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At least 811 records · Page 45Linked to original sources
PubMed1991

Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens.

Feline infectious peritonitis virus (FIPV) causes a mostly fatal, immunologically mediated disease in cats. Previously, we demonstrated that immunization with a recombinant vaccinia virus expressing the FIPV spike protein (S) induced early death after challenge with FIPV (Vennema et al., 1990, J. Virol. 64, 1407-1409). In this paper we describe similar immunizations with the FIPV membrane (M) and nucleocapsid (N) proteins. The genes encoding these proteins were cloned and sequenced. Comparison of the amino acid sequences with the corresponding sequences of porcine transmissible gastroenteritis virus revealed 84.7 and 77% identity for M and N, respectively. Vaccinia virus recombinants expressing the cloned genes induced antibodies in immunized kittens. Immunization with neither recombinant induced early death after challenge with FIPV, strongly suggesting that antibody-dependent enhancement is mediated by antibodies against S only. Immunization with the N protein recombinant had no apparent effect on the outcome of challenge. However, three of eight kittens immunized with the M protein recombinant survived the challenge, as compared to one of eight kittens of the control group.

Amino Acid Sequence↗
PubMed1980

Enzyme linked immunoassay and fluorescent antibody techniques in the diagnosis of viral diseases using staphylococcal protein-A instead of anti-gamma-globulins.

Staphylococcal protein-A (SpA) is known to interact with the crystallizable fragment (Fc) of IgG molecules from several species. In the present study, SpA coupled to either fluorescein isothiocyanate (FITC) or peroxidase was used in place of antisera to IgG for the fluorescent antibody (FA) techniques and the enzyme linked immunoassay (ELISA). The SpA conjugates produced low background staining when applied in these techniques, and provide a rapid, highly specific and sensitive means for the identification of viral isolates and the detection of serum antibodies. Moreover, SpA is a single reagent that replaces various preparations of anti-gamma globulin against many species. SpA-FITC conjugate was successfully applied for the identification of pseudorabies virus, hog cholera virus, swine vesicular disease virus, transmissible gastroenteritis virus, porcine parvovirus and porcine enteroviruses. Antibody titers against the mentioned viruses could be determined semi-quantitatively in the indirect FA test with SpA-FITC. In our laboratory the ELISA became a routinely practicable serological test for the detection of antibodies only after we introduced SpA-peroxidase as a marker for the IgG.

Animals↗
PubMed1995

Immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies.

Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location.

Animals↗
PubMed1992

Sequence analysis of human coronavirus 229E mRNAs 4 and 5: evidence for polymorphism and homology with myelin basic protein.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.

Amino Acid Sequence↗
PubMed1995

Adventitious pestivirus RNA in live virus vaccines against bovine and swine diseases.

Live virus vaccines against bovine and porcine diseases were examined for the presence of adventitious pestivirus RNA or pestiviruses by reverse transcription-polymerase chain reaction (PCR). Pestivirus RNA was detected in the live virus vaccines against Akabane disease, Ibaraki disease, infectious bovine rhinotracheitis, porcine parvovirus infection, transmissible gastroenteritis and Japanese encephalitis. Pestivirus RNA or pestivirus in the fetal bovine serum used to grow the host cells used to prepare the bovine and swine viral vaccines is a likely source of the contamination. Nucleotide sequence analysis of the PCR products suggests that modified live virus vaccines being used for immunization of cattle against bovine viral diarrhoea was not responsible for the contamination of the vaccines examined.

Animals↗
PubMed1994

Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences.

The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites. The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli.

Amino Acid Sequence↗
PubMed1988

Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets.

Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC). Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05). Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal. Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E. coli and the number of bacteria adhering to 250 micron villus brush border was counted. Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection. Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001). The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E. coli.

Animals↗
PubMed1989

Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV).

The porcine epidemic coronavirus (PEDV), tentatively classified as a coronavirus, was adapted to Vero cells and a plaque test developed for infectivity titration, allowing us to test the biological and biophysical properties of the virus. Growth kinetics showed peak titers of 10(5.5) plaque-forming units ml-1 15 h after infection. Filtration experiments and electron microscopy revealed a particle diameter between 100 and 200 nm. The buoyant density of the virus was 1.18. The particle lost its infectivity on treatment with lipid solvents. Virus replication could not be inhibited by 5-iodo-2'-deoxyuridine. PEDV was moderately stable at 50 degrees C, but heat sensitivity was not altered by divalent cations. At 4 degrees C, the virus was stable between pH 5.0 and 9.0, but at 37 degrees C stability was restricted to the pH range 6.5-7.5. Viral infectivity was not impaired by ultrasonication or by multiple freezing and thawing. PEDV was not neutralized by transmissible gastroenteritis virus antiserum. On the basis of the tests carried out, PEDV is a pleomorphic, enveloped RNA virus with a particle diameter of approximately 150 nm and a buoyant density of 1.18. Infectivity depends on the presence of trypsin, and infected cells show a tendency to fuse and to form syncytia. All of these properties, as well as its physicochemical characteristics, allow PEDV to be classified as a coronavirus.

Animals↗
PubMed1990

Contribution of molecular biology to the study of the porcine interferon system.

We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time of implantation in the uterus. By probing porcine genomic libraries with human and porcine IFN-alpha probes to isolate related genes, we have shown that the porcine IFN-alpha multigene family included, like several other mammalian species, two subfamilies of related but distinct genes. Class I subfamily contains at least 11 loci, located on chromosome no. 1, among which nine have been cloned and two (potentially functional) sequenced. Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned. One of the sequenced class I loci: PoIFN-alpha 1 encodes a 189 amino acids (AA) preprotein. After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-alpha 1 (methionyl IFN-alpha 1). Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E. coli and immunodetection of clones expressing high amounts of recombinant protein. The E. coli strain obtained produced high levels of a 18,000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN). However, it had a stronger antiviral effect on porcine cells than LeuIFN. After immunoaffinity purification to a specific activity of 5-10 x 10(7) International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-alpha in the pig. These experiments revealed no major toxic effects in newborn (given 5 x 10(6) IU/kg) or adult (1 X 10(6) IU/kg) pigs. A significant pyrogenic effect (+ 1.5 degrees C) was noted only in the adults.

Animals↗
PubMed1993

Coronavirus immunogens.

Coronaviruses (CV) infect a variety of livestock, poultry and companion animals. They belong to at least five antigenic groups. CV cause localized infections of the respiratory and/or intestinal tracts, with the exception of feline infectious peritonitis virus (FIPV) and hemagglutinating encephalomyelitis (HEV) which cause systemic infections. The enteropathogenic CV infect the villous enterocytes resulting in villous atrophy leading to malabsorptive diarrhea. Several CV (bovine CV-BCV, porcine respiratory CV-PRCV, infectious bronchitis virus-IBV) cause respiratory disease. Current evidence indicates that protection against enteric and respiratory CV infections is mediated by passive or active immunity at the primary site of CV replication. Maternal vaccination approaches to induce passive immunity include the use of inactivated and modified live viral vaccines. Modified live viruses and a Ts mutant CV (FIPV) are also used as oral or intranasal vaccines to induce active mucosal immunity. The success of these vaccines in the field is often compromised by a number of potential problems. Coronaviruses are spherical, enveloped viruses, ranging from 80-160 nm in diameter and containing a positive-stranded RNA genome. They possess prominent surface spikes and some species display a fringe of smaller surface projections believed to be the hemagglutinin (HE). Coronaviruses possess 3 to 4 structural proteins: the spike (S) glycoprotein (150-200 kDa), the integral membrane glycoprotein (M; 20-30 kDa) and the nucleocapsid phosphoprotein (N; 43-50 kDa). A subset of CV (BCV, HEV, turkey CV) possess a third glycoprotein on the virion surface, the HE (60-65 kDa). These proteins can be quantitated using pooled monoclonal antibodies (mAb) to distinct epitopes of each protein in ELISA. Most research has focused on the S protein as a candidate antigen for CV vaccines since it induces virus neutralizing (VN) antibodies. However the HE protein stimulates the production of VN and HE inhibiting antibodies and the M protein induces antibodies that neutralize virus in the presence of complement. Attempts to correlate in vitro VN antibody activity with in vivo protection have shown that the passive transfer of VN mAb to the S or HE protein conferred passive protection against CV challenge in some studies, but not others. Additional research has implicated a possible role for other CV proteins in immunity. Studies of mAb to the M protein of transmissible gastroenteritis (TGEV) have provided evidence for a direct role of the M protein in the induction of alpha IFN by porcine blood leukocytes. The potential significance of this phenomenon to immunity to TGEV is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV.

Previously, we have identified a truncated antigenic fragment named protein C [441 to 700 amino acids (a.a.)] as the immunodominant fragment of Spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We have now successfully expressed protein C using the baculovirus system in S. frugiperda (Sf-9) cells. This recombinant baculovirus expressing protein C was first characterized using five SARS convalescent human sera and five normal human sera. The results showed that protein C is an authentic antigen against SARS-CoV antibody. Our Spike protein-based immunoflourescence assay (IFA) based on this recombinant baculovirus-Sf-9 system was further assessed with a panel of 163 clinical samples collected during the SARS epidemic in Singapore, which include samples from 21 clinically confirmed SARS, 42 non-SARS patient sera, and 100 normal sera. The results were compared to a commercial SARS IFA kit (EUROIMMUN, Germany) and a conventional IFA test performed in Singapore General Hospital. All of the 21 SARS-positive serum samples could be recognized by our IFA, giving a specificity and sensitivity of 100%, which was compatible with both whole virus-based IFA assays. No cross-reactivity with serum samples against infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV) were detected in our assays. Thus, our Spike protein-based IFA could offer a safer procedure which can be performed in a BSL-2 laboratory as it could mimic the whole virus based-IFA without any loss of sensitivity and specificity. It is also more user-friendly and cost-effective than the whole virus-based IFA.

Animals↗
PubMed2004

Immunopurification applied to the study of virus protein composition and encapsidation.

A protocol for obtaining small amounts of highly pure virus preparations starting from reduced volumes (<5 ml) of infected tissue culture supernatants is described. This procedure was adapted to the study of transmissible gastroenteritis coronavirus (TGEV) protein composition and RNA encapsidation. This protocol relies on virion capture by monoclonal antibodies specific for a virion membrane protein. These antibodies were bound to protein A-coated ELISA wells armed with rabbit anti-mouse IgG antibodies. As an example, the purification of (35)S-labelled TGEV virions was performed. The quality of virus preparations was assessed by quantifying common contaminating RNAs by real-time RT-PCR. The most critical factors influencing the purity degree are analysed and discussed.

Animals↗
PubMed2005

Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus.

The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlighted the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying on existing broadly active antiviral compounds. The development of rapid screening assays is essential for antiviral drug discovery. Thus, a screening system for anti-SARS-CoV agents was developed, which evaluated compound potency, specificity and cytotoxicity at the initial screening phase. Cell lines were engineered to constitutively express an enhanced green fluorescent protein (EGFP) and used to detect (1) antiviral potency in SARS-CoV infection tests; (2) antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus (TGEV); and (3) cytotoxicity in the same assays without virus challenge. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. The suitability of this assay system in drug discovery was demonstrated by screening of 3388 small molecule compounds. The results show that these assays can be applied to high-throughput screening for identification of inhibitors selectively active against SARS-CoV.

Antiviral Agents↗
PubMed2004

An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces.

Monoclonal antibodies to porcine epidemic diarrhoea virus (PEDV) membrane protein M were prepared and used for the comparative assessment of three blocking ELISA variants to detect PEDV. The competitive blocking ELISA (CB-ELISA) format showed the highest sensitivity, allowing detection of 10(2.5) plaque-forming units of PEDV/ml in culture medium. Its specificity was verified by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and rotavirus A in each analysis. Eighty porcine field samples of faeces obtained from 38 herds affected with diarrhoea were examined, and PEDV was found in 15 (19%) samples from 6 (16%) herds. The suitability of the CB-ELISA for the screening herds in epizootiologic situations is discussed.

Animals↗
PubMed2006

Coronavirus nucleocapsid protein is an RNA chaperone.

RNA chaperones are nonspecific nucleic acid binding proteins with long disordered regions that help RNA molecules to adopt its functional conformation. Coronavirus nucleoproteins (N) are nonspecific RNA-binding proteins with long disordered regions. Therefore, we investigated whether transmissible gastroenteritis coronavirus (TGEV) N protein was an RNA chaperone. Purified N protein enhanced hammerhead ribozyme self-cleavage and nucleic acids annealing, which are properties that define RNA chaperones. In contrast, another RNA-binding protein, PTB, did not show these activities. N protein chaperone activity was blocked by specific monoclonal antibodies. Therefore, it was concluded that TGEV N protein is an RNA chaperone. In addition, we have shown that purified severe acute respiratory syndrome (SARS)-CoV N protein also has RNA chaperone activity. In silico predictions of disordered domains showed a similar pattern for all coronavirus N proteins evaluated. Altogether, these data led us to suggest that all coronavirus N proteins might be RNA chaperones.

Chaperonins↗
PubMed1988

Sequence analysis of the 3'-end of the feline coronavirus FIPV 79-1146 genome: comparison with the genome of porcine coronavirus TGEV reveals large insertions.

The genetic information, carried on mRNA 6 of feline infectious peritonitis virus (FIPV) strain 79-1146, was determined by sequence analysis of cDNA clones derived from the 3' end of the FIPV genome. Two ORFs were found, encoding polypeptides of 11K (ORF-1) and 22K (ORF-2). The FIPV sequence was compared to the 3' end sequence of transmissible gastroenteritis virus (TGEV). ORF-1 has a homologous counterpart (ORF-X3) in the TGEV genome; both ORFs are located at the same position relative to the nucleocapsid gene. However, as a result of an in-frame insertion or deletion, ORF-1 is 69 nucleotides larger than ORF-X3. A similar event has occurred immediately downstream of ORF1: a 624-nucleotide segment, containing the complete ORF-2, is absent in the TGEV sequence. Most sequence similarity (98.5%) was found in the 3' noncoding sequences. ORF-X3 and ORF-1 are preceded by the sequence AACTAAAC, which is assumed to be the transcription-initiation signal in FIPV and TGEV (P.A. Kapke and D.A. Brian (1986) Virology 151, 41-49). By S1 nuclease analysis, the 5' end of FIPV RNA 6 was mapped immediately upstream of this sequence. A 700-nucleotide TGEV-specific RNA was found by cross-hybridization with an FIPV 3' end probe, suggesting that TGEV ORF-X3 is also carried on a separate mRNA. The differences at the 3' ends of the FIPV and TGEV genomes may be the result of RNA recombination events.

Amino Acid Sequence↗
PubMed1999

Selection for high immune response: an alternative approach to animal health maintenance?

To test the hypothesis that variation in ability to respond immunologically correlates with health, Yorkshire pigs were bred for high (HIR) and low (LIR) antibody (Ab) and cell-mediated immune response (CMI). Selection was based on standardized measures of Ab (secondary response to hen egg white lysozyme, serum IgG concentration) and CMI (cutaneous delayed-type hypersenstivity to purified protein derivative of tuberculin after immunization with bacillus Calmette-Guérin and in vitro lymphocyte response to Con-A). Differences in Ab and CMI by line were not restricted to the antigens used in the selection. Antibody response to vaccines was highest in HIR and non-responders were restricted to LIR pigs. The HIR pigs had the best rate of weight gain. After infection with Mycoplasma hyorhinis, HIR developed more severe arthritis and less polyserositis. Differences were associated with variation in cytokine message in joint-related cells. Following exposure to attenuated transmissible gastroenteritis virus, natural killer cells of the LIR pigs but not of HIR or control lines, were unresponsive. Genetic selection for Ab and CMI may provide health and productivity advantages and complement traditional health-maintenance methods.

Animals↗