An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine.

The full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 micrograms per 10(6) cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 micrograms per 10(6) cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S' and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.

Expression and immunogenicity of the spike glycoprotein of porcine respiratory coronavirus encoded in the E3 region of adenovirus.

The full length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen to an amount of approximately 33 micrograms per 10(6) cells, as determined by ELISA. The antigen was cell-associated except in the late phase of the infection, when a low amount (4 micrograms per 10(6) cells) was released in the culture supernatant. The cell-associated antigen consisted of 2 polypeptides of 160 K and 175 K, respectively. The 160 K polypeptide comigrated with the authentic S' precursor from PRCV-infected cells. The 175 K polypeptide had the same mobility as the authentic mature S protein from PRCV-infected cells and from PRCV released in the supernatant. The extracellular recombinant antigen corresponded with the 175 K mature protein. Immunofluorescent staining gave evidence that some recombinant S protein was exposed on the cell surface; it also showed that the protein was recognized by conformation-specific anti-S monoclonal antibodies. Piglets, immunized oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV-challenge.

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.

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.

Susceptibility of hares and rabbits to a Belgian isolate of European brown hare syndrome virus.

Signs and pathologic changes of European brown hare syndrome (EBHS) were reproduced in four hares (Lepus europaeus) after experimental inoculation of a liver suspension from hares from Belgium, which naturally died of EBHS. Virus particles were demonstrated by electron microscopy in the liver of an experimentally infected hare. They were spherical with a diameter of 28 to 30 nm. After density gradient centrifugation, virus particles were detected in the density zone of 1.34 g/ml. Based on immunoelectron microscopy with a convalescent serum, we identified the virus as the cause of EBHS. Clinical signs were not produced in three seronegative domestic rabbits after subcutaneous inoculation of the EBHS virus. Although low levels of antibodies against EBHS virus were found in the serum of these three rabbits 4 weeks after the inoculation of EBHS virus, the rabbits were not protected when challenged with viral hemorrhagic disease (VHD) virus. The different pathogenicity of the Belgian EBHS and VHD virus isolates in rabbits and the lack of protection in rabbits with circulating EBHS virus antibodies against a challenge with VHD virus indicates that both Belgian virus isolates form two different virus populations.

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)

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

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).

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

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 greater than or equal to 10(3) 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 occurred 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.

Antigenic homology among coronaviruses related to transmissible gastroenteritis virus.

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

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 competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus.

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.

Characterization of the structural proteins of porcine epizootic diarrhea virus, strain CV777.

Pig epizootic diarrhea virus cannot be grown in cell culture; for its characterization, intestinal perfusate material from a pig infected with the strain CV777 had to be used. In isopyknic sucrose gradients, a peak of virus-specific ELISA activity was detected at a density of 1.17 g/ml. Using immunoprecipitation of radioiodinated-purified virus material followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 3 proteins of low molecular weight (20,000 to 32,000 daltons [D]) were found; after blotting nitrocellulose and glycoprotein identification with concanavalin A and horseradish peroxidase, 1 of the proteins (23,000 D) gave a signal. Another protein of 58,000 D was encountered, which was the only protein binding an RNA probe. Finally, a protein of 85,000 D was visible, associated with minor bands of about 110,000 and 135,000 D in most experiments. Using the concanavalin A-blotting technique, the same bands were visualized. The demonstration of a polydisperse cluster of proteins from 20,000 to 32,000 D (of which at least 1 is glycosylated), of glycosylated proteins from 85,000 to 135,000 D, and of an RNA-binding protein of 58,000 D is taken as structural evidence that pig epizootic diarrhea virus should be classified with the Coronaviridae, irrespective of the apparent lack of an antigenic relationship with other members of that family.

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.

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.

Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the coronavirus-like agent in feces of pigs naturally affected with porcine epidemic diarrhea (PED) or experimentally infected with the CV777 isolate. The assay was specific and more sensitive than electron microscopy. An ELISA blocking assay is described for the detection and titration of antibodies. Specific antibody formation was demonstrated in pigs experimentally infected with CV777 and in swine naturally affected in PED.

A seroepizootiologic study of vomiting and wasting disease virus in pigs.

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.

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.

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.