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H Laude

Publications and source records attributed to H Laude.

13 recordsLinked to original sources
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↗
PubMed1992

TGEV corona virus ORF4 encodes a membrane protein that is incorporated into virions.

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).

Baculoviridae↗
PubMed1992

Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus.

Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-alpha synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly E1) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30- to 300-fold lower than that of the parental virus. The transcription of IFN-alpha genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic determinant resides in the N-terminal, exposed part of the molecule and provided further evidence for the direct role of M protein in the induction of IFN-alpha by transmissible gastroenteritis virus. The acronym VIP (viral interferogenic protein) is proposed as a designation for this particular class of proteins.

Amino Acid Sequence↗
PubMed1979

Properties of Border disease virus as studied in a sheep cell line.

A fetal lamb muscle cell line has been isolated in which two strains of Border disease virus replicate well and induce a clear-cut cytopathic effect, thus providing a sensitive and practical assay system for both the virus and neutralizing antibodies. This system enabled us to study some characteristics of Border disease virus in comparison with two other agents responsible for hog cholera and bovine viral diarrhea. Our results indicate that the last virus and the Border disease agent are indistinguishable in vitro.

Animals↗
PubMed1978

A direct plaque assay for hog cholera virus.

Direct plaque formation with representative strains of hog cholera virus (HCV) has been obtained using several pig kidney cell lines under agar overlay. HCV-infected cells appear as hazy plaques when viewed against an indirect light source, and as white plaques after neutral red staining. HCV assay by direct plaque procedure is rapid and convenient and gives infectivity titres identical to the fluorescent focus assay technique.

Cell Line↗
PubMed1977

Improved method for the purification of hog cholera virus grown in tissue culture.

A method for purification of Hog Cholera Virus (HCV) is presented. Cell-associated virus was extracted from PK15 cells 17 hours p.i. by fluorocarbon treatment. The virus was concentrated by polyethylene glycol precipitation and partially purified by pelleting on a fluorocarbon cushion. Final purification was achieved by rate zonal centrifugation in a 7--35 per cent sucrose gradient. This procedure permits a recovery of approximately 40 per cent of viral infectivity. A specific infectivity of about 2 X 10(7) PFU/microgram of protein was achieved. An apparent density d=1.13 g/ml in sucrose and a Sw20 value of about 150 was determined for purified HC virions.

Animals↗
PubMed1977

Isoelectric focusing of hog cholera virus.

The isoelectric point of Hog Cholera virus (Alfort strain) has been determined (pHi = 4.8). A clear stimulation of its infectivity can be recorded at this pH. The isoelectrophoretic profiles of strains isolated in the field show no significant differences.

Animals↗
PubMed1977

Hog cholera virus : sensitivity to hydrolytic enzymes.

The actions of trypsin and phospholipase C on th infectivity of Hog Cholera virus (HCV) were studied. Inactivation kinetics reveal a marked decrease of the infectivity of HC virus in the presence of these two agents. The particular sensitivity of this virus towards proteolytic action sheds light on certain of its behavior characteristics in the field. Virus infectivity seems to be dependent on the integrity of membrane phospholipids. No relation was observed between the rate of inactivation and the pathogenicity of the strains.

Animals↗
PubMed1976

Sub-clinical swine fever: a survey of neutralizing antibodies in ther sera of pigs from herds having reproductive failures.

Sera harvested from breeding farms where reproductive failures were observed but where swine fever vaccination was not carried out, where tested for the presence of neutralizing antibodies specific for swine fever virus. Neutralization tests were performed in tissue culture using two viruses strains: the american serological variant "331" strain isolated by MENGELING (1969) and the virulent "normal strain" Alfort. For comparison, sera harvested from healthy, vaccinated and unvaccinated breeding farms were also tested. Results show absence of antibodies against these two strains in healthy and unvaccinated animals. All sera harvested from healthy and vaccinated breeding farms have high levels of neutralizing activity against the two strains. From 1,251 harvested unvaccinated breeding farms have reproductive failures, 6.2 p. 100 had neutralizing antibodies against the "Alfort" strain and 17.1 p. 100 contained neutralizing antibodies against the "331" strain. In 87 out of these 189 breeding farms, positive serological reactions were observed. Considering the respective intensity of neutralizing activity against the two strains , the vaccination with live virus vaccines ("Chinese strain" or "Thiverval" strain) induces high levels of antibodies but a higher titer against "Alfort" strain. Inversely, low virulent strains responsible for sub-clinical swine fever induce low or moderate levels against the "331" strain and nil or low levels against the "Alfort" strain. These results suggest that low virulent strains having common antigenic properties with the serological variant "331" strain are circulating among pigs in unvaccinated breeding farms having reproductive failures. These features are important for future development and establishment of routine serological checking for diagnosis and herd testing.

Animals↗
PubMedDate not supplied

[Isolation of a cytolytic strain of hog cholera virus from IB-RS2 cells (author's transl)].

An infectious agent, able to induce a definite cytopathic effect in pig kidney cell monolayers, was isolated from the IB-RS2 cell line. Immunofluorescence, seroneutralization and ultracentrifugation studies have permitted to identify this agent as hog cholera virus (HCV). Representative HCV-strains being devoid of pathogenicity in tissue culture, the practical and theoretical interest of such an isolation is discussed.

Animals↗