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G C Millson

Publications and source records attributed to G C Millson.

At least 19 recordsLinked to original sources
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

Disinfection studies with two strains of mouse-passaged scrapie agent. Guidelines for Creutzfeldt-Jakob and related agents.

A variety of disinfection procedures were tested on two strains of scrapie agent, treated either as brain macerates (autoclaving) or as 10% homogenates (chemical treatments). It is suggested that a given treatment should produce a titre loss, of both strains of scrapie, of at least 10(4) units before it be regarded as useful for the disinfection of the agents of scrapie and Creutzfeldt-Jakob disease (CJD). By this criterion, treatment at room temperature with about 4% Hycolin (0.6% chlorinated phenols), 0.2% permanganate, 5% Tego (dodecyl-di(aminoethyl)-glycine) or 5% sodium dodecyl sulphate (SDS) are unsuitable. However, data indicate that SDS might be used to reduce the heat stability of scrapie agent. Hypochlorite (Sterilex) was the only satisfactory chemical reagent tested. At least 10(4)-10(5) units of infectivity were lost by treatment with hypochlorite containing 1,000 ppm available chlorine after a 4-16 h exposure, or containing 10,000 ppm available chlorine after a half-hour exposure. The latter result points to the use of concentrated hypochlorite (about 2% available chlorine; approximately 20% Sterilex) to decontaminate surfaces. We suggest that the cleaning action of SDS, or other strong detergents, might also help to decontaminate surfaces, but studies on this are needed. Autoclaving at 126 degrees C for 1-2 h reduced titres by 10(3)-10(7) units, depending on the strain of agent. However, total disinfection of brain containing high titres of infectivity was approached only at 136 degrees C when titre losses of about 10(6) units were obtained by autoclaving for 4-32 min. Further studies are needed before we can make simple, general recommendations for the disinfection of CJD agents in hospital practice.

Animals↗
PubMed1983

Can scrapie titres be calculated accurately from incubation periods?

Since endpoint titrations of scrapie material are costly and time-consuming, several workers have estimated titre from the correlation between the incubation period of the disease and the infectivity titre. However, we show here that the relationship between incubation period and titre cannot be assumed to be constant for all scrapie preparations. Our results indicate that sodium deoxycholate treatment of scrapie preparations does not reduce the titre, but can lengthen the incubation period by about 10 days. This is equivalent to a discrepancy of 1 log LD50 unit if the estimation of titre was based on the incubation period.

Deoxycholic Acid↗
PubMed1983

Infectivity of liposomally encapsulated nucleic acids isolated from EMC virus and scrapie-infected mouse brain.

The ability of liposomes to enhance nucleic acid infectivity in vivo was studied. Encephalomyocarditis (EMC) virus RNA was extracted and encapsulated within liposomes. The infectivity of EMC RNA was increased by liposomal entrapment after intraperitoneal injection of mice, but decreased after intracerebral injection. In contrast, when nucleic acids from scrapie-infected brains were entrapped in liposomes and injected into mice by one of four routes, no cases of scrapie were observed. This is the first report of the enhancement of nucleic acid infectivity by liposomes in vivo.

Animals↗
PubMed1983

Involvement of protein in scrapie agent infectivity.

The nature of the causative agent of scrapie is not known. Previous work has demonstrated that nuclease digestion does not inactivate scrapie infectivity, but there are conflicting reports about the effects of proteases. It is shown here that the broad range protease, proteinase K, reduces scrapie infectivity under all conditions tested. Control experiments demonstrated that the loss of infectivity is not artefactual and results from protein breakdown. Proteolytic digestion in the presence of detergent greatly increases proteolysis, but does not lead to a further loss of infectivity. This suggests that the protein involved may be a surface component, but whether the component is an integral or secondary part of the agent is not known.

Animals↗
PubMed1980

Sensitivity of scrapie infectivity to detergents and 2-mercaptoethanol.

Exposure of fractions obtained from scrapie-infected mouse brain to Triton X-100 resulted in no change in its infectivity except after sedimentation. Infectivity was reduced after exposure to 2-mercaptoethanol but only in the presence of SDS. These results support the view that protein is an important component of the scrapie agent and that disulfide bonds may play a part in maintaining the structural integrity of the infectious agent.

Animals↗
PubMed1977

Genetic markers in Herdwick sheep: no correlation with succeptibility or resistance to experimental scrapie.

Genetic markers were studied in Herdwick sheep, bred at Compton, with special reference to genetically selected lines which differ in susceptibility or resistance to experimentally produced scrapie. There were no correlations between susceptibility to the disease and albumin, pre-albumin, esterase and haemoglobin phenotypes, and between reduced glutathione levels and alpha-mannosidase isoenzymes, the latter possibly representing a previously undescribed genetic marker in sheep.

Animals↗
PubMed1976

The membrane location of scrapie infectivity.

Using 5'-nucleotidase and NADPH: cytochrome c reductase as respective enzyme markers for the plasma membrane and endoplasmic reticulum, a satisfactory separation of these two membrane fractions from a cell line (SMB) derived from a scrapie mouse brain has been achieved. The coincident distribution of scrapie infectivity and 5'-nucleotidase in various fractions isolated from these cells indicates that most of the scrapie infectivity present in this cell line is associated with the plasma membrane.

Animals↗
PubMed1976

Search for scrapie-specific RNA and attempts to detect an infectious DNA or RNA.

Preparations of in vivo labelled RNA from normal and scrapie infected mouse brains were fractionated by polyacrylamide gel electrophoresis. No appreciable differences were detected between the two preparations. The biological activity of preparations of RNA and DNA from scrapie brain was also examined. In each case the nucleic acid was obtained by one method envolving the use of phenol and one without phenol. Attempts were made to bind the nucleic acid to normal membrane by three different methods. In no instance was a single case of scrapie obtained by inoculating nucleic acid extracts from scrapie brain either alone or associated with normal membranes. It is concluded that a putative scrapie-specific nucleic acid must either be present in scrapie brain in such an unusual from that it is not extracted readily by methods suitable for the preparation of most virus nucleic acids; or, if it is extracted, that it has no detectable biological activity. In either case, the scrapie agent differs substantially in its properties from the plant viroids.

Animals↗
PubMed1975

Transferrin polymorphism in Herdwick sheep.

The transferrin system in Herdwick sheep, bred at Compton, was investigated with special reference to the susceptibility of the flock to experimentally produced scrapie. No significant correlations were observed between susceptibility and transferrin phenotypes.

Animals↗