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D J Garwes

Publications and source records attributed to D J Garwes.

At least 19 recordsLinked to original sources
PubMed1992

Antigenic structure of transmissible gastroenteritis virus nucleoprotein.

A group of 11 monoclonal antibodies (MAbs) raised against transmissible gastroenteritis virus (TGEV) was used to study the antigenic structure of the virus nucleoprotein (N). To identify the regions recognized by MAbs, DNA fragments derived from the N-coding region of the TGEV strain FS772/70 were cloned into pUR expression plasmids and the antigenicity of the resulting fusion proteins was analyzed by immunoblotting. A major antigenic domain was identified, covering the first 241 amino acid residues of N, within which an epitope (residues 57-117) was also found. A second antigenic domain extended from residues 175 to 360 of the nucleoprotein, within which a subsite was characterized within the region covering residues 241-349. MAb DA3 recognized a linear epitope which mapped within residues 360 and 382 at the carboxy terminus of the nucleoprotein. The binding of the majority of the MAbs (8 out of 11) to large fusions, but not to smaller fragments included in them, suggests a conformational dependence of the MAb binding sites. Our data show that the use of fusions in Western blot experiments is a useful approach to map not only linear epitopes but more complex antigenic structures found in the nucleoprotein of the TGEV.

Antibodies, Monoclonal↗
PubMed1989

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice.

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.

Animals↗
PubMed1989

The polypeptide of Mr 14,000 of porcine transmissible gastroenteritis virus: gene assignment and intracellular location.

Synthetic oligopeptides, corresponding to an amino acid sequence encoded by a potential Mr 9000 product's open reading frame (ORF-4) at the 3' terminus of the transmissible gastroenteritis virus genome, were used to generate rabbit antiserum. These antibodies produced immune complexes with an Mr 14,000 (14K) polypeptide in infected cells. The 14K product was shown by immune fluorescence to become associated with the cell nucleus, correlating with the onset of nuclear vacuolation, and suggesting a role in pathogenesis for the ORF-4 gene.

Amino Acid Sequence↗
PubMed1989

Genome typing of three serotypes of porcine adenovirus.

Porcine enteric adenovirus serotypes 1-3, previously shown to be distinct antigenically, were purified by density gradient centrifugation, and the DNA was extracted. Fragments generated by digestion with restriction endonucleases BamHI, EcoRI, HindIII, and PaeR7 were separated by electrophoresis and compared with human adenovirus type 2 as a control. The results demonstrated that the three porcine viruses were closely related to each other, although distinguishable, and distinct by restriction endonuclease analysis from human, murine, canine, bovine, and fowl adenovirus.

Adenoviridae↗
PubMed1988

Transmissible gastroenteritis.

Transmissible gastroenteritis (TGE) virus, a coronavirus, causes an acute infection of the small intestine of the pig. The disease is rarely fatal in adult animals but can cause extensive mortality in the neonate. Since its first description in Britain in the 1950s, the disease has been epizootic but recently it has become established on some farms and an antigenically related respiratory virus has become endemic in the national herd over the past two years. Piglet immunity to TGE, which relies on passive protection from milk, has been impossible to achieve with vaccines and research has aimed at understanding the nature of the interaction between virus and the pig. Following infection of the pregnant sow, antibody-producing cells migrate to the mammary gland where they secrete virus neutralising antibodies into the milk; prospective vaccines will need to stimulate a similar response. The location and number of antigenic sites on the virus particle associated with neutralisation have been established with monoclonal antibodies and the role of the other viral genes in pathogenesis and immunity is being studied with genetic engineering techniques.

Animals↗
PubMed1988

The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli.

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29,459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29,459 polypeptide and beta-galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

Amino Acid Sequence↗
PubMed1988

Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae.

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.

Amino Acid Sequence↗
PubMed1984

Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line.

During a search for established cell lines to produce large quantities of porcine transmissible gastroenteritis virus (TGEV), we observed bright immunofluorescent staining 6- 12h after infection of pig kidney derived LLC-PK1 line. Infectious virus yield was, however, 2 log10 lower than that from secondary adult pig thyroid (APT/2) cell cultures, although small plaques were visible by three days in cultures maintained under agarose, suggesting limited replication. Attempts to adapt TGEV to the LLC-PK1 cell line by 10 serial 20h passes were unsuccessful. Procedures to purify virions from infected LLC-PK1 cells produced less than 1% of the particles isolated from parallel APT/2 cultures. Examination of intracellular viral RNA in actinomycin-D treated cells revealed similar amounts of genomic RNA and the 4 major subgenomic species in both cell types, suggesting that there was no defect in viral RNA replication. In vitro translation of polyadenylated RNA from infected APT/2 and LLC-PK1 cells, followed by immune precipitation of the products, showed similar profiles of precursors to structural polypeptides, confirming the functional integrity of the viral messengers in the restrictive cell. Comparison of the viral polypeptides synthesised following infection of the two cell types showed that similar species were synthesised in both, corresponding to a group of 28-30,000 mol. wt. envelope glycopolypeptides, a 47,000 mol. wt. nucleoprotein and peplomer glycopolypeptides of about 200,000 mol. wt. The rate of viral polypeptide synthesis in LLC-PK1 cells was reproducibly higher than in APT/2, resulting in the earlier detection of bands and greater incorporation of isotope. Tunicamycin at 1 microgram/ml had a similar effect in both cells, preventing glycosylation of the 26,000 mol. wt. precursor of the envelope glycopolypeptides and synthesis of the 200,000 peplomer glycoprotein. Degradation of the nucleoprotein from 47,000 to 42,000 mol. wt. although detectable in both cells was more marked in the LLC-PK1 cultures. Phosphorylation of these proteins was readily demonstrated in both cells, although phosphorylation of host proteins and, to some extent, viral envelope proteins was considerably greater in the LLC-PK1. The significance of this finding with respect to virus maturation is being investigated.

Animals↗
PubMed1983

Some properties of mycoplasma virus Br 1.

Morphologically mycoplasma virus Br 1 is a typical contractile-tailed bacteriophage with a head 77 nm in diameter and a tail 104 nm long. Its type of nucleic acid was not determined. Br 1 was closely associated with its host cell and assays reflected infectious centres. During growth of Br 1 in mycoplasma cultures at multiplicities of infection (MOI) greater than 0.001, there was a lag period: this was up to 23 hours at an MOI of 35. The mean generation time of a mycoplasma culture infected at MOI up to 235 was 2 hours, compared with 1 hour for an uninfected culture. However in these infected cultures there were viable mycoplasmas all of which appeared to be fully susceptible to Br 1 infection and did not seem to be carrying the virus. Br 1 formed plaques on M. bovirhinis but failed to produce plaques on strains of 8 Mycoplasma, 2 Acholeplasma and 4 bacterial species.

Anti-Bacterial Agents↗
PubMed1981

The polypeptide structure of canine coronavirus and its relationship to porcine transmissible gastroenteritis virus.

Canine coronavirus (CCV) isolate 1-71 was grown in secondary dog kidney cells and purified by rate zonal centrifugation. Polyacrylamide gel electrophoresis revealed four major structural polypeptides with apparent mol. wt. of 203800 (gp204), 49800 (p50), 31800 (gp32) and 21600 (gp22). Incorporation of 3H-glucosamine into gp204, gp32 and gp22 indicated that these were glycopolypeptides. Comparison of the structural polypeptides of CCV and porcine transmissible gastroenteritis virus (TGEV) by co-electrophoresis demonstrated that TGEV polypeptides corresponded closely, but not identically, with gp204, p50 and gp32 of CCV and confirmed that gp22 was a major structural component only in the canine virus. The close similarities in structure of the two coronaviruses augments the relationship established by serology.

Animals↗
PubMed1979

Comparison of mycoplasmatales virus MV-Lg-pS2-L172 with plasmavirus MV-L2 and the other mycoplasma viruses.

Mycoplasma virus MV-Lg-pS2-L172 was sensitive to heat (56 degrees C/30 minutes), Nonidet-P40 and ether. In these respects it resembled Plasmavirus MB-L2. However, it differed from MV-L2 (and the other mycoplasma viruses, MV-L1, MV-L3 and BN1 virus) in reciprocal plaque inhibition and serum neutralization tests (MV-L2 only). By plaque formation on host lawns resistant to the different mycoplasma viruses, including MV-Lg-pS2-L172, this latter virus was shown to be distinct from the other viruses, including MV-L2. Both MV-Lg-pS2-L172 and MV-L2 possessed one polypeptide band (out of 10) that was not common to the heterologous virus.

Acholeplasma laidlawii↗