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PubMed1981

Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses.

The infectivity and pathogenicity to newborn pigs of antigenically related coronaviruses from pigs (transmissible gastroenteritis virus; TGEV), cats (feline infectious peritonitis virus; FIPV), and dogs (canine gastroenteritis virus; CGEV) were studied by light, scanning electron, and immunofluorescence microscopy. Hysterectomy-derived, 12-hour-old pigs were orally given tissue culture or frozen preparations of 6 coronavirus strains (3 porcine, 2 feline, and 1 canine). The pigs were killed at regular intervals between 24 and 144 hours after exposure. Virulent TGEV and virulent FIPV produced necrosis of villous epithelium, resulting in villous atrophy in the jejunum and the ileum. Similar, but less extensive and severe lesions, were produced by the 4 other viruses. Coronaviral antigens were identified by immunofluorescence in villous epithelial cells of pigs that had been inoculated with virulent TGEV, attenuated TGEV, virulent FIPV, and tissue culture-adapted FIPV. In contrast, coronaviral antigens were not induced by the small plaque variant TGEV and virulent CGEV in the villous epithelium, but rather in cells of the lamina propria and crypt epithelium.

Animals↗

PubMed1993

Enrichment of coronavirus-induced interferon-producing blood leukocytes increases the interferon yield per cell: a study with pig leukocytes.

Porcine peripheral blood mononuclear cells, which secrete IFN alpha in response to a coronavirus, transmissible gastroenteritis virus, were detected by a filter immunoplaque assay (ELISPOT). IFN alpha-producing cells (IPC), which are present at a low frequency in the blood, could be enriched up to 100-fold by sequential depletion of plastic-adherent cells and cell fractionation on metrizamide density gradients. IPC were present in the non-adherent low-density cell subpopulation. Cell selection experiments using antibody (Ab)-coated immunomagnetic beads revealed that porcine IPC could be positively selected by anti-CD4 or -SLA-class-II Ab, but not by anti-CD2 or -CD8 Ab. The estimated IFN yield per IPC was found to increase when IPC were assayed at higher concentrations. These data suggest that IPC represent a unique and distinct cell population in the blood, which could secrete higher amounts of IFN following its accumulation at a site of viral infection.

Animals↗

PubMed2005

Coronavirus reverse genetics and development of vectors for gene expression.

Knowledge of coronavirus replication, transcription, and virus-host interaction has been recently improved by engineering of coronavirus infectious cDNAs. With the transmissible gastroenteritis virus (TGEV) genome the efficient (>40 microg per 106 cells) and stable (>20 passages) expression of the foreign genes has been shown. Knowledge of the transcription mechanism in coronaviruses has been significantly increased, making possible the fine regulation of foreign gene expression. A new family of vectors based on single coronavirus genomes, in which essential genes have been deleted, has emerged including replication-competent, propagation-deficient vectors. Vector biosafety is being increased by relocating the RNA packaging signal to the position previously occupied by deleted essential genes, to prevent the rescue of fully competent viruses that might arise from recombination events with wild-type field coronaviruses. The large cloning capacity of coronaviruses (>5 kb) and the possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, has increased the potential of coronaviruses as vectors for vaccine development and, possibly, gene therapy.

Animals↗

PubMed1993

Age-related increase of porcine natural interferon alpha producing cell frequency and of interferon yield per cell.

Porcine blood mononuclear cells (PBMC) were shown to secrete interferon alpha (IFN-alpha) after induction by a coronavirus, the transmissible gastroenteritis virus (TGEV). IFN-alpha producing cells, referred to as natural interferon alpha producing (NIP) cells, were detected by an ELISPOT assay using anti-porcine IFN-alpha monoclonal antibodies. The frequency of NIP cells among blood cells is low, at most 40-110 per 10(5) PBMC and each NIP cell was found to produce several units of IFN. We have shown that NIP cell frequency and IFN yield per cell gradually increased with the age of the donor animals, from the neonatal period to the adult age, with a significant increase around puberty. Our present results also indicate that NIP cells may be influenced by physiological and genetic factors; thus (1) NIP cell frequency and IFN yield per cell were decreased during lactation; (2) Chinese (Meishan) pigs were found to have higher NIP cell frequency and IFN yield per cell than European (Large White) animals.

Aging↗

PubMed1991

Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683.

We prepared 31 monoclonal antibodies (MAbs) against either FIPV strain 79-1146 or FECV strain 79-1683, and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay (IFA). Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses, also reacted with canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). In many of them, the polypeptide specificity was the recognition of transmembrane (E1) protein of the virus. We succeeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses (showing cell-associated growth) but reacted with FIPV Type II (79-1146, KU-1) and/or FECV Type II (79-1683) (showing non-cell associated growth). These MAbs also reacted with CCV or TGEV. These MAbs recognized peplomer (E2) glycoprotein, and many antigenic differences were found in this E2 protein. These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses. Furthermore, the MAb prepared in this study has enabled discrimination between FIPV strain 79-1146 and FECV strain 79-1683, which was thought to be impossible by the previous serological method.

Animals↗

PubMed1996

Comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline, canine and porcine coronaviruses.

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79-1683. Only M and N genes were analyzed in strain KU-2 and strain 79-1683 which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79-1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.

Amino Acid Sequence↗

PubMed1985

Effect of virus-induced destruction of villous epithelium on intestinal secretion induced by heat-stable Escherichia coli enterotoxins and prostaglandin E1 in swine.

Villous atrophy and crypt hyperplasia were induced in the jejunal epithelium of thirteen 3-week-old pigs by inoculation with transmissible gastroenteritis virus. The responses (changes in net fluid movement) induced in ligated intestinal loops of these pigs by intraloop injections of prostaglandin E1 (PGE1) or Escherichia coli broth culture filtrates containing either or both E coli heat-stable enterotoxins (STa and STb) were compared with the responses induced by these preparations in littermates not inoculated with virus. Villous atrophy was associated with a marked decrease in response to preparations containing STa, STb, or STa + STb, but the response to PGE1 was undiminished. These results were consistent with the reports of others that the response to cyclic adenosine monophosphate-mediated secretogogues (PGE1) is a function of crypt epithelium; however, the present results also suggest that the secretory response to STa and to STb is dependent on the integrity of the villous epithelium. In the present study, loss of villous epithelium was associated with loss of response to STa and STb, but not to PGE1.

Alprostadil↗

PubMed1991

Characterization of monoclonal antibodies against feline infectious peritonitis virus type II and antigenic relationship between feline, porcine, and canine coronaviruses.

Seven monoclonal antibodies (MAbs) with neutralizing activity against feline infectious peritonitis virus (FIPV) strain 79-1149 (type II) were prepared. When the polypeptide specificity recognized by these monoclonal antibodies (MAbs) was investigated by Western immunoblotting, all of the MAbs reacted with peplomer glycoprotein (S) of the virus. By competitive binding assay these MAbs were found to recognize at least 3 different epitopes. The reactivity of these MAbs with 6 viruses classified as FIPV type I (UCD-1, UCD-2, UCD-3, UCD-4, NW-1, and Black), feline enteric coronavirus (FECV) type II strain 79-1683, canine coronavirus (CCV) strain 1-71, and transmissible gastroenteritis virus (TGEV) strains TO-163 and SH was examined by neutralization tests. All MAbs neutralized FECV strain 79-1683, CCV strain 1-71, and TGEV strains TO-163 and SH, while they did not neutralize the 6 FIPV type I viruses. Moreover, the MAb against TGEV strain TO-163, which has strong neutralizing activity against 7 TGEV viruses, neutralized CCV strain 1-71, FECV strain 79-1683, and FIPv strain 79-1146, but did not neutralize the 6 FIPV type I viruses. These results demonstrated that there are at least 3 epitopes involved in the neutralization of FIPV type II strain 79-1146, and that these epitopes are not present in FIPV type I viruses but are present in FECV strain 79-1683 which does not induce feline infectious peritonitis, TGEV strains TO-163 and SH, and CCV strain 1-71. These results suggest the presence of 2 serotypes of FIPV which can be clearly distinguished by the neutralization test using MAbs.

Animals↗

PubMed1989

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice.

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.

Animals↗

PubMedDate not supplied

Enteric coronavirus TGEV: partial sequence of the genomic RNA, its organization and expression.

The sequence of the 3'-most 8300 nucleotides of the genome RNA of the Purdue-115 strain of the transmissible gastroenteritis virus TGEV, a porcine coronavirus, was determined from cDNA clones. The available sequence corresponds to the part of the genome (total length greater than 20 kb) expressed through subgenomic mRNAs. The 5 subgenomic and the genomic RNA species detected in TGEV-infected cells form a 3'-coterminal 'nested' structure, a unique feature of Coronaviridae. The transcription initiation site of the TGEV subgenomic RNAs appears to involve the hexameric sequence 5'CTAAAC, which is present upstream from each coding region. In addition to the previously identified genes encoding the three structural proteins, E2, E1 and N, two regions, X1 and X2, corresponding to the non-overlapping portion of mRNAs 4 and 3, may code for so far unidentified non-structural polypeptides. The predicted X1 polypeptide (9.2 kDa) is highly hydrophobic. The sequence of the X2 region allows the translation of two non-overlapping products, i.e., X2a (7.7 kDa) and X2b (18.8 kDa). No RNA species liable to express the extreme 3' open reading frame X3 was found.

Base Sequence↗

PubMed1998

Interference of coronavirus infection by expression of IgG or IgA virus neutralizing antibodies.

Mouse immunoglobulin gene fragments encoding the variable modules of the heavy (VH) and light (VL) chains of a transmissible gastroenteritis coronavirus (TGEV) neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. Chimeric immunoglobulin genes with the variable modules from the murine MAb and constant modules of human gamma 1 and kappa chains were constructed using RT-PCR. These chimeric immunoglobulins were stably or transiently expressed in murine myelomas and COS cells, respectively. The secreted recombinant antibodies had radioimmunoassay (RIA) titers higher than 10(3) and reduced the infectious virus more than 10(4)-fold. Recombinant dimeric IgA showed a 50-fold enhanced neutralization of TGEV relative to a recombinant monomeric IgG1 which contained the identical antigen binding site. Epithelial cell lines stably-transformed with these constructs and expressing either recombinant IgG or IgA TGEV neutralizing antibodies reduced virus production by > 10(5)-fold after infection with homologous virus, although a residual level of virus production (< 10(2) PFU/ml) remained in less than 0.1% of the cells.

Animals↗

PubMed2001

Enhanced immune responses to viral epitopes by combining macrophage-inducible expression with multimeric display on a Salmonella vector.

In this study, the immunogenicity of chimeric 987P fimbriae on a Salmonella vaccine strain was improved by optimizing fimbrial expression. The constitutive tetA promoter and the in vivo activated nirB and pagC promoters were evaluated for their use to express two epitopes of the transmissible gastroenteritis virus (TGEV) spike protein carried by fimbriae which were displayed on a Salmonella vaccine strain. Constructs with the pagC promoter were shown to drive increased expression of chimeric 987P fimbriae in macrophages as well as in Mg(2+)-poor media, mimicking a major environmental signal found in Salmonella-containing endocytic vacuoles of macrophages. Mice immunized orally with a Salmonella vaccine strain which expressed chimeric fimbriae from the pagC promoter elicited significantly higher mucosal and systemic immune responses to both the 987P fimbriae and the TGEV epitopes than mice immunized with the same strain hosting a tetA or nirB promoter-driven expression plasmid. Moreover, only the Salmonella vaccine strains harboring a plasmid with the pagC promoter, with or without an additional tetA promoter in tandem, elicited neutralizing antibodies to TGEV. This indicated that the pagC promoter can be used successfully to improve epitope-display by chimeric fimbriae on Salmonella vaccine strains for the induction of a desired immune response.

Animals↗

PubMed1990

Assembly of coronavirus spike protein into trimers and its role in epitope expression.

The folding and oligomerization of coronavirus spike protein were explored using a panel of monoclonal antibodies. Chemical cross-linking and sedimentation experiments showed that the spike of transmissible gastroenteritis virus is a homotrimer of the S membrane glycoprotein. The spike protein was synthesized as a 175,000-apparent-molecular-weight (175K) monomer subunit that is sensitive to endo-beta-N-acetylglucosaminidase H. Assembly of monomers into a trimeric structure was found to occur on a partially trimmed polypeptide and to be a rate-limiting step, since large amounts of monomers failed to trimerize 1 h after completion of synthesis. Terminal glycosylation of newly assembled trimers, resulting in the biosynthesis of three 220K oligomers, occurred with a half time of approximately 20 min. Monomeric (230K to 240K) processed forms were also observed in cells and in virions. The 175K monomeric form expressed four major antigenic sites previously localized within the amino-terminal half of the S polypeptide chain; however, two classes of trimer-restricted epitopes (borne by three 220K and/or three 175K oligomers) were identified. The S glycoprotein of coronavirus might be a valuable model system for discovering new aspects of the maturation of membrane glycoproteins.

Animals↗

PubMed1992

Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.

Coronaviruses, like many animal viruses, are characterized by a restricted host range and tissue tropism. Transmissible gastroenteritis virus (TGEV), a major pathogen causing a fatal diarrhoea in newborn pig, replicates selectively in the differentiated enterocytes covering the villi of the small intestine. To investigate the molecular determinants of the infection, we characterized the surface molecule used by the virus for binding and entry into host cells. Here we report that aminopeptidase N, an ectoenzyme abundantly expressed at the apical membrane of the enterocytes, serves as a receptor for TGEV. Monoclonal antibodies were selected for their ability to block infection by TGEV of porcine cell lines. They recognized a brush-border membrane protein of M(r) 150K, which was identified as aminopeptidase N by amino-terminal sequencing. Two lines of evidence supported the view that the peptidase itself acts as a receptor. First, virions bound specifically to aminopeptidase N that was purified to homogeneity. Second, recombinant expression of aminopeptidase N conferred infectivity by TGEV to an otherwise non-permissive cell line.

Amino Acid Sequence↗

PubMed1989

Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons.

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.

Animals↗

PubMed2002

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, M(pro), a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus M(pro) is reported. The structure was refined to 1.96 A resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that M(pro) self-processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin-like fold that is connected by a 16 residue loop to an extra domain featuring a novel alpha-helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of M(pro) substrates. Interactions involving the N-terminus and the alpha-helical domain stabilize the loop in the orientation required for trans-cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

Amino Acid Sequence↗

PubMed1979

A seroepizootiologic study of five viruses in a swine-evaluation station.

This serologic study was done to gain information on the spread, maintenance, and effect upon performance of five porcine viruses. Blood samples were taken from two groups of 8- to 11-week-old pigs from a large number of Indiana swine herds in a performance-testing station 1 week after entry, 7 weeks after entry (one group only), and at slaughter. The sera were tested by indirect fluorescent antibody tests for antibodies to transmissible gastroenteritis virus (TGEV), swine influenza virus (SIV), hemagglutinating encephalomyelitis virus (HEV), porcine adenovirus (PAV), and pseudorabies virus (PRV). Seroconversions to TGEV, HEV, and PAV occurred in a group of pigs entered in May and slaughtered in August (group 1). In the group that was entered in October and slaughtered in January (group 2), pigs developed antibodies to SIV, HEV, and PAV, but not to TGEV. Only 1 of the 434 pigs tested had antibodies to PRV, and there were no seroconversions to this virus. The only statistically valid effect of infection on performance was found in group 1 pigs, which had seroconverted to TGEV during the first 7 weeks of their stay. These pigs gained 0.077 kg less per day than pigs that did not develop antibodies to TGEV during that period. The pattern of serologic reactions was indicative of a relatively slow spread of these viruses in the groups. We interpret this as supporting the concept that a relatively slow spread of these viruses through large groups of pigs kept under conditions that are less than optimum for virus spread may be an important means of their interepizootic survival.

Adenoviridae↗

PubMed1997

Pathogenicity and sequence analysis studies suggest potential role of gene 3 in virulence of swine enteric and respiratory coronaviruses.

Coronaviruses have been commonly associated with enteric and respiratory diseases. Two of the swine coronaviruses, namely transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), have been extensively studied. TGEV replicates in both the enteric and respiratory tracts and causes enteric disease, whereas, PRCV replicates in the respiratory tract with limited to no replication in the enteric tract. We have isolated PRCV from swine herds with respiratory disease and have reproduced moderate pneumonia in gnotobiotic and conventionally reared pigs with two of the PRCV isolates. We have also identified two PRCV isolates with low virulence. One consistent difference that we have observed between PRCV isolates of different pathogenicities is in gene 3. The gene 3 is intact in the two virulent PRCV isolates, whereas gene 3 is altered in the two low virulence isolates. A similar observation has been reported for TGEV as a nonpathogenic TGEV mutant with a small plaque morphology had a deletion in gene 3. We have also observed that one of the low virulence PRCV isolates, IA 1894, which has a deletion in gene 3, replicates poorly in cell cultures. Collectively these studies suggest that gene 3 may be an important determinant for in vivo virulence and in vitro replication of coronaviruses.

Animals↗