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.

The coding potential of the open reading frame ORF4 (82 amino acids) of transmissible gastroenteritis virus (TGEV) has been confirmed by expression using a baculovirus vector. Five monoclonal antibodies (MAbs) raised against the 10K recombinant product immunoprecipitated a polypeptide of a similar size in TGEV-infected cells. Immunofluorescence assays performed both on insect and mammalian cells revealed that ORF4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in ORF4 sequence. Two epitopes were localized within the last 21 C-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated ORF4 recombinant protein. Since the relevant MAbs were found to induce a cell surface fluorescence, these data suggest that ORF4 may be an integral membrane protein having a Cexo-Nendo orientation. Anti-ORF4 MAbs were also used to show that ORF4 polypeptide may be detected in TGEV virion preparations, with an estimated number of 20 molecules incorporated per particle. Comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to TGEV ORF4. Our results led us to propose that ORF4 represents a novel minor structural polypeptide, tentatively designated SM (small membrane protein).

This review considers the potential reduction of embryo mortality in vitro and in vivo in ruminants. Data on cytokines provided by different fields of reproductive immunology and biology were collated. Because of the crucial importance of the local interactions between the embryo and its dam, the expression of growth-factor and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and endometrium by reverse transcriptase polymerase chain reaction in sheep and in cattle during the pre- and periimplantation periods. Many deleterious cytokines, such as tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth factor-beta, leukaemia inhibiting factor, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and IFN-tau appeared to be involved in embryo survival in ruminants and other species. Their administration is efficient in a murine experimental model (CBA/J x DBA/2) of embryonic and fetal mortality. For instance, recombinant ovine IFN-tau (roIFN-tau) injected at the moment of implantation drastically reduces embryonic mortality in this model. In ruminants, roIFN-tau and recombinant bovine IFN-tau are very efficient in maintaining progesterone luteal secretion in cyclic animals. The involvement of IFN-tau in the mechanisms of maternal pregnancy recognition are particularly detailed in relation to inhibition of 13,14 dihydro-15-keto-prostaglandin F2 alpha (PGFM) pulses and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic effects of IFN-tau on the endometrial cell is proposed. Finally, the particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60, pregnancy-associated glycoprotein) for monitoring embryo survival, with examples given for cattle and sheep is underlined.

Summary. Most toxigenic strains of Clostridium di#iciie produce two toxins : an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. dzficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in citro when examined in three distinct immunoassays. Nevertheless, all the strains produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. dz3ciie strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C . dificiie strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. dtficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C . dzflcile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. diflcile strains with regard to toxin production.

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