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PubMed1993

Molecular mimicry between S peplomer proteins of coronaviruses (MHV, BCV, TGEV and IBV) and Fc receptor.

In previous studies we have demonstrated molecular mimicry between the S peplomer protein of Mouse Hepatitis Virus (MHV) and Fc gamma Receptor (Fc gamma R) of IgG. Rabbit IgG, but not its F(ab')2 fragments, monoclonal rat and mouse IgG and the rat 2.4G2 anti-mouse Fc gamma R monoclonal antibody (mab) immunoprecipitated natural and recombinant MHV S protein. On the basis of a number of criteria, MHV S peplomer protein exhibits Fc IgG binding ability. We report here a molecular mimicry between the S peplomer protein of Bovine Coronavirus (BCV) and Fc gamma R. BCV S peplomer protein which belongs to the same antigenic subgroup as MHV also binds Fc portion of rabbit IgG and is immunoprecipitated by the 2.4G2 anti-Fc gamma R mab. In contrast, Transmissible Gastroenteritis Coronavirus (TGEV) and Infectious Bronchitis Virus (IBV) S peplomer proteins which represent two distinct antigenic subgroups of Coronaviridae do not bind rabbit IgG and do not react with anti-Fc gamma R mab. However, homologous swine IgG, but not its F(ab')2 fragments, immunoprecipitated from TGEV-infected cells a polypeptide chain with molecular mass of 195 kDa, identical to that immunoprecipitated by the T36 mab anti-TGEV S peplomer protein.

Animals↗

PubMed2026

SCAN: A sample-to-answer cross-priming isothermal assay for on-site virus detection with RT-qPCR sensitivity and genomically similar virus differentiation specificity.

Genomically similar viruses often differ in pathogenicity and host tropism due to specific mutations, and failure to distinguish them risks misdiagnosis and ineffective control. Molecular methods can differentiate such viruses but require laboratory settings and skilled personnel, while field-deployable immunological methods suffer from cross-reactivity. To address this challenge, we developed SCAN (Sample-to-answer Cross-priming isothermal amplification Assay with Nucleic acid strip), a general framework for on-site detection of genomically similar viruses. Comparative bioinformatics of isolation and sequencing data identifies key conserved differential determinants for primer design, ensuring specificity and reducing non-specific amplification. A one-tube cross-priming isothermal amplification (CPA) enables rapid target amplification without thermal cycling, and the products are visually detected on a nucleic acid strip. All steps are integrated into a handheld, lightweight device (9.9 × 4.4 × 3.3 cm, <200 g) that also prevents aerosol contamination. Using transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), the latter a natural mutant of TGEV, as a model, SCAN achieves a detection limit of 102 copies/μL with sensitivity comparable to RT-qPCR and supports sample-to-answer testing within 80 min and simple operations. With verified high sensitivity, specificity, and accuracy, as well as field usability, SCAN provides a generalizable route for developing point-of-care tests (PoCT) that require precise field differentiation of closely related pathogens.

Cross-priming isothermal amplification↗

PubMed2006

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation.

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.

Animals↗

PubMed1994

Sequence of the spike protein of the porcine epidemic diarrhoea virus.

The complete sequence of the spike (S) gene of the Br1/87 isolate of porcine epidemic diarrhoea virus (PEDV) was determined from cDNA clones. The predicted polypeptide was 1383 amino acids long, contained 29 potential N-linked glycosylation sites and showed structural features similar to those of the coronavirus spike protein. The PEDV S protein, like that of the members of the transmissible gastroenteritis virus (TGEV)-related subset, lacks a proteolytic site to yield cleaved amino and carboxy subunits S1 and S2. Viral polypeptide species of the expected M(r), i.e. 170K/190K, were observed in PEDV-infected cells. Sequence comparison confirmed that, within the subset, PEDV was most closely related to the human respiratory coronavirus HCV 229E. However, PEDV S protein has an additional 250 residue N-terminal domain which is absent from HCV 229E and porcine respiratory coronavirus, the respiratory variant of TGEV. Alignment of the S1 regions revealed a second domain of about 90 residues with increased sequence divergence which might possibly express virus-specific determinants.

Amino Acid Sequence↗

PubMed1995

Frequency of interferon-alpha-secreting blood leukocytes in irradiated and bone-marrow-grafted pigs.

The effects of irradiation were studied on porcine interferon-alpha (IFN-alpha) secreting cells (IFN-alpha SC). IFN-alpha SC were characterized by an ELISPOT assay on non-adherent PBMC following incubation with the transmissible gastroenteritis coronavirus. In vitro irradiation of PBMC was followed by a decrease in the number of IFN-alpha SC while IFN-gamma production and cell viability were not affected. These data indicate that porcine IFN-alpha SC are relatively radiosensitive. Indeed, the frequency of blood IFN-alpha SC decreased markedly and rapidly after in vivo whole body or partial lymphoid irradiation. In addition, within several days of compatible bone-marrow engraftment in the irradiated animals, the number of blood IFN-alpha SC returned to normal values. These data demonstrate that circulating porcine IFN-alpha SC are derived from bone-marrow progenitors.

Animals↗

PubMed2005

Design of wide-spectrum inhibitors targeting coronavirus main proteases.

The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M(pro)s), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M(pro)s, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

Animals↗

PubMedDate not supplied

Screening of a small set of random peptides: a new strategy to identify synthetic peptides that mimic epitopes.

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10(6)-10(7) random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC50s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest.

Animals↗

PubMed1997

In vivo induction of interferon-alpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alpha-producing cells.

A low frequency peripheral blood mononuclear cell (PBMC) subpopulation, referred to as natural interferon-producing (NIP) cells, is described as producing interferon-alpha (IFN-alpha) following contact with non-infectious viral structures, namely viral glycoproteins. These cells are characterized in vitro as non-T, non-B, MHC class II+ and CD4+ cells. In this study, NIP cells were analysed in vivo after an intravenous injection of UV-inactivated transmissible gastroenteritis virus in newborn piglets, which resulted in strong serum IFN-alpha production. Splenocytes, but not PBMC, were the IFN-alpha producers in vivo. Using double immunohistochemical labelling for both IFN-alpha and leukocyte markers, we established that splenic NIP cells were not T or B cells. The majority were MHC class II+ and only a minority expressed a macrophage marker. NIP cells were localized in contact with MHC class II-expressing cells and T cells, which suggested that NIP cells might modulate the antiviral immune response.

Animals↗

PubMed1993

Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid.

Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar. Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo). The IPI-1 cells were found of fibroblastic lineage. The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells. A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency. Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers. Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells. Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning. These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm. The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line. All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus). The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig. In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells.

Animals↗

PubMed1992

Isolation of sequences from a random-sequence expression library that mimic viral epitopes.

We describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa- or octapeptides attached to a beta-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site III and one for site IV. The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed.

Amino Acid Sequence↗

PubMed1998

Characterization of determinants involved in the feline infectious peritonitis virus receptor function of feline aminopeptidase N.

Feline aminopeptidase N (fAPN) is a major cell surface receptor for feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV), human coronavirus 229E (HCV 229E) and canine coronavirus (CCV). By using chimeric molecules assembled from porcine, human and feline APN we have analysed the determinants involved in the coronavirus receptor function of fAPN. Our results show that amino acids 670-840 of fAPN are critically involved in its FIPV and TGEV receptor function whereas amino acids 135-297 are essential for the HCV 229E receptor function. We also demonstrate that a chimeric molecule assembled from human and porcine APN is able to act as a receptor for FIPV. This is surprising as neither human nor porcine APN by themselves mediate FIPV infection. These results suggest that different determinants in the APN protein are involved in mediating the coronavirus receptor function.

Animals↗

PubMed2003

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.

A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Differences in virus receptor for type I and type II feline infectious peritonitis virus.

Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120-130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs.

Amino Acid Sequence↗

PubMed1991

Processing and antigenicity of entire and anchor-free spike glycoprotein S of coronavirus TGEV expressed by recombinant baculovirus.

The S gene of transmissible gastroenteritis virus (TGEV) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer plasmid pVL941. Infection of Sf9 insect cells with the recombinant virus resulted in the synthesis of a 175K polypeptide which was able to trimerize and was transported to the cell surface as is the authentic TGEV S protein. Despite the lack of complete carbohydrate processing, the recombinant S protein exhibited antigenic properties similar to TGEV S and induced high levels of neutralizing antibodies in immunized rats. Engineering a deletion (70 amino acids) into the carboxy-terminus containing the membrane anchor of the polypeptide allowed its secretion. The oligomerization process and the antigenic profile of the anchor-free S protein were shown to be partially altered.

Antibodies, Monoclonal↗

PubMed1993

Genomic organization and expression of the 3' end of the canine and feline enteric coronaviruses.

The genomic organization at the 3' end of canine coronavirus (CCV) and feline enteric coronavirus (FECV) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine. Comparison of the latter two has previously revealed an extra open reading frame (ORF) at the 3' end of the FIPV genome, lacking in TGEV, now designated ORF 6b. Both CCV and FECV possess 6b-related ORFs. The CCV ORF 6b is colinear with that of FIPV, but the predicted amino acid sequences are only 58% identical. The FECV ORF 6b contains a large deletion compared to that of FIPV, reducing the colinear part to 60%. The sequence homologies were highest between CCV and TGEV on the one hand and between FECV and FIPV on the other. The expression product of the CCV and the FECV ORF 6b can be detected in infected cells by immunoprecipitation.

Animals↗

PubMed1993

Identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity.

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibody-dependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (K-cELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Animals↗

PubMed1997

Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens.

A diagnostic test for feline infectious peritonitis virus (FIPV) infection based on a nested PCR (nPCR) assay was developed and tested with FIPV, feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV) and clinical fluid samples from cats with effusive feline infectious peritonitis (FIP). The target sequence for the assay is in the S1 region of the peplomer protein E2 gene. A vaccine strain of FIPV and two wild-type FIPV strains tested positive, but FECV, TGEV, and CCV tested negative. Preliminary tests with 12 cats with clinical evidence of effusive FIP and 11 cats with an illness associated with effusions, but attributed to other causes, were performed. Eleven of the 12 cats with effusive FIP tested positive, while 1 was negative. Ten of the 11 cats ill from other causes tested negative, while 1 was positive. On the basis of clinical laboratory and histopathologic criteria, the preliminary sensitivity and specificity of the assay were 91.6 and 94%, respectively.

Animals↗

PubMed1991

Histocompatible miniature pig (d/d haplotype): generation of hybridomas secreting A or M monoclonal antibody.

Two Hypoxanthine/Aminopterin/Thymidine-sensitive cell sublines (L142 and L231) have been derived from independent lymphoblastoid cell lines of B lineage. After propagation for more than 100 population doublings (1 year) in culture, these cells still retained a doubling time between 19 to 20 hours, near diploïdy and relatively low (L142) and high (L231) secretion rate of M immunoglobulins. Near diploid hybrid cells were easily generated with leukocytes from the spleen, the gut lamina propria or the mesenteric lymph nodes of pigs immunized against the transmissible gastroenteritis virus. Both the tumor sublines and the B cells were derived from histocompatible miniature pigs (d/d haplotype). Demonstration of fusion between the tumor sublines and B-cells was supported by the selection of hybridomas making the antigen-specific heavy (alpha isotype) and light chains from the B cell parent as well as the heavy and light chains of the lymphoblastoid parent. Moreover, some hybridomas were found to secrete only class A (dimeric) or class M immunoglobulins (0.2-10 micrograms/ml). Forty hybridomas secreted antibodies reactive in a virus-enzyme-linked cell immunoassay against cell-bound antigens and two were found to produce an antibody active only against the infected cell monolayer. Construction of intraspecies hybridoma can be used to perpetuate lymphocyte subsets useful for the study of the porcine immune system.

Animals↗