Identification and characterization of a Golgi retention signal in feline coronavirus accessory protein 7b.
ABSTRACT: 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.
Project description:Feline infectious peritonitis (FIP) is the most frequent lethal infectious disease in cats. However, understanding of FIP pathogenesis is still incomplete. Mutations in the ORF 3c/ORF 7b genes are proposed to play a role in the occurrence of the fatal FIPV biotype. Here, we investigated 282 tissue specimens from 28 cats that succumbed to FIP. Within one cat, viral sequences from different organs were similar or identical, whereas greater discrepancies were found comparing sequences from various cats. Eleven of the cats exhibited deletions in the 3c gene, resulting in truncated amino acid sequences. The 7b gene was affected by deletions only in one cat. In three of the FIP cats, coronavirus isolates with both intact 3c genes as well as 7b genes of full length could also be detected. Thus, deletions or stop codons in the 3c sequence seem to be a frequent but not compelling feature of FIPVs.
Project description:Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.
Project description: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.
Project description:Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/?L, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.
Project description:The genes encoding accessory proteins 3a, 3b, 3c, 7a and 7b, the S2 domain of the spike (S) protein gene and the membrane (M) protein gene of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) samples were amplified, cloned and sequenced. For this faeces and/or ascites samples from 19 cats suffering from feline infectious peritonitis (FIP) as well as from 20 FECV-infected healthy cats were used. Sequence comparisons revealed that 3c genes of animals with FIP were heavily affected by nucleotide deletions and point mutations compared to animals infected with FECV; these alterations resulted either in early termination or destruction of the translation initiation codon. Two ascites-derived samples of cats with FIP which displayed no alterations of ORF3c harboured mutations in the S2 domain of the S protein gene which resulted in amino acid exchanges or deletions. Moreover, changes in 3c were often accompanied by mutations in S2. In contrast, in samples obtained from faeces of healthy cats, the ORF3c was never affected by such mutations. Similarly ORF3c from faecal samples of the cats with FIP was mostly intact and showed only in a few cases the same mutations found in the respective ascites samples. The genes encoding 3a, 3b, 7a and 7b displayed no mutations linked to the feline coronavirus (FCoV) biotype. The M protein gene was found to be conserved between FECV and FIPV samples. Our findings suggest that mutations of 3c and spike protein genes correlate with the occurrence of FIP.
Project description:Feline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studies in vivo To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grown in vitro Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterization in vivo are currently hampered by the lack of in vitro propagation systems.IMPORTANCE The availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequent in vivo studies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitable in vitro culture systems are not available.
Project description:Feline infectious peritonitis virus (FIPV), an alpha Coronavirus, is the causative agent of a fatal immune mediated disease in cats. It is currently unclear if this virus circulates in the field or develops in felines that are infected with Feline enteric coronavirus. To better understand the genomic changes associated with viral adaptation, we sequenced the complete genomes of FIPV WSU 79-1146 at different tissue passage levels: passage 1, passage 8, and passage 50 tissue culture. Twenty-one amino acid differences were observed in the polyprotein 1a/ab between the different passages. Only one residue change was observed in the spike glycoprotein, which reverted back on subsequent passages, four changes were observed in the 3c protein, and one change was observed in each 3a, small membrane, nucleocapsid and 7a proteins. The mutation rate was calculated to be 5.08-6.3 × 10(-6) nucleotides/site/passage in tissue culture suggesting a relatively stable virus. Our data show that FIPV has a low mutation rate as it is passed in cell culture but has the capacity for change specifically in nsp 2, 3c, and 7b as it is passed in cell culture.
Project description:Feline infectious peritonitis virus (FIPV) causes a fatal disease called FIP in Felidae. The effusion in body cavity is commonly associated with FIP. However, the exact mechanism of accumulation of effusion remains unclear. We investigated vascular endothelial growth factor (VEGF) to examine the relationship between VEGF levels and the amounts of effusion in cats with FIP. Furthermore, we examined VEGF production in FIPV-infected monocytes/macrophages, and we used feline vascular endothelial cells to examine vascular permeability induced by the culture supernatant of FIPV-infected macrophages. In cats with FIP, the production of effusion was related with increasing plasma VEGF levels. In FIPV-infected monocytes/macrophages, the production of VEGF was associated with proliferation of virus. Furthermore, the culture supernatant of FIPV-infected macrophages induced hyperpermeability of feline vascular endothelial cells. It was suggested that vascular permeability factors, including VEGF, produced by FIPV-infected monocytes/macrophages might increase the vascular permeability and the amounts of effusion in cats with FIP.
Project description:Purpose:We investigated sequences of the feline coronaviruses (FCoV), which include feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), from China and other countries to gain insight into the adaptive evolution of this virus. Methods:Ascites samples from 31 cats with suspected FIP and feces samples from 8 healthy cats were screened for the presence of FCoV. Partial viral genome sequences, including parts of the nsp12-nsp14, S, N, and 7b genes, were obtained and aligned with additional sequences obtained from the GenBank database. Bayesian phylogenetic analysis was conducted, and the possibility of recombination within these sequences was assessed. Analysis of the levels of selection pressure experienced by these sequences was assessed using methods on both the PAML and Datamonkey platforms. Results:Of the 31 cats investigated, two suspected FIP cats and one healthy cat tested positive for FCoV. Phylogenetic analysis showed that all of the sequences from mainland China cluster together with a few sequences from the Netherlands as a distinct clade when analyzed with FCoV sequences from other countries. Fewer than 3 recombination breakpoints were detected in the nsp12-nsp14, S, N, and 7b genes, suggesting that analyses for positive selection could be conducted. A total of 4, 12, 4, and 4 positively selected sites were detected in the nsp12-nsp14, S, N, and 7b genes, respectively, with the previously described site 245 of the S gene, which distinguishes FIPV from FECV, being a positive selection site. Conversely, 106, 168, 25, and 17 negative selection sites in the nsp12-14, S, N, and 7b genes, respectively, were identified. Conclusion:Our study provides evidence that the FCoV genes encoding replicative, entry, and virulence proteins potentially experienced adaptive evolution. A greater number of sites in each gene experienced negative rather than positive selection, which suggests that most of the protein sequence must be conservatively maintained for virus survival. A few of the sites showing evidence of positive selection might be associated with the more severe pathology of FIPV or help these viruses survive other harmful conditions.
Project description:The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.