Biomedical subjects
Publications and source records attributed to D J Garwes.
Transmissible gastroenteritis virus was administered orally to cats. No clinical disease resulted but infectious virus was isolated from faeces for up to 22 days after infection and serum antibody was detected by neutralisation and immunofluorescence tests.
Exposure of transmissible gastroenteritis virus of pigs to unheated normal heterotypic serum resulted in a drop in infectivity, an effect which was lost after heating the serum to 56 degrees C for 30 min or by treatments inactivating complement. Analysis of virus proteins, RNA and lipids, and centrifugation studies showed little difference between inactivated and control virus, but electron microscopy of negatively stained particles after treatment with serum revealed holes in the virus envelope, characteristic of those caused by complement in the presence of antibody.
High titres of neutralizing activity to transmissible gastroenteritis virus (TGEV), a porcine coronavirus, were found in sera and peritoneal fluids from cats infected with feline infectious peritonitis (FIP). A small proportion of cats, from a hospital population unaffected by FIP, also had neutralising activity. Procedures to remove non-specific viral inhibitors, including treatment by heat inactivation, trypsin, sulphydryl reagent and kaolin absorption were unsuccessful. The active component was unable to neutralise another porcine coronavirus, haemagglutinating encephalomyelitis virus or the porcine enterovirus, Talfan. Gel filtration of feline sera and peritoneal fluid demonstrated high levels of the neutralising activity in the area corresponding to 7S IgG, which could be removed by absorption with specific anti-IgG serum and these properties are suggested to be consistent with those of antibody. These findings imply that there is a coronavirus in cats which is antigenically related to TGEV and its possible nature is discussed.
The lipids of two cell types (primary pig kidney and secondary adult pig thyroid) and those of transmissible gastroenteritis virus (TGEV) grown in these cells were studied using 14C-palmitic acid. Differences were demonstrated between the incorporation of isotopically labelled lipid precursors in the two cell types and it was found that the phospholipid and glycolipid profiles of purified TGEV closely resembled those of the host cell in which it was grown.
Exposure of purified transmissible gastroenteritis virus, a porcine coronavirus, to non-ionic detergents resulted in the removal of the surface projections and greater than 98% of the virus lipid. Virus RNA was associated with a subviral particle which had a sedimentation coefficient of 650S, compared with 495S for the intact virion, and which banded in Cs2SO4 gradients at 1-295 g/ml. Negatively stained preparations of subviral particles were shown by electron microscopy to contain spherical particles of 60 to 70 nm diam., similar in appearance to those derived from oncornaviruses. Polyacrylamide gel electrophoresis of the polypeptides from isolated subviral particles showed that these structures contained three of the four major virus structural proteins, the arginine-rich polypeptide VP2 and the two membrane glycopolypeptides VP2 and 4. The detergent-liberated surface projections, composed of a single species of sulphated glycopolypeptide, VPI, were isolated by rate-zonal centrifugation through sucrose gradients followed by precipitation with ammonium sulphate in the presence of bovine serum albumin.
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The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5-8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37 degrees C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4 degrees C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0.
The proportion of cells absorbing trypan blue (tb-+ character) can be used to measure the late c.p.e. of wild-type poliovirus (ts-+. tb-+), which was the same at restrictive (39-2 to 39-6 degrees C) or permissive (37 degrees C) temperatures. Of twenty ts mutants, seven showed normal c.p.e. at 37 degrees C but were defective in C.P.E. (TB) AT 39-5 degrees C; all seven tb mutants have previously been shown (Cooper et al. 1971) to give evidence of a primary defect in replicase 1 activity (to make the complementary or minus strand of virus RNA). The remainder (tb-+) have all previously been shown to give evidence of a primary defect either in replicase II activity (to make progeny plus strands) or in structural protein. Thus, the late c.p.e. is dependent on a product of the replicase I gene, of which the in vivo effector is probably double-stranded RNA. Late c.p.e. is not caused by prevention of host protein, RNA or DNA synthesis and is not necessarily correlated with lysosomal enzyme release. The tb mutants were also defective in inducing early changes in chromatin (chr) and in prevention of thymidine incorporation (pti), but the tb and pti/chr characters are probably independent expressions of replicase I activity. Virus growth does not depend on repression of DNA synthesis. Poliovirus represses the activities of host DNA-dependent RNA polymerase I and II to an equal extent. There is no evidence that repression of DNA or RNA synthesis results from direct interaction of virus protein with the DNA.
Purified preparations of Mycoplasmatales virus-laidlawii 3 were negatively stained and studied by electron microscopy. They were seen to consist of uniform sized particles having a polyhedral head, 57 nm by 61 nm, and a short tail, 25 nm long, joined to the head at one vertex by a collar. The particles were shown to have buoyant densities of 1-477 g/ml in CsC1, 1-32 g/ml and 1-26 g/ml in potassium tartrate and a sedimentation coefficient, S20,W, OF 290 +/- 13. They are composed of 35-2% double-stranded DNA and five structural polypeptides with approximate mol. wt. of 172000, 81000 73000, 68000 and 43000. The classification of the virus from its morphology and chemical properties is discussed.
The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have beem examined by polyacrylamide gel electrophoresis. Four major polypeptides, VPI (mol. wt. 200000), VP2 (50 000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VPIa (105000) and VPIb (80500) have been reproducibly demonstrated in the virion, of which VPI, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VPI, thus identifying this glycopolypeptide as the major structural component of the projection.
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