Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats.
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ABSTRACT:
SUBMITTER: Borschensky CM
PROVIDER: S-EPMC7111757 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Project description:Feline infectious peritonitis (FIP) is a fatal infectious disease of wild and domestic cats, and the occurrence of FIP is frequently reported in China. To trace the evolution of type I and II feline coronavirus in China, 115 samples of ascetic fluid from FIP-suspected cats and 54 fecal samples from clinically healthy cats were collected from veterinary hospitals in China. The presence of FCoV in the samples was detected by RT-PCR targeting the 6b gene. The results revealed that a total of 126 (74.6%, 126/169) samples were positive for FCoV: 75.7% (87/115) of the FIP-suspected samples were positive for FCoV, and 72.2% (39/54) of the clinically healthy samples were positive for FCoV. Of the 126 FCoV-positive samples, 95 partial S genes were successfully sequenced. The partial S gene-based genotyping indicated that type I FCoV and type II FCoV accounted for 95.8% (91/95) and 4.2% (4/95), respectively. The partial S gene-based phylogenetic analyses showed that the 91 type I FCoV strains exhibited genetic diversity; the four type II FCoV strains exhibited a close relationship with type II FCoV strains from Taiwan. Three type I FCoV strains, HLJ/HRB/2016/10, HLJ/HRB/2016/11 and HLJ/HRB/2016/13, formed one potential new clade in the nearly complete genome-based phylogenetic trees. Further analysis revealed that FCoV infection appeared to be significantly correlated with a multi-cat environment (p < 0.01) and with age (p < 0.01). The S gene of the three type I FCoV strains identified in China, BJ/2017/27, BJ/2018/22 and XM/2018/04, exhibited a six nucleotide deletion (C4035 AGCTC4040 ). Our data provide evidence that type I and type II FCoV strains co-circulate in the FIP-affected cats in China. Type I FCoV strains exhibited high prevalence and genetic diversity in both FIP-affected cats and clinically healthy cats, and a multi-cat environment and age (<6 months) were significantly associated with FCoV infection.
| S-EPMC7168551 | biostudies-literature
Project description:It has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (FIP). However, only a few studies on the B-cell activation mechanism after FIP virus (FIPV) infection have been reported. The present study shows that: (1) the ratio of peripheral blood sIg(+) CD21(-) B-cells was higher in cats with FIP than in SPF cats, (2) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sIg(+) CD21(-) B-cell, (3) cells strongly expressing mRNA of the plasma cell master gene, B-lymphocyte-induced maturation protein 1 (Blimp-1), were increased in peripheral blood in cats with FIP, (4) mRNA expression of B-cell differentiation/survival factors, IL-6, CD40 ligand, and B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), was enhanced in macrophages in cats with FIP, and (5) mRNAs of these B-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ADE)-induced macrophages. These data suggest that virus-infected macrophages overproduce B-cell differentiation/survival factors, and these factors act on B-cells and promote B-cell differentiation into plasma cells in FIPV-infected cats.
| S-EPMC7087278 | biostudies-literature
Project description:Feline coronaviruses (FCoVs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. These proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. In the current study, we produced and characterized 7b-specific monoclonal antibodies (mAbs). A recombinant form of the 7b protein was expressed as a fusion protein in Escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. Two hybridoma lines, 5B6 and 14D8, were isolated that expressed mAbs that recognized 7b proteins of both FCoV serotypes. Using an extensive set of N- and C-terminally truncated 7b proteins expressed in E. coli and a synthetic peptide, the binding sites of mAbs 5B6 and 14D8 were mapped to an 18-residue region that comprises the only potential N-glycosylation site of the FCoV 7b protein. The two mAbs were suitable to detect a 24-kDa protein, which represents the nonglycosylated form of 7b in FCoV-infected cells. We speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site.
| S-EPMC7117465 | biostudies-literature
Project description:Uveitis is common in cats, and is often a feature of feline infectious peritonitis (FIP). We evaluated 3 tools for detection of feline coronavirus (FCoV) in aqueous humor: 1) a 7b gene reverse-transcription real-time PCR (7b-RT-rtPCR) assay to detect FCoV RNA, 2) a spike gene mutation RT-rtPCR (S-RT-rtPCR) assay to detect 2 point mutations in the spike gene of FCoV in cats positive by 7b-RT-rtPCR, and 3) immunocytochemistry (ICC) for detection of FCoV antigen in aqueous humor macrophages. We studied 58 cats, including 31 cats with FIP and 27 control cats. FIP was excluded by postmortem examination and negative immunohistochemistry (IHC). Aqueous humor samples obtained postmortem were assessed using 7b-RT-rtPCR in all cats, and positive samples were evaluated with S-RT-rtPCR. ICC evaluation of aqueous humor samples from 36 of the 58 cats was done using an avidin-biotin complex method and monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, and negative and positive predictive values were calculated including 95% CIs. 7b-RT-rtPCR had a specificity of 100.0% (95% CI: 87.2-100.0) and sensitivity of 35.5% (95% CI: 19.2-54.6). Specificity of S-RT-rtPCR could not be determined because there were no FCoV 7b-RT-rtPCR-positive samples in the control group. Sensitivity of S-RT-rtPCR was 12.9% (95% CI 3.6-29.8). Sensitivity and specificity of ICC were 62.5% (95% CI: 40.6-81.2) and 80.0% (95% CI: 44.4-97.5), respectively. The combination of 7b-RT-rtPCR and IHC could be useful in diagnosing FIP; S-RT-rtPCR did not add value; and ICC of aqueous humor samples cannot be recommended for the diagnosis of FIP.
| S-EPMC7438658 | biostudies-literature
Project description:(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.
| S-EPMC9412601 | biostudies-literature
Project description:As previously demonstrated by our research group, the oral multicomponent drug Xraphconn® containing GS-441524 was effective at curing otherwise fatal feline infectious peritonitis (FIP) in 18 feline coronavirus (FCoV)-infected cats. The aims of the current study were to investigate, using samples from the same animals as in the previous study, (1) the effect of treatment on fecal viral RNA shedding; (2) the presence of spike gene mutations in different body compartments of these cats; and (3) viral RNA shedding, presence of spike gene mutations, and anti-FCoV antibody titers in samples of 12 companion cats cohabitating with the treated cats. Eleven of the eighteen treated FIP cats (61%) were shedding FCoV RNA in feces within the first three days after treatment initiation, but all of them tested negative by day 6. In one of these cats, fecal shedding reoccurred on day 83. Two cats initially negative in feces were transiently positive 1-4 weeks into the study. The remaining five cats never shed FCoV. Viral RNA loads in feces decreased with time comparable with those in blood and effusion. Specific spike gene mutations linked to systemic FCoV spread were consistently found in blood and effusion from treated FIP cats, but not in feces from treated or companion cats. A new mutation that led to a not yet described amino acid change was identified, indicating that further mutations may be involved in the development of FIP. Eight of the twelve companion cats shed FCoV in feces. All but one of the twelve companion cats had anti-FCoV antibodies. Oral treatment with GS-441524 effectively decreased viral RNA loads in feces, blood, and effusion in cats with FIP. Nonetheless, re-shedding can most likely occur if cats are re-exposed to FCoV by their companion cats.
| S-EPMC9147249 | biostudies-literature
Project description:Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a-c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.
| S-EPMC4868892 | biostudies-literature
Project description:Coronaviruses (CoVs) are important human and animal pathogens that cause respiratory and gastrointestinal diseases. Porcine epidemic diarrhoea (PED), characterized by severe diarrhoea and vomiting in pigs, is a highly lethal disease caused by porcine epidemic diarrhoea virus (PEDV) and causes substantial losses in the swine industry worldwide. However, currently available commercial drugs have not shown great therapeutic effects. In this study, a fluorescence resonance energy transfer (FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds, 3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone, that target the 3C-like protease (3CLpro) of PEDV. These compounds are of low molecular weight (MW) and greatly inhibited the activity of this enzyme (IC50 values were obtained in this study). Furthermore, these compounds exhibited antiviral capacity against another member of the CoV family, feline infectious peritonitis virus (FIPV). Here, the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified (with EC50 values) and cell viability assays were performed. The results of putative molecular docking models indicate that these compounds, labeled compound 1 and compound 2, contact the conserved active sites (Cys144, Glu165, Gln191) of 3CLpro via hydrogen bonds. These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.
| S-EPMC8258280 | biostudies-literature
Project description:ObjectivesFeline infectious peritonitis (FIP) emerges when feline coronaviruses (FCoVs) mutate within their host to a highly virulent biotype and the immune response is not able to control the infection. FCoV spike (S) gene mutations are considered to contribute to the change in virulence by enabling FCoV infection of and replication in macrophages. This study investigated the presence of FCoV with and without S gene mutations in cats with FIP using two different real-time RT-PCRs on different samples obtained under clinical conditions.MethodsFine-needle aspirates (FNAs) and incisional biopsies (IBs) of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys (each n = 20), EDTA blood (n = 13), buffy coat smears (n = 13), serum (n = 11), effusion (n = 14), cerebrospinal fluid (n = 16), aqueous humour (n = 20) and peritoneal lavage (n = 6) were obtained from 20 cats with FIP diagnosed by immunohistochemistry. Samples were examined by RT-PCR targeting the FCoV 7b gene, detecting all FCoV, and S gene mutation RT-PCR targeting mutations in nucleotides 23531 and 23537. The prevalence of FCoV detected in each sample type was calculated.ResultsIn 20/20 cats, FCoV with S gene mutations was present in at least one sample, but there was variation in which sample was positive. FCoV with mutations in the S gene was most frequently found in effusion (64%, 95% confidence interval [CI] 39-89), followed by spleen, omentum and kidney IBs (50%, 95% CI 28-72), mesenteric lymph node IBs and FNAs (45%, 95% CI 23-67), and FNAs of spleen and liver and liver IBs (40%, 95% CI 19-62).Conclusions and relevanceIn these 20 cats with FIP, FCoVs with S gene mutations were found in every cat in at least one tissue or fluid sample. This highlights the association between mutated S gene and systemic FCoV spread. Examining a combination of different samples increased the probability of finding FCoV with the mutated S gene.
| S-EPMC7206566 | biostudies-literature
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.
| S-EPMC7117521 | biostudies-literature