Identification of peptide domains involved in the subcellular localization of the feline coronavirus 3b protein.

Feline coronavirus (FCoV) has been identified as the aetiological agent of feline infectious peritonitis (FIP), a highly fatal systemic disease in cats. FCoV open reading frame 3 (ORF3) encodes accessory proteins 3a, 3b and 3 c. The FCoV 3b accessory protein consists of 72 amino acid residues and localizes to nucleoli and mitochondria. The present work focused on peptide domains within FCoV 3b that drive its intracellular trafficking. Transfection of different cell types with FCoV 3b fused to enhanced green fluorescent protein (EGFP) or 3×FLAG confirmed localization of FCoV 3b in the mitochondria and nucleoli. Using serial truncated mutants, we showed that nucleolar accumulation is controlled by a joint nucleolar and nuclear localization signal (NoLS/NLS) in which the identified overlapping pat4 motifs (residues 53-57) play a critical role. Mutational analysis also revealed that mitochondrial translocation is mediated by N-terminal residues 10-35, in which a Tom20 recognition motif (residues 13-17) and two other overlapping hexamers (residues 24-30) associated with mitochondrial targeting were identified. In addition, a second Tom20 recognition motif was identified further downstream (residues 61-65), although the mitochondrial translocation evoked by these residues seemed less efficient as a diffuse cytoplasmic distribution was also observed. Assessing the spatiotemporal distribution of FCoV 3b did not provide convincing evidence of dynamic shuttling behaviour between the nucleoli and the mitochondria.

Cellular localisation of the proteins of region 3 of feline enteric coronavirus.

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflect in vivo stability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

Identification and characterization of a Golgi retention signal in feline coronavirus accessory protein 7b.

Feline coronaviruses encode five accessory proteins with largely elusive functions. Here, one of these proteins, called 7b (206 residues), was investigated using a reverse genetic approach established for feline infectious peritonitis virus (FIPV) strain 79-1146. Recombinant FIPVs (rFPIVs) expressing mutant and/or FLAG-tagged forms of 7b were generated and used to investigate the expression, processing, glycosylation, localization and trafficking of the 7b protein in rFIPV-infected cells, focusing on a previously predicted ER retention signal, KTEL, at the C-terminus of 7b. The study revealed that 7b is N-terminally processed by a cellular signalase. The cleavage site, 17-Ala|Thr-18, was unambiguously identified by N-terminal sequence analysis of a 7b processing product purified from rFIPV-infected cells. Based on this information, rFIPVs expressing FLAG-tagged 7b proteins were generated and the effects of substitutions in the C-terminal 202KTEL206 sequence were investigated. The data show that (i) 7b localizes to and is retained in the medial- and/or trans-Golgi compartment, (ii) the C-terminal KTEL sequence acts as a Golgi [rather than an endoplasmic reticulum (ER)] retention signal, (iii) minor changes in the KTEL motif (such as KTE, KTEV, or the addition of a C-terminal tag) abolish Golgi retention, resulting in relocalization and secretion of 7b, and (iv) a KTEL-to-KDEL replacement causes retention of 7b in the ER of rFIPV-infected feline cells. Taken together, this study provides interesting new insights into an efficient Golgi retention signal that controls the cellular localization and trafficking of the FIPV 7b protein in virus-infected feline cells.

Determination of the cell tropism of serotype 1 feline infectious peritonitis virus using the spike affinity histochemistry in paraffin-embedded tissues.

Unlike for serotype II feline coronaviruses (FCoV II), the cellular receptor for serotype I FCoV (FCoV I), the most prevalent FCoV serotype, is unknown. To provide a platform for assessing the pattern by which FCoV I attaches to its host receptor(s), HEK293 cell lines that stably express the ectodomains of the spike (S) proteins derived from a FCoV I feline enteric coronavirus strain UU7 (FECV UU7) and a feline infectious peritonitis virus strain UU4 (FIPV UU4) were established. Using the recombinant S proteins as probes to perform S protein affinity histochemistry in paraffin-embedded tissues, although no tissue or enteric binding of FECV UU7 S protein was detected, it was found that by immunohistochemistry that the tissue distribution of FIPV UU4 S protein-bound cells correlated with that of FIPV antigen-positive cells and lesions associated with FIP and that the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of host cell-surface sialic acid with neuraminidase. These findings suggest that a factor(s) other than sialic acid contribute(s) to the macrophage tropism of FIPV strain UU4. This approach allowed obtaining more information about both virus-host cell interactions and the biological characteristics of the unidentified cellular receptor for FCoV I.

Interferon-γ/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Receptor Binding Domains by the T Cells from COVID-19-Vaccinated Humans and FCoV1-Infected Cats.

The receptor binding domain (RBD) of SARS-CoV-2 (SCoV2) has been used recently to identify the RBD sequences of feline coronavirus serotypes 1 (FCoV1) and 2 (FCoV2). Cats naturally infected with FCoV1 have been shown to possess serum reactivities with FCoV1 and SCoV2 RBDs but not with FCoV2 RBD. In the current study, COVID-19-vaccinated humans and FCoV1-infected laboratory cats were evaluated for interferon-gamma (IFNγ) and interleukin-2 (IL-2 ELISpot responses by their peripheral blood mononuclear cells (PBMC) to SCoV2, FCoV1, and FCoV2 RBDs. Remarkably, the PBMC from COVID-19-vaccinated subjects developed IFNγ responses to SCoV2, FCoV1, and FCoV2 RBDs. The most vaccinated subject (five vaccinations over 2 years) appeared to produce hyperreactive IFNγ responses to all three RBDs, including the PBS media control. This subject lost IFNγ responses to all RBDs at 9 months (9 mo) post-last vaccination. However, her IL-2 responses to FCoV1 and FCoV2 RBDs were low but detectable at 10 mo post-last vaccination. This observation suggests that initially robust IFNγ responses to SCoV2 RBD may be an outcome of robust inflammatory IFNγ responses to SCoV2 RBD. Hence, the T-cell responses of vaccine immunity should be monitored by vaccine immunogen-specific IL-2 production. The PBMC from chronically FCoV1-infected cats developed robust IFNγ responses to SCoV2 and FCoV2 RBDs but had the lowest IFNγ responses to FCoV1 RBD. The constant exposure to FCoV1 reinfection may cause the IFNγ responses to be downregulated to the infecting virus FCoV1 but not to the cross-reacting epitopes on the SCoV2 and FCoV2 RBDs.

An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. Recently, an FIP outbreak from an animal shelter was confirmed in Taiwan. FCoV from all the cats in this shelter were analyzed to determine the epidemiology of this outbreak. Thirteen of 46 (28.2%) cats with typical signs of FIP were identified. Among them, seven cats were confirmed by necropsy and/or histopathological examinations. Despite the fact that more than one FCoV was identified in this multi-cat environment, the eight FIP cats were invariably found to be infected with a type II FCoV. Sequence analysis revealed that the type II FIPV detected from fecal samples, body effusions and granulomatous tissue homogenates from the cats that succumbed to FIP all harbored an identical recombination site in their S gene. Two of the cats that succumbed to FIP were found to harbor an identical nonsense mutation in the 3c gene. Fecal shedding of this type II virus in the effusive form of FIP can be detected up to six days before death. Taken together, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment.

Spike protein fusion peptide and feline coronavirus virulence.

Coronaviruses are well known for their potential to change their host or tissue tropism, resulting in unpredictable new diseases and changes in pathogenicity; severe acute respiratory syndrome and feline coronaviruses, respectively, are the most recognized examples. Feline coronaviruses occur as 2 pathotypes: nonvirulent feline enteric coronaviruses (FECVs), which replicate in intestinal epithelium cells, and lethal feline infectious peritonitis viruses (FIPVs), which replicate in macrophages. Evidence indicates that FIPV originates from FECV by mutation, but consistent distinguishing differences have not been established. We sequenced the full genome of 11 viruses of each pathotype and then focused on the single most distinctive site by additionally sequencing hundreds of viruses in that region. As a result, we identified 2 alternative amino acid differences in the putative fusion peptide of the spike protein that together distinguish FIPV from FECV in >95% of cases. By these and perhaps other mutations, the virus apparently acquires its macrophage tropism and spreads systemically.

Feline infectious peritonitis virus with a large deletion in the 5'-terminal region of the spike gene retains its virulence for cats.

In this study, the Japanese strain of type I feline infectious peritonitis virus (FIPV), C3663, was found to have a large deletion of 735 bp within the gene encoding the spike (S) protein, with a deduced loss of 245 aa of the N-terminal region of the S protein. This deletion is similar to that observed in porcine respiratory coronavirus (PRCoV) when compared to transmissible gastroenteritis virus, which correlates with reduced virulence. By analogy to PRCoV, we expected that the pathogenicity of C3663 may be attenuated in cats. However, two of four cats inoculated with C3663 died of FIP, and a third C3663-inoculated cat showed FIP lesions at 91 days after challenge. These results indicate that the 5'-terminal region of the S gene is not essential for the development of FIP.

Expression of Toll-like receptors 3, 7, 9 and cytokines in feline infectious peritonitis virus-infected CRFK cells and feline peripheral monocytes.

BACKGROUND: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood. OBJECTIVES: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-ß, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes. METHODS: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-ß, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence. RESULTS: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-ß expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-ß was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time. CONCLUSION: TLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-ß needs to be explored further.

Surface Display of Peptides Corresponding to the Heptad Repeat 2 Domain of the Feline Enteric Coronavirus Spike Protein on Bacillus subtilis Spores Elicits Protective Immune Responses Against Homologous Infection in a Feline Aminopeptidase-N-Transduced Mouse Model.

Although feline coronavirus (FCoV) infection is extremely common in cats, there are currently few effective treatments. A peptide derived from the heptad repeat 2 (HR2) domain of the coronavirus (CoV) spike protein has shown effective for inhibition of various human and animal CoVs in vitro, but further use of FCoV-HR2 in vivo has been limited by lack of practical delivery vectors and small animal infection model. To overcome these technical challenges, we first constructed a recombinant Bacillus subtilis (rBSCotB-HR2P) expressing spore coat protein B (CotB) fused to an HR2-derived peptide (HR2P) from a serotype II feline enteric CoV (FECV). Immunogenic capacity was evaluated in mice after intragastric or intranasal administration, showing that recombinant spores could trigger strong specific cellular and humoral immune responses. Furthermore, we developed a novel mouse model for FECV infection by transduction with its primary receptor (feline aminopeptidase N) using an E1/E3-deleted adenovirus type 5 vector. This model can be used to study the antiviral immune response and evaluate vaccines or drugs, and is an applicable choice to replace cats for the study of FECV. Oral administration of rBSCotB-HR2P in this mouse model effectively protected against FECV challenge and significantly reduced pathology in the digestive tract. Owing to its safety, low cost, and probiotic features, rBSCotB-HR2P is a promising oral vaccine candidate for use against FECV/FCoV infection in cats.

Mind the feline coronavirus: Comparison with SARS-CoV-2.

Both feline coronavirus (FCoV) and SARS-CoV-2 are coronaviruses that infect cats and humans, respectively. However, cats have been shown to be susceptible to SARS-CoV-2, and FCoV also had been shown to infect human. To elucidate the relationship between FCoV and SARS-CoV-2, we highlight the main characteristics of the genome, the receptor usage, and the correlation of the receptor-binding domain (RBD) of spike proteins in FCoV and SARS-CoV-2. It is demonstrated that FCoV and SARS-CoV-2 are closely related to the main characteristics of the genome, receptor usage, and RBD of spike proteins with similar furin cleavage sites. In particular, the affinity of the conserved feline angiotensin-converting enzyme 2 (fACE2) receptor to the RBD of SARS-CoV-2 suggests that cats are susceptible to SARS-CoV-2. In addition, cross-species of coronaviruses between cats and humans or other domesticated animals are also discussed. This review sheds light on cats as potential intermediate hosts for SARS-CoV-2 transmission, and cross-species transmission or zoonotic infection of FCoV and SARS-CoV-2 between cats and humans was identified.

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses.

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity.

The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap ((7Me)GpppAC(3-6)) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2'O position of the first transcribed nucleotide, thus converting (7Me)GpppAC(3-6) into (7Me)GpppA(2')(O)(Me)C(3-6). The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2'O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2'O methylation follows an order of events in which 2'O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

Feline Infectious Peritonitis Virus Nsp5 Inhibits Type I Interferon Production by Cleaving NEMO at Multiple Sites.

Feline infectious peritonitis (FIP), caused by virulent feline coronavirus, is the leading infectious cause of death in cats. The type I interferon (type I IFN)-mediated immune responses provide host protection from infectious diseases. Several coronaviruses have been reported to evolve diverse strategies to evade host IFN response. However, whether feline infectious peritonitis virus (FIPV) antagonizes the type I IFN signaling remains unclear. In this study, we demonstrated that FIPV strain DF2 infection not only failed to induce interferon-ß (IFN-ß) and interferon-stimulated gene (ISG) production, but also inhibited Sendai virus (SEV) or polyinosinic-polycytidylic acid (poly(I:C))-induced IFN-ß production. Subsequently, we found that one of the non-structural proteins encoded by the FIPV genome, nsp5, interrupted type I IFN signaling in a protease-dependent manner by cleaving the nuclear factor κB (NF-κB) essential modulator (NEMO) at three sites-glutamine132 (Q132), Q205, and Q231. Further investigation revealed that the cleavage products of NEMO lost the ability to activate the IFN-ß promoter. Mechanistically, the nsp5-mediated NEMO cleavage disrupted the recruitment of the TRAF family member-associated NF-κB activator (TANK) to NEMO, which reduced the phosphorylation of interferon regulatory factor 3 (IRF3), leading to the inhibition of type I IFN production. Our research provides new insights into the mechanism for FIPV to counteract host innate immune response.

Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function.

Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein.

A novel glycoprotein of feline infectious peritonitis coronavirus contains a KDEL-like endoplasmic reticulum retention signal.

A new protein of the feline infectious peritonitis virus (FIPV) was discovered in lysates of infected cells. Expression of the gene encoding open reading frame (ORF) 6b of FIPV in recombinant vaccinia virus infected cells was used to identify it as the 6b protein. It is a novel type of viral glycoprotein whose function is not clear. It is a soluble protein contained in microsomes; its slow export from the cell is caused by the presence of an ER-retention signal at the C-terminus. This amino acid sequence, KTEL, closely resembles the consensus KDEL-signal of soluble resident ER proteins. A mutant 6b protein with the C-terminal sequence KTEV became resistant to digestion by endo-beta-N-acetylglucosaminidase H with a half-time that was reduced threefold. In contrast, a mutant with the sequence KDEL was completely retained in the ER. The FIPV 6b protein is the first example of a viral protein with a functional KDEL-like ER-retention signal.

Feline aminopeptidase N is a receptor for all group I coronaviruses.

Human coronavirus HCV-229E and porcine transmissible gastroenteritis virus (TGEV), both members of coronavirus group I, use aminopeptidase N (APN) as their cellular receptors. These viruses show marked species specificity in receptor utilization as they can only use APN of their respective species to initiate virus infection. Feline and canine coronaviruses are also group I coronaviruses. To determine whether feline APN could serve as a receptor for feline coronaviruses (FCoVs), we cloned the cDNA encoding feline APN (fAPN) by PCR from feline cells and stably expressed it in FCoV-resistant mouse or hamster cells. These became susceptible to infection with either of several biotypes of FCoVs. The expression of recombinant fAPN also made hamster and mouse cells susceptible to infection with other group I coronaviruses, including several canine coronavirus strains, transmissible gastroenteritis virus (TGEV), and human coronavirus HCV-229E. Thus, fAPN served as a functional receptor for each of these coronaviruses in group I. As expected, fAPN could not serve as a receptor for mouse hepatitis virus (MHV), a group II coronavirus which uses murine biliary glycoproteins as receptors. Thus, fAPN acts as a common receptor for coronaviruses in group I, in marked contrast to human and porcine APN glycoproteins which serve as receptors only for human and porcine coronaviruses, respectively. These observations suggest that cats could serve as a "mixing vessel" in which simultaneous infection with several group I coronaviruses could lead to recombination of viral genomes.

Further characterization of aminopeptidase-N as a receptor for coronaviruses.

We recently reported that porcine aminopeptidase-N (pAPN) acts as a receptor for transmissible gastroenteritis virus (TGEV). In the present work, we addressed the question of whether TGEV tropism is determined only by the virus-receptor interaction. To this end, different non-permissive cell lines were transfected with the porcine APN cDNA and tested for their susceptibility to TGEV infection. The four transfected cell lines shown to express pAPN at their membrane became sensitive to infection. Two of these cell lines were found to be defective for the production of viral particles. This suggests that other factor(s) than pAPN expression may be involved in the production of infectious virions. The pAPN-transfected cells were also tested for their susceptibility to several viruses which have a close antigenic relationship to TGEV. So far, we failed to evidence permissivity to the feline infectious peritonitis coronavirus FIPV and canine coronavirus CCV. In contrast, we found clear evidence that porcine respiratory coronavirus PRCV, a variant of TGEV which replicates efficiently in the respiratory tract but to a very low extent in the gut, may also utilise APN to gain entry into the host cells. This suggests that the switch between TGEV and PRCV tropisms in vivo may involve other determinant(s) than receptor recognition.

Cloning and expression of FECV spike gene in vaccinia virus. Immunization with FECV S causes early death after FIPV challenge.

The spike gene of the feline enteric coronavirus (FECV), strain FECV-1683, was PCR amplified from total RNA extracted from FECV-infected cells and its sequence determined. A primary translation product of 1454 amino acids is predicted from the nucleotide sequence, containing a N-terminal signal sequence, a C-terminal transmembrane region and 33 potential N-glycosylation sites. The sequence shares 92% homology with the previously published feline infectious peritonitis virus, strain WSU-1146; however, several regions were identified that distinguished FECV from Feline Infectious Peritonitis virus, FIPV. The full length FECV S gene was cloned and expressed in vaccinia virus. Recombinants produced a 200 kD protein which was recognized by sera from cats infected with FIPV. When kittens were immunized with the vaccinia/FECV S recombinant, neutralizing antibodies to FIPV were induced. After challenge with a lethal dose of FIPV, the recombinant vaccinated animals died earlier than control animals immunized with vaccinia virus alone.

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

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.