Paul Callebaut

Isolation of a porcine respiratory, non‐enteric coronavirus related to transmissible gastroenteritis

A porcine respiratory, non-enteric virus which is related to the coronavirus transmissible gastroenteritis virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83. It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract.

Antigenic Differentiation between Transmissible Gastroenteritis Virus of Swine and a Related Porcine Respiratory Coronavirus

The antigenic relationship between a recently isolated porcine respiratory coronavirus (TLM 83) and transmissible gastroenteritis (TGE) virus of swine was studied by neutralization, immunoblotting and radioimmunoassay (RIA) using TGE virus-specific monoclonal antibodies (MAbs) and polyclonal antibodies specific for both viruses. A complete two-way neutralization activity between the two viruses was found. Immunoblotting revealed cross-reactions between TLM 83 and TGE virus antigens at the level of the envelope protein (E1), the nucleoprotein (N) and the peplomer protein (E2). By virus neutralization assays and RIA with TGE virus-specific MAbs, the presence of similar epitopes in the E1 and N proteins and in the neutralization-mediating antigenic site of the E2 protein were demonstrated. E2 protein-specific MAbs, without neutralizing activity and reacting with antigenic sites B, C and D (previously defined), failed to recognize TLM 83. These results indicated a close antigenic relationship and structural similarity between TLM 83 and TGE viruses and also suggested potential ways of differentiating between the two viruses.

Intestinal replication of a porcine respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus

Abstract One-week-old piglets were inoculated with the porcine respiratory coronavirus (PRCV) either intravenously or directly into the lumen of the gastrointestinal tract. Both inoculation routes resulted in the isolation of virus from the caudal small intestine. Viral replication, however, was only observed upon inoculation into the digestive tract in quantities of ≥ 103 TCID50. Replication remained limited to a few unidentified cells located in or underneath the epithelial layer at villus- or crypt-sites. Virus was excreted in the faeces for several days but infection of the respiratory tract occured rarely in the same pigs. The results of this study indicate that small changes in molecular structure between PRCV and transmissible gastroenteritis virus have resulted in important changes in host cell tropism.

Comparative study of a blocking enzyme-linked immunosorbent assay and the immunoperoxidase monolayer assay for the detection of antibodies to the porcine reproductive and respiratory syndrome virus in pigs.

Abstract A blocking enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to the porcine reproductive and respiratory syndrome virus (PRRSV) in pig sera was developed and compared with the immunoperoxidase monolayer assay (IPMA), the most widely used serological diagnostic test in Europe. The blocking ELISA was specific and more sensitive than the IPMA when applied to field sera and to sera which were collected early after an experimental infection with PRRSV. Problems with high background activity as observed in IPMA or indirect ELISA were not encountered.

Characterization and isolation of structural polypeptides in haemagglutinating encephalomyelitis virus.

Haemagglutinating encephalomyelitis virus (HEV), a member of the coronavirus family, was purified and analysed by SDS-polyacrylamide gel electrophoresis. It was shown to contain eight polypeptides, seven of which were glycosylated. They had apparent mol. wt. of 180,000 (GP 180), 130,0000 (GP 130), 120,000 (GP 120) 76,000 (GP 76), 64,000 (VP 64), 54,000 (GP 54), 32,000 (GP 32) and 31,000 (GP 31). Electrophoresis of virus samples dissociated under varying conditions showed that GP 54 and GP 120 could be interpreted as larger products of GP 31 and GP 32 and of GP 76, respectively. GP 76 also appeared as a dimer with a mol. wt. of 140,000 (GP 140) in the absence of beta-mercaptoethanol. Subviral particles, obtained by treatment with bromelain, banded at a slightly lower density than the intact virus and lacked surface projections. Analysis of these particles indicated that GP 180, GP 130 and GP 76 are associated with the virus projections. A small part of GP 31 and GP 32 also appeared to protrude from the lipid envelope since 20% of each molecule was sensitive to digestion. Two glycoproteins, GP 130 and GP 76, were solubilized with the detergent Triton X-100 and separated by rate zonal centrifugation. According to its activity-in indirect haemagglutination tests, GP 76 was considered to be a monovalent haemagglutinin subunit.

Intestinal protection against challenge with transmissible gastroenteritis virus of pigs immune after infection with the porcine respiratory coronavirus

Abstract An infection of pigs with the porcine respiratory coronavirus (PRCV) induces antibodies which neutralize the enteropathogenic transmissible gastroenteritis virus (TGEV) and PRCV to the same titre. In the present study, 10-week-old seronegative pigs (n = 8), pigs immune following TGEV inoculation (n = 4) or pigs immune following aerosol (n = 8) or intragastric inoculation (n = 4) with PRCV were challenged with TGEV. Whereas TGEV-immune pigs were completely protected against challenge, all PRCV-immune pigs showed serological evidence of TGEV replication. Nevertheless, the aerosol or intragastric inoculation with PRCV primed the humoral immune system against TGEV and the TGEV challenge induced a secondary antibody response in most PRCV-immune pigs. Furthermore, all PRCV-immune pigs showed a decrease in the duration of the excretion of infectious TGEV (0–4 days) in comparison with the duration of the virus excretion by seronegative pigs (5–6 days).

A sero-epizootiological study of porcine respiratory coronavirus in Belgian swine.

A porcine respiratory coronavirus (PRCV), antigenically closely related to transmissible gastroenteritis virus (TGEV), appeared in the European swine population in 1984. The present serological study was performed to obtain insight into the epizootiology of PRCV and of TGEV. PRCV-induced neutralizing antibodies were found in 90.6 per cent of the 160 sera collected from sows at slaughter, demonstrating the enzootic appearance of PRCV in the Belgian swine population. A serological study of fattening swine on 33 farms revealed that 11 farms situated in an area with a high farm density (all farms within 4 km2) and 11 on 22 closed breeding-fattening farms situated in areas with a low farm density (only one to four farms per 12 km2) were infected with PRCV throughout the year, whereas the other 11 closed breeding-fattening farms were temporarily free of PRCV. PRCV disappeared from the farms mainly in spring and summer. All the 11 farms became reinfected in autumn or winter, indicating that PRCV is regularly reintroduced in farms in the colder seasons. There was no correlation between the herd size and the temporary disappearance of PRCV from farms. It was observed on some farms that PRCV could infect pigs shortly after weaning in the presence of declining maternal antibodies, indicating that PRCV can persist on a farm by regularly infecting newly weaned pigs. TGEV-specific antibodies were found in 7.6 per cent of the 160 sera from the slaughterhouse sows. TGEV-specific antibodies were also detected in sera from fattening swine of 5 of the above mentioned 33 farms. TGEV-outbreaks were not observed on these farms.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of a Recombinant Adenovirus for the Expression of the Glycoprotein S Antigen of Porcine Respiratory Coronavirus

Efforts to develop respiratory and enteric vaccines focus on the efficient delivery of protective antigens to the mucosal lymphoid tissue. One approach is to use recombinant viral vectors, i.e. apathogenic viruses with a tropism for mucosal surfaces and genetically manipulated to express the protective antigens. Adenoviruses, particularly the human adenovirus type 5 (Ad5), are promising candidates for use as vectors.

Induction of Milk IgA Antibodies by Porcine Respiratory Coronavirus Infection

An ELISA was developed to examine the prevalence of TGEV-specific immunoglobulin (Ig)A in the milk of sows, infected in the field with PRCV or with TGEV. It was shown that previous PRCV-infections can induce the secretion of IgA antibodies in the milk. However, only 9 out of 28 PRCV-infected sows had IgA in their milk whereas 11 TGEV-infected sows all secreted IgA. On farms where a reinfection with PRCV occurred, the number of IgA-secreting sows increased from 2 to 11 on a total of 13 sows. This showed that the presence of IgA antibodies in the milk may depend upon the occurrence of reinfection with PRCV. As demonstrated by density gradient analysis, the milk IgA induced by PRCV was 11S secretory IgA and had the capacity to neutralize TGEV.

A seroepizootiologic study of vomiting and wasting disease virus in pigs

Summary Neutralizing antibodies to Vomiting and Wasting Disease virus were found in 95 per cent of the sera collected from Belgian sows at slaughter. Piglets suckled by immune sows and kept in isolation acquired maternal antibodies; these had disappeared in all the animals at the age of 15 weeks. Most pigs had lost their maternal antibodies at the age of 11 or 12 weeks (respectively 57 per cent or 86 per cent). A serologic study on two conventional breeding farms showed that this passive immunity was replaced by active immunity between the ages of 8 and 16 weeks. No clinical disturbances appeared to be associated with the infection. The present data indicate that Vomiting and Wasting Disease virus persists on the majority of the conventional breeding farms.