Paul M. Osterrieth

Changes in Morphology, Infectivity and Haemagglutinating Activity of Semliki Forest Virus Produced by the Treatment with Caseinase C from Streptomyces albus G.

SUMMARY: Electron micrographs of negatively stained Semliki Forest virus were made before and after treatment of the virus with caseinase C from Streptomyces albus G. The virus appeared as roughly spherical (diameter 550–590 A), covered with projections and sometimes bearing an appendix, which seemed to be a fold of the envelope. Treated with caseinase C in tris buffered saline, the virus lost its projections and its haemagglutinating activity but remained infectious. The site of haemagglutination, then, is probably located on the projections. Treated with caseinase C in phosphate buffer, the virus lost its infectivity as well as its haemagglutinating activity; the number of the remaining viral particles was not significantly modified. These structures had no projections; their envelope was degraded and sometimes completely destroyed. In this case, they had a smaller diameter (385–390 A) than the projectionless particles obtained by treatment in tris buffered saline (410–460 A). As the virus particles with degraded envelope still contained as much infectious RNA as the controls, it was thought that some degree of integrity of the envelope was necessary for the particle infectivity. Thus the site for infectivity appeared to be different from that for haemagglutination.

Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Abstract Milk fat globule membranes and mammary tumour virus particles ( d = 1.17 g/cm 3 ) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.

Peroral infection of suckling mice with milk-borne mouse mammary tumour virus: uptake of the main viral antigens by the gut.

Persistence of mouse mammary tumour virus (MMTV) components in the digestive tract of suckling mice was investigated by immunoperoxidase staining of the main viral antigens and micro-immunoenzyme assays of gp52 and p28; these latter assays were also performed after ingestion of milk enriched in viral antigens using Cr2O3 as a marker for the alimentary bolus migration. When compared to the ingested antigens, the amounts of gp52 and p28 decreased during transit, p28 being more rapidly digested than gp52. The antigens were, however, destroyed to a much larger extent in the gut of the adult than in that of the newborn mouse. A fraction of the marker remained for a long time in the stomach; a prolonged retention was also observed with gp52 and especially with p28. Significant amounts of viral antigens were detected in the intestinal walls: both p28 and gp52 were found in the duodenum and small intestine. Moreover, the four viral antigens gp52, gp36, p28 and p8 were clearly observed in very large supranuclear vacuoles inside the epithelial cells of the distal part of the gut. Total particles can reach the intestine; the viral material could then be either destroyed or taken up in the epithelial cells by endocytosis, so that the intestinal epithelium might serve as a portal of entry for MMTV in the suckling mouse.

Typing of nosocomial strains of Serratia marcescens: comparison of pulsed-field gel electrophoresis of macrorestriction fragments with biotyping, esterase typing and ribotyping

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with XbaI. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRI and HindIII. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymotypes) proved to be the most discriminating, followed by ribotyping (28 ribotypes) and biotyping (10 biochemical profiles). Biotyping would serve as a screen to identify isolates, due to its accessibility. Esterase typing provided a reliable tool to make subdivisions within biotypes because of congruence between biochemical groups and esterase patterns. Additional discrimination was still achieved by ribotyping and PFGE. It is concluded that the combined results of these four markers were useful for distinguishing all epidemic and sporadic isolates.

Distribution of Mouse Mammary Tumour Virus Antigens in RIII Mice as Detected by Immunofluorescence on Tissue Sections and by Immunoassays in Sera and Organ Extracts

Organs of RIII mice at various physiological stages were tested for mouse mammary tumour virus (MMTV) antigen expression. Indirect immunofluorescence was used with three monospecific antisera to localize one envelope glycoprotein, gp47, and two core proteins, p28 and p8. These virus-specific antigens gave characteristic fluorescent patterns in the mammary tissues and were also detected in thymus and salivary gland sections of some mice. The amounts of antigens gp47 and p28 were measured by immunoassay in sera and organ extracts of corresponding samples of mice. Sera of mice of both sexes contained virus antigens from the suckling age onwards. Although ingested virus could be traced in suckling mice, weanlings were characterized by the absence of virus expression except in lymph nodes. This location points to the possible role of lymphoid tissue in producing the antigens of the blood and in disseminating the infection. In adult animals, virus antigens were present in salivary glands, digestive tract, lymph nodes, male genital organs and female mammary glands. Antigen expression, even found in the mammary glands of virgin mice, was strikingly increased by pregnancy, lactation and (or) ageing, the highest values being found in mammary tumours. The results for milk-borne MMTV infection in RIII mice are compared with those obtained previously in Swiss albino mice.

Electron microscopy studies on Banzi virus particle and its development in the suckling mice brains.

Banzi virus [ Flavivirus (Togaviridae)] was purified from infected mice brains. Negatively stained, the particle appears roughly spherical (525 in diameter), its envelope bears thin projections, its central core (270 in diameter) has a polygonal outline suggesting a cubic symmetry. Hexagonal structures (120 in diameter) might represent the capsomeres. In thin sections, the sizes of the total particle and of the core are 440 and 285 , respectively. The viral development in suckling mice brains is cytoplasmic. An enlargement of the endoplasmic reticulum cysternae is observed, which is followed by an extensive hypertrophy of the intracytoplasmic membranous material. This latter material consists of numerous vesicles located inside the cysternae and a peculiar assembly of concentric lamellae. Mature virus particles appear in the extracytoplasmic spaces delimited by these membranes. They are also found outside the cells, either in groups or isolated and fixed to a plasma membrane.

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. I. Localization by immunofluorescence of four antigens in mammary tissues and other organs.

The indirect immunofluorescence technique was used to investigate the expression of virus antigens in a Swiss albino mouse strain infected by mouse mammary tumour virus (MMTV). The antisera used were monospecific for: the glycopolypeptides gp160 and gp47, and the internal polypeptides p28 and p8. In mice infected with milk-borne MMTV, all four antigens were detected in the mammary gland during the first pregnancy and then throughout life, and in mammary tumours. Antigen gp160, located at the secretory border of all glandular cells, is shared by the plasma membrane of the mammary cell, whether virus-producing or not, and by the virus envelope, as shown by the use of absorbed antisera. The other three antigens are virus-specific. Anti-p47 serum stained the secretory border of cells whereas the fluorescence related to p28 and p8 consisted of a few large intracytoplasmic spots. The specific antibodies for p28 and p8 were only absorbed by disrupted virions, confirming that these polypeptides are internal antigens. With serial sections of glands from pregnant and old female mice, the alveoli stained with anti-p28 or anti-p8 serum were found to react also with anti-gp47 serum. However, during lactation, the presence of p28 and p8 could not be detected in the alveoli stained with anti-gp47 serum. Thus, infected alveoli express virus antigens differently. In the mammary glands of all mice studied, some alveoli did not display any staining when the latter three antisera were used. The specific antigenic pattern, maintained in differentiated mammary tumours, became a diffuse and unlocalized staining after transformation into the undifferentiated state. With the exception of the kidney, where gp47 was found in the glomeruli, all other organs of the mouse did not stain with any of the antisera. The presence of gp47 in the glomeruli, probably related to immune-complex deposits, increases with the age of the mouse and is most noticeable in tumour-bearing females. In mice free of milk-borne MMTV, examined during the first week of lactation, antigen gp47 could not be detected. Internal antigens p28 and p8 were detected in nearly all mammary cells as numerous small cytoplasmic dots. This suggests a limited expression of an endogenous virus genome; this expression would be modified when milk-borne virus is produced.

Natural infection of Swiss mice with mouse mammary tumor virus (MMTV): viral expression in milk and transmission of infection.

SummaryQuantitative determinations of gp52, the main envelope glycoprotein, and p28, the main core protein, of MMTV, have been performed in about 1000 individual samples of milk of breeding females from our colony of MMTV-infected Swiss mice, a line characterized by a moderate incidence of mammary tumors. A computer analysis of the results showed: 1— an important individual variation, ranging from 0 to 120µg per ml of milk for p28, and from 0 to 320 µg per ml of milk for gp52; 2— a variation of the release of both antigens during a single lactation, with a maximum on the 7–8th day of nursing; 3— an increase of the release of both antigens with parity up to the 6th lactation, followed by a marked decrease during later lactations; 4— a higher degree of infection in the offspring of 2nd and 3rd litters. The possible dependence of viral expression and transmission of infection upon factors such as cyclic activity of the mammary gland and progressive immunization of mice against MMTV is analyzed. The status of our laboratory line of MMTV infected Swiss mice is discussed in comparison with high and low tumor incidence strains.

Detection of Virus Antigens in Swiss Albino Mice Infected by Milk-borne Mouse Mammary Tumour Virus: the Effect of Age, Sex and Reproductive Status. II. Radioimmunoassay of Two Virus Components, gp47 and p28 in Serum and Organ Extracts

Extracts of various organs, mammary tumours and sera from milk-borne MMTV infected Swiss albino mice of different age, sex and physiological conditions were tested by radioimmunoassay for the presence of gp47, the main envelope polypeptide, and p28, the main core protein of the virus. Except in brain, ovaries and testes, both antigens were found in all organs of old animals and of females after the onset of their first pregnancy. Antigens were not present in organs of weanlings or in whole foetuses. Higher values were found in mammary glands, mammary tumours, epididymis and seminal vesicles. These organs also harboured a greater amount of gp47 than p28. The serum generally contained gp47 but rarely p28. This indicates that gp47 is not virion-bound in blood. Pregnancy, lactation and especially the presence of mammary tumours increased the concentration of gp47 in serum. The results do not allow localization of target organs of MMTV infection in the interval between ingestion of the virus by the suckling mouse and the first pregnancy. Moreover, results obtained with one group of mice devoid of exogenous virus show that, as endogenous MMTV genome expresses p28, it might account for part of the p28 detected exogenous MMTV-infected mice.

Enzymoimmunoassay of the main core protein (p28) of mouse mammary tumour virus (MMTV)

Abstract An enzymoimmunoassay was developed to detect p28, the main core protein of mouse mammary tumour virus. It is an ELISA test (enzyme-linked immunosorbent assay) using polystyrene tube as solid phase. The method was assessed according to two techniques: the one-step technique in which tubes coated with anti-p28 antibodies are incubated only once after simultaneous addition of both antigen (sample or standard) and enzymatic tracer (anti-p28 antibodies conjugated to glucose oxidase); and the two-step technique in which coated tubes are incubated first with antigen, then, after washing, submitted to a second incubation with the enzymatic tracer. In both techniques, after the last incubation, tubes are washed and submitted to enzymic detection. The dose-response curves of the reaction are investigated and appear as different according to the technique used. As regards sensitivity, precision, accuracy and reproducibility, these methods were equal to competition radioimmunoassay and the results obtained by enzymoimmunoassay and radioimmunoassay for a series of tissue extracts are closely related. The advantages of the enzymoimmunoassay over radioimmunoassay are simplicity, speed, absence of radioactive material and stability of the tracer.