequence identity	Status of 3c	GenBank Accession #
Lucy	FECV-UCD3a	E,M,N,3a-c,7a,b	47/4554	98.97	Intact	FJ9943761
	FECV-UCD3b	E,M, 3a-c	53/2517	97.89	Intact	FJ9943762
Simba	FECV-UCD4	E, M, N, 3c, 7a,b	15/4166b	99.65	Intact	FJ9943763
Toby	FIPV-UCD12	3a,b,7a,b	0/2954	100	Deletion	FJ9943765
						FJ9943766
Tux	FIPV-UCD13c	E,M,N,3a-c,7a,b	7/4486	99.84	Deletion	FJ9943764
Red	Unnamedd	3a,b,7b	7/1516	99.54	Unknown	FJ9943767
						FJ9943768
388210	FIPV-UCD15b	3a-c,7a,b	14/2498	99.44	Deletion	FJ9943769
						FJ9943770
388406	FECV-UCD6	E,M, 3a-c,7a,b	44/3595	98.88	Intact	FJ9943771
						FJ9943772
Cat-T	Unnamedd	7a,b	0/1177	100	Unknown	FJ9943773

^a # SNP differences between fecal and diseased tissue isolates from the same cat;

^b # SNP differences between fecal virus and consensus sequence of FECVs from other related cats.

^c There were 7 SNPs between FIPVs found in colonic scraping and the diseased omentum.

^d The biotypes of the virus isolated from the feces of these cats were not determined due to an inability to amplify the 3c gene.

A similar finding was found for the cats that were unrelated to those described above and that were from disparate geographic regions. Three of four fecal samples (388210, 388406 and Cat-T) contained amplifiable RNA and complete 3c genes were sequenced in 2/3 of these cats (388210 and 388406). The 3c gene sequence of the fecal virus of cat 388406 was intact and ≥99% related to the FIPV found in diseased tissue (Table 2). This fecal isolate was designated FECV-UCD6. The 3c gene of 388210 fecal virus contained a deliterious two-nucleotide deletion near the end of the 3c gene and was designated FIPV-UCD15b. This same deletion was also detected in the lesional FIPV-UCD15a. However, FIPV-UCD15a did not contain the 48-nucleotide deletion involving 3b and 3c of FIPV-UCD15b (Figure 2). Only the 7a, b genes were sequenced from the feces of Cat-T and the sequence was 100% identical to the 7a, b sequence from the omental FIPV-UCD17.

This study of lesional and/or fecal coronaviruses from nine cats both supported and modified the previous conclusions of Vennema et al. [17]. Viruses from diseased tissues from all eight cats in this study had truncating mutations, either in the form of deletions leading to frame shifts or coding changes causing premature stop codons in the 3c gene. Such damaging mutations were not present in other accessory and structural genes in this or in a previous study [17]. As with the earlier study [17], all or almost all of the fecal isolates from diseased cats and a healthy contact control animal had intact 3c genes. Taken as a whole, the present study supported a role for deleterious 3c gene mutations in the genesis of FIPVs from FECVs. However, not all FIPV isolates have deleterious 3c gene mutations. Although 8/8 (100%) of lesional isolates in the present study had functional mutations in their 3c genes, only 11/13 (85%) of the FIPVs reported by Vennema et al. [17] had deliterious 3c gene mutations. We have also recently observed what appeared to be intact 3c genes in 12/31 random breed cats that were adopted from a large shelter in Northern California and died of FIP. However, several of these isolates contained mutated 3c genes as minor variants, and without animal inoculation studies it is not possible to say whether or not these or the remaining isolates were capable of causing FIP. The existence of helper/defective virus replication in the latter situation also needs to be considered. Animal inoculation studies to determine the biotype of a given feline coronavirus are critical for determining the ultimate biotype of any isolate, regardless of its sequence regularities or irregularities. It was therefore important to demonstrate herein that an isolate from the four related cats reported herein was capable of causing FIP.

Since some FIPVs appear to have intact 3c genes, it may be premature to ascribe the FIP biotype solely to deleterious mutations in the 3c gene. However, what are the alternatives? It can be argued that mutations in the conserved replicase/transcriptase genes may have a similar effect; that small mutations in other structural and accessory genes, collectively or singly, will have the same effect; that FIPV and FECV are identical viruses; or that deleterious 3c gene mutations are an effect of the disease and not its cause. Involvement of the replicase/transcriptase genes is unlikely, because the replicase/transcriptase region is highly conserved among feline coronaviruses and unlikely to be involved in cell tropism or evasion of the host’s immune response. One study of a natural serotype I FIPV isolate (C1Je) showed a high degree of sequence conservation within the replicase/transcriptase genes compared to other feline coronaviruses, while a premature stop codon limited the 3c gene product to the first 16 amino acids [19]. It is also unlikely that mutations in other accessory or structural genes are involved, even though such mutations have been frequently found in feline coronaviruses. Firstly, 3c gene mutations in FIPVs occur significantly out of proportion to mutations in other structural or accessory genes. Secondly, there is little scientific evidence, especially based on animal inoculation, that other accessory genes are involved in FIP. In the original report that proposed the internal mutation theory, 11/13 of the FIPVs had 3c mutations, while 2/13 isolates had only 7b mutations [17]. However, both of the latter cats were related and had been experimentally infected with an identical FECV (FECV-RM); a third sibling cat from this group had the same 7b mutation but with a unique functional 3c mutation. Variants were not tested at the time and it is possible that 3c mutants would have been present if the two discordant isolates had been adequately sequenced. Earlier studies have also demonstrated an absence of 7b mutations in almost all FECVs and other FIPVs and indicate that such mutations are most likely tissue culture artifacts [17,20]. Yet other studies suggest that 7a and 7b mutations occur in nature in both FIP and enteric infections and are therefore not directly linked to pathogenicity [20,21,23].

There is a general belief that host and environmental factors, and not virus mutation, are the basic determinants of whether a cat develops FIP or just a mild enteritis following exposure to the common feline coronavirus [24-28]. For such a theory to be correct, FECVs and FIPVs would have to be identical in both genetic structure and virulence. The evidence that FECVs and FIPVs cause very different diseases is strong [10-13]. Even though environmental and host factors are admittedly important in FIP [29,30], lesional viruses from the eight FIP cats in this study, even though highly related to fecal isolates, were easily differentiated from each other based on deliterious 3c gene mutations alone. Moreover, an infectious inoculum made from the diseased omentum of one of the FIP cats induced FIP in 3 of 12 cats that were experimentally infected (see section 2.2.). Confirmation of biotype by animal inoculation, such as described herein, is rarely done in published reports concerned with feline coronavirus infection [reviewed in 11].

The possibility that deleterious 3c gene mutations are an effect of the disease and not a cause also has to be considered. However, there is little precedence for this and given the ability of a lesional isolate from the present study to produce FIP, it is counterintuitive for a functional 3c gene mutant to be both a cause and effect of its own disease. This theory would also not explain why all non-tissue culture adapted FECV strains used for experimental inoculation studies have intact 3c genes, while all tissue culture adapted and non-adapted strains have mutated 3c genes [17].

The existence of feline coronavirus variants was not a novel observation [23,31,32], but their frequency and fate has not been previously addressed. Variant forms were found in both extraintestinal tissues and feces of the 9 cats in this study, but only one variant became predominant upon experimental passage from one cat to another (see section 2.2). The infecting variant may have been merely the first virus into a macrophage, or its selection may have involved more complex host/virus interactions. We also found that subtle, and sometimes significant, genetic mutations (usually SNPs and deletions) occurred upon primary replication in a new host. Therefore, genetic variation among feline coronaviruses occurs both within and between host cats. Selective infection with a single variant can also rapidly lead to genetically distinct clades of coronavirus, especially when combined with a high intrinsic and extrinsic mutation rate.

2.2. Experimental infection of laboratory cats establishes that FIPV-UCD11a, b possess the FIP biotype and that co-infection with both variants leads to infection with one or the other variant but not both

Twelve laboratory cats were inoculated intraperitoneally with a cell-free inoculum prepared from the diseased omentum of Red, which contained two variant forms of the virus (FIPV-UCD11a and -UCD11b). Three of these cats developed effusive FIP within 2-4 weeks. Viral RNA was isolated from the omentum of each experimentally infected cat at the time of necropsy. The S (one cat) and E, M, N and 3a-c, 7a, b genes (all three cats) were sequenced. One of the cats was found to be infected with FIPV-UCD11a, while two of the cats were each infected with FIPV-UCD11b. Each of these cats had a nearly identical variant of UCD-11a or UCD-11b in its diseased omentum (Table 4). The premature stop codon of parental 3c gene was preserved in FIPV isolates from all three cats. However, FIPV-UCD11b.2 isolated from one of the three cats had acquired two additional large deletions affecting both the 3b and 3c genes that were not in the infecting virus (Table 4 and Figure 2).

Table 4. Name and biotype designation of coronavirus isolates from three cats dying of experimentally induced FIP. The genes that were sequenced, their mutability, degree of relatedness to the consensus sequence of FIPV-UCD11a, b, and nature of the functional mutation in the 3c gene are given for each cat.

Table 4. Name and biotype designation of coronavirus isolates from three cats dying of experimentally induced FIP. The genes that were sequenced, their mutability, degree of relatedness to the consensus sequence of FIPV-UCD11a, b, and nature of the functional mutation in the 3c gene are given for each cat.

Cat #	Isolate	Genes sequenced	#SNPs/nts sequenced a	Type of mutation in 3c	GenBank Accession #
07-036	FIPV-UCD11a.1ab	E,M,N,3a-c,7a,b	5/6711	Stop codon same as FIPV-UCD11a	FJ917530
	FIPV-UCD11a.1bc		7/6711		FJ917531
05-243	FIPV-UCD11b.1ab	E,M,N,3a-c,7a,b	6/4680	Stop codon same as FIPV-UCD11b	FJ917532
	FIPV-UCD11b.1bc		7/4680		FJ917533
98-272	FIPV-UCD11b.2ab	S,E,M,N,3a-c,7a,b	5/8943	Stop codon same as FIPV-UCD11b, plus deletions in 3b,c	FJ917534
	FIPV-UCD11b.2bc		8/8943		FJ917535

^a # SNPs detected when compared to their parental FIPV-UCD11a or FIPV-UCD11b viruses.

^bVariant strain isolated from experimentally infected cat.

^cVariant strain used for SNP comparison.

It is important to determine by animal inoculation studies the true biotype of a feline coronavirus that is being reported, rather than always referring to a generic feline coronavirus [reviewed in 11]. Feline coronaviruses that possess the FIP biotype, such as FIPV-UCD11a,b, will readily induce FIP in from 25-100% of infected individuals, depending on the strain being tested [reviewed in 11]. However, bonifed (cat-to-cat passaged, non-tissue culture adapted) FECV strains will rarely induce FIP in healthy cats [9,12,15,17].

2.4. All of the feline coronavirus isolates in this study were serotype I as based on both S and 3a gene sequences

The S sequences of FIPV-UCD11 to 14 were compared to that of previously reported FIPVs and to a purported FECV (WSU-79-1683) (data not shown). The S protein shared 98% sequence identity among the four FIPV isolates, and 87-91% sequence identity to other published serotype I FIPVs. However, when compared to the S protein of serotype II feline coronaviruses WSU-79-1146 and WSU-79-1683, there was only 43-44% sequence identity (data not shown). Based on the comparison of S proteins, FIPV-UCD11 to 14 were classified as serotype I feline coronaviruses. However, these studies demonstrate that serotype designation of feline coronaviruses can be more easily made from comparisons of the much smaller 3a rather than significantly larger S sequences (71 vs 1471 amino acids in the respective gene products) (Figure 3). Similar to the S protein comparison, the 3a sequence of all four FIPVs from the related cats shared 98-100% sequence identity to each other, and 84-94% sequence identity to the four FIPVs from unrelated cats and from published serotype I FIPVs (Figure 3). However, when compared to the 3a protein of serotype II feline coronaviruses WSU-79-1146 and WSU-79-1683 or to 3a of Canine coronavirus, there was only 65-70% sequence identity. These results indicate all eight FIPVs from this study clearly belonged to serotype I based on their 3a protein sequences, while known serotype II viruses and the canine coronavirus formed a separate group (Figure 3).

Figure 3. A comparison of the amino acid sequences of 3a proteins from several different FIPVs and a canine coronavirus (CCoV). The reference sequence is from FIPV-UCD11. GenBank accession numbers for additional FIPV isolates are EU186072(Black), DQ160294(NTU2/R), DQ848678(C1Je), NC_007025(WSU-79/1146), X80799(WSU-79/1683), and DQ112226(CCoV). ^aFIPV serotype I; ^b FIPV serotype II. ^cCanine coronavirus.

3. Experimental Section

3.5. cDNA synthesis and PCR amplification of viral sequence

The published sequences of feline coronaviruses in GenBank were used to design the primers for a reverse transcripase polymerase chain reaction (RT-PCR). Three primer pairs were designed from highly conserved regions and used to amplify three overlapping fragments containing the nine structural and accessory genes of feline coronavirus (Figure 4 and Table 5). The RT-PCR was carried out with QIAgen LongRange 2Step RT-PCR kit (QIAgen, USA). The viral RNA was first denatured by incubating at 65°C for 5 min and then chilled on ice. The reverse transcription was carried out in 20 μl reaction mixture containing 10 units of LongRange reverse transcriptase, 0.8 unit of RNase inhibitor, 1 mM dNTP, 1 mM Oligo dT , and 5 μL of denatured viral RNA in 1x reaction buffer. The mixture was incubated at 42°C for 2 hr followed by 85°C for 5 min. The reverse transcribed cDNA was stored at -20°C or used immediately in PCR amplification. The viral cDNA was amplified in 20 μl reaction mixture containing 2 μl cDNA, 1 unit LongRange PCR enzyme mix, 0.5 mM dNTP, 0.25 mM forward primer, and 0.25 mM reverse primer in 1x PCR buffer. The mixture was then incubated at 93°C for 3 min and amplified for 30 cycles at 93°C for 30 s, 60°C for 30 s, and 68°C for 1 min per kb of PCR product, followed by a final extension for 10 min at 68°C. The reverse transcribed viral RNA from feces was amplified for 40 cycles under the same condition. The PCR products were electrophoresed in TAE buffer on a 0.8% agarose gel. The PCR product was purified using a QIAgen gel purification kit (QIAgen, USA).

Table 5. Primer sequences used for the amplification of accessory and structural genes of feline coronavirus isolates.

Table 5. Primer sequences used for the amplification of accessory and structural genes of feline coronavirus isolates.

Primer name	Nucleotide sequence	Genome Positiona
Forward primer for PCR fragment A	5’TCGTTGGATTACTAAGGAAGG-3’	20121
Reverse primer for PCR fragment A	5'CTGTGATATTAAACAGTCGTATG-3'	24854
Forward primer for PCR fragment A	5'GGCCTTGGTATGTGTGGCTAC-3'	24380
Reverse primer for PCR fragment B	5'CTATTCCAATAACCAATTTGTTGATC-3'	26996
Forward primer for PCR fragment C	5'GCTACACCTAGTAGAACCATCG-3'	26605
Reverse primer for PCR fragment C	5'GTGTATCACTATCAAAAGGAAAATTTTC-3'	29125

^a defined by the genome of FIPV-WSU-79/1146 (NC_0007025).

Figure 4. A cartoon shows the structural (S, E, M, and N) and accessory (3a, 3b, 3c, 7a, and 7b) genes of feline coronavirus. The PCR primers used for PCR amplifications are shown as arrows. The PCR products are represented by a black line joining the forward and reverse PCR primers.

4. Conclusions

References

1. Holzworth, J.E. Some important disorders of cats. Cornell Vet. 1963, 53, 157–160. [Google Scholar] [PubMed]
2. Feldman, B.F.; Jortner, B.S. Clinico-pathology conference. J. Am. Vet. Med. Assoc. 1964, 144, 1409–1411. [Google Scholar] [PubMed]
3. Rohrbach, B.W.; Legendre, A.M.; Baldwin, C.A.; Lein, D.H.; Reed, W.M.; Wilson R.B. Epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals. J. Am. Vet. Med. Assoc. 2001, 218, 1111–1115. [Google Scholar] [CrossRef] [PubMed]
4. Wolfe, L.G.; Griesemer, R.A. Feline infectious peritonitis. Pathol. Vet. 1966, 3, 255–270. [Google Scholar] [PubMed]
5. Zook, B.C.; King, N.W.; Robinson. R.L.; McCombs H.L. Ultrastructural evidence for the viral etiology of feline infectious peritonitis. Pathol. Vet. 1968, 5, 91–95. [Google Scholar]
6. Ward, J. Morphogenesis of a virus in cats with experimental feline infectious peritonitis. Virol. 1970, 41, 191–194. [Google Scholar] [CrossRef]
7. Montali, R.J.; Strandberg, J.D. Extraperitoneal lesions in feline infectious peritonitis. Vet. Pathol. 1972, 9, 109–121. [Google Scholar] [PubMed]
8. Pedersen, N.C.; Ward, W. Antigenic relationship of the feline infections peritonitis virus to coronaviruses of other species. Arch. Virol. 1978, 58, 45–53. [Google Scholar] [CrossRef] [PubMed]
9. Pedersen, N. C.; Black, J. W.; Boyle, J. F.; Evermann, J. F.; McKeirnan, A. J.; Ott, R. L. Pathogenic differences between various feline coronavirus isolates. Coronaviruses; molecular biology and pathogenesis. Adv. Exp. Med. Biol. 1984, 173, 365–380. [Google Scholar] [PubMed]
10. Pedersen, N.C.; Boyle, J.F.; Floyd, K.; Fudge, A.; Barker J. An enteric coronavirus infection of cats and its relationship to feline infectious peritonitis. Am. J. Vet. Res. 1981, 42, 368–377. [Google Scholar] [PubMed]
11. Pedersen, N.C. A review of feline infectious peritonitis virus infection: 1963-2008. J. Feline Med. Surg. 2009, 11, 225–258. [Google Scholar] [CrossRef] [PubMed]
12. Pedersen, N.C.; Allen, C.E.; Lyons L.A. Pathogenesis of feline enteric coronavirus infection. J. Feline Med. Surg. 2008, 10, 529–541. [Google Scholar] [CrossRef] [PubMed]
13. Pedersen, N.C. Virologic and immunologic aspects of feline infectious peritonitis virus infection. Adv. Exp. Med. Biol. 1987, 218, 529–550. [Google Scholar] [PubMed]
14. Horzinek, M.C.; Herrewegh, A.; de Groot, R.J. Perspectives on feline coronavirus evolution. Feline Pract. 1995, 23, 34–39. [Google Scholar]
15. Hickman, M.A.; Morris, J.G.; Rogers, Q.R.; Pedersen, N.C. Elimination of Feline Coronavirus Infection from a Large Experimental Specific Pathogen-Free Cat Breeding Colony by Serologic Testing and Isolation. Feline Pract. 1995, 23, 96–102. [Google Scholar]
16. Poland, A.M.; Vennema, H.; Foley, J.E.; Pedersen, N.C. Two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus. J. Clin. Microbiol. 1996, 34, 3180–3184. [Google Scholar] [PubMed]
17. Vennema, H.; Poland, A.; Foley, J.; Pedersen, N.C. Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virol. 1998, 243, 150–157. [Google Scholar] [CrossRef]
18. Rottier, P. J.; Nakamura, K. Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein. J. Virol. 2005, 79, 14122–14130. [Google Scholar] [CrossRef] [PubMed]
19. Dye, C.; Siddell, S. G. Genomic RNA sequence of feline coronavirus strain FCoV C1Je. J Feline Med. Surg. 2007, 9, 202–213. [Google Scholar] [CrossRef]
20. Herrewegh, A.A.; Mähler , M.; Hedrich, H.J.; Haagmans, B.L.; Egberink, H.F.; Horzinek, M.C.; Rottier, P.J.; de Groot, R.J. Persistence and evolution of feline coronavirus in a closed cat-breeding colony . Virol. 1997, 4   , 349–363. [Google Scholar]
21. Lin, C-N.; Su, B-L.; Huang, H-P.; Lee, J-J.; Hsieh, M-W.; Chueh, L-L. Field strain feline coronaviruses with small deletions in ORF7b associated with both enteric infection and feline infectious peritonitis. J. Feline Med. Surg. 2009, 11, 413–419. [Google Scholar] [CrossRef] [PubMed]
22. Kennedy, M.; Boedeker, N.; Gibbs, P.; Kania, S. Deletions in the 7a ORF of feline coronavirus associated with an epidemic of feline infectious peritonitis. Vet. Microbiol. 2001, 8, 227–234. [Google Scholar] [CrossRef]
23. Kiss, I.; Kecskeméti, S.; Tanyi, J.; Klingeborn, B.; Belák S. Preliminary studies on feline coronavirus distribution in naturally and experimentally infected cats. Res. Vet. Sci. 2000, 68, 237–242. [Google Scholar] [CrossRef] [PubMed]
24. Addie, D. D.; Kennedy, L. J.; Nart, P ; Radford, A. D. Feline leucocyte antigen class II polymorphism and susceptibility to feline infectious peritonitis. J. Feline Med. Surg. 2004, 6, 59–62. [Google Scholar] [CrossRef] [PubMed]
25. Kipar, A.; Baptiste, K.; Barth, A.; Reinacher, M. Natural FCoV infection: cats with FIP exhibit significantly higher viral loads than healthy infected cats. J. Feline Med. Surg. 2006, 8, 69–72. [Google Scholar] [CrossRef]
26. Kipar , A.; Meli, M. L.; Failing, K.; Euler, T.; Gomes-Keller, M. A.; Schwartz, D.; Lutz, H.; Reinacher, M. Natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection. Vet. Immunol. Immunopathol. 2006, 112, 141–155. [Google Scholar] [CrossRef] [PubMed]
27. Meli, M.; Kipar, C.; Müller, A.; Jenal, K.; Gönczi, E.; Borel, N.; Gunn-Moore, D.; Chalmers, S.; Linn, F.; Rinacher, M.; Lutz, H. High viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (FCoV) type I and in naturally FCoV-infected cats. J. Feline Med. Surg. 2004, 6, 69–81. [Google Scholar] [CrossRef] [PubMed]
28. Paltrinieri, S.; Gelain, M.E.; Ceciliani, F.; Ribera, A.M.; Battilani, M. Association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation. J. Feline Med. Surg. 2008, 10, 514–518. [Google Scholar] [CrossRef] [PubMed]
29. Foley, J. E.; Pedersen, N. C. Inheritance of susceptibility of feline infectious peritonitis in purebred catteries. Feline Pract. 1996, 24, 14–22. [Google Scholar]
30. Foley, J. E.; Poland, A.; Carlson, J.; Pedersen, N.C. Risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus. J. Am. Vet. Med. Assoc. 1997, 210, 1313–1318. [Google Scholar] [PubMed]
31. Battilani, M.; Coradin, T.; Scagliarini, A.; Ciulli, S.; Ostanello, F.; Prosperi, S.; Morganti, L. Quasispecies composition and pylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats. FEMS 32. Immunol. Med. Microbiol. 2003, 39, 141–147. [Google Scholar] [CrossRef]
32. Mochizuki, M.; Mitsutake, Y.; Miyanohara, Y. Antigenic and plaque variations of serotype II feline infectious peritonitis coronaviruses. J. Vet. Med. Sci. 1997, 59, 253–258. [Google Scholar] [CrossRef] [PubMed]
33. Addie, D.D.;Schaap; Nicolson, L; Jarrett, O.    Persistence and transmission of natural type I feline coronavirus infection. J. Gen. Virol. 2003, 84, 2735–2744. [Google Scholar] [CrossRef] [PubMed]
34. Herrewegh, A.A.; Vennema, H.; Horzinek, M.C.; Rottier, P.J.; de Groot, R.J. The molecular genetics of feline coronaviruses: comparative sequence analysis of the ORF7a/7b transcription unit of different biotypes. Virol. 1995, 212, 622–631. [Google Scholar] [CrossRef]
35. Hardy, Jr., W.D.; Hurvitz, L. Feline infectious peritonitis: Experimental studies. J Am. Vet. Med. Assoc. 1971, 158, 994–1002. [Google Scholar] [PubMed]
36. Can-Sahna, K.; Soydal Ataseven, V.; Pinar, D.; Oğuzoğlu, T. C. The detection of feline coronaviruses in blood samples from cats by mRNA RT-PCR. J. Feline Med. Surg. 2004, 9, 369–372. [Google Scholar] [CrossRef]
37. Oostra, M.; de Haan, C.A.M.; de Groot, R.J.; Rottier P.J.M. Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M. J. Virol. 2006, 80, 2326–2336. [Google Scholar] [CrossRef] [PubMed]
38. Ito, N.; Mossel, E. C.; Narayanan, K.; Popov, V. L.; Huang, C.; Inoue, T.; Peters, C. J.; Makino, S. Severe acute respiratory syndrome coroanvirus 3a protein is a viral structural protein. J. Virol. 2005, 79, 3182–3186. [Google Scholar] [CrossRef] [PubMed]
39. Zhong, X.; Guo, Z.; Yang, H.; Peng, L.; Xie, Y.; Wong, T. Y.; Lai, S. T.; Guo, Z. Amino terminus of the SARS coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients. J. Gen. Virol. 2006, 87, 369–373. [Google Scholar] [CrossRef] [PubMed]
40. Tan, Y. J. The Severe Acute Respiratory Syndrome (SARS)-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein. Virol. J. 2005, 10, 2–5. [Google Scholar]