Pertti Väänänen

Fusion and haemolysis of erythrocytes caused by three togaviruses: Semliki Forest, Sindbis and rubella.

Semliki Forest, Sindbis and rubella viruses can fuse erythrocytes from several different species. Large fusion vesicles consisting of tens to hundreds of red blood cells were seen under optical conditions. For the haemagglutination and cell fusion to occur the adsorption of virus and further incubation had to be carried out at pH 5.8. Haemagglutination took place over a wide temperature range (0 to 40 degrees C) whereas fusion required temperatures between 37 and 42 degrees C. Haemolysis of red blood cells induced by togaviruses also required initial incubation at pH 5.8 to enable attachment of the virus to occur after which the pH of the buffer could be raised to neutrality without inhibiting the haemolysis. The amount of togaviruses and Sendai virus required to fuse red blood cells was about the same [I haemagglutinating unit (HAU)/ml] but different ionic conditions were required for fusion.

Haemolysis by two alphaviruses: Semliki Forest and Sindbis virus.

Purified preparations of Semliki Forest (SFV) and Sindbis virus haemolyse red blood cells from several species of animals and birds. The optimal haemolysis by SFV was obtained at pH 5.8 with 1-day-old chick erythrocytes incubated at room temperature. Considerable variation in haemolytic activity was observed between different virus preparations purified by different methods. The haemolytic activity of SFV was inhibited by antisera against whole virus or isolated envelope proteins but not with antiserum against virus capsid protein. Neither lipid and detergent-free envelope protein octamers with high haemaggluinating titre, nor isolated nucleocapsids caused haemolysis. Fresh, unpurified SFV and Sindbis virus preparations did not haemolyse unless they were exposed for repeated cycles of freezing and thawing. It appears that the haemolytic activity resides in the virus glycoproteins(s) but can only be manifested in slightly damaged whole virus particles.

Purification of Rubella Virus Particles

Summary A procedure for the purification of rubella virus from infected suspensions of BHK 21 cells resulted in preparations containing 1 to 3 × 1010 p.f.u./ml. In sucrose gradient centrifugation the area of maximal infectivity (buoyant density of 1.175 g./ml.) coincided with peaks in haemagglutinating activity, and in E 260. It also coincided with presence of virus-like particles, and a sharp band visible by naked eye. Specific activities of the order 30 × 106 p.f.u./µg. protein, 106 HAU/µg. protein, and 5 to 10 × 105 p.f.u./HAU were achieved. Glutaraldehyde-fixed negatively stained rubella virus preparations showed round particles with rough surfaces measuring 50 to 73 nm. (average 61 nm.) in diameter. Unfixed viruses had a greater size variation, 55 to 89 nm. (average 74 nm.) in diameter, apparently due to deformability and fragility of the particles. Spontaneous and deoxycholate-induced breakdown of the particles showed rupture of the envelope but revealed no characteristic inner structure.

Fusion of Semliki forest virus with red cell membranes

Abstract The interaction of Semliki Forest virus (SFV) and red cells was studied using biochemical and immunological methods. When [ 3 H]uridine-labeled SFV was adsorbed at 0°, pH 5.8, to ribonuclease-loaded human red cell ghosts, the viral RNA became sensitive to ribonuclease within 2 to 5 min at 20–37°, but not at 0°. Maximal digestion of RNA was achieved within 30 min at 37° suggesting that the nucleocapsids had penetrated the red cell membranes. When the red cells that had been fused with [ 35 S]methionine-labeled SFV were treated with trypsin, about 70% of E2 protein and 50% of E1 protein was digested, demonstrating that the viral envelope proteins remained on the surface of the red cell. This was also shown by radioimmunoassay, by complement-dependent immune hemolysis assay, and by the ability of the red cells to bind Staphylococcus aureus bacteria to the cell surface after treatment with anti-envelope antiserum. When the red cells carrying SFV glycoproteins on their surfaces were transferred to pH 5.8, the cells were readily fused after the temperature had been raised to 42°. This shows that the fusion of virus envelope with red cell membranes precedes the fusion of red cells.

Effect of low level immunity on response to live rubella virus vaccine

Serum specimens of 1075 young adults representing nursing and medical staff were collected for determination of rubella immunity using the radial haemolysis (RH) technique, also know as the haemolysis in gel (HIG) test. Of the sera 84 (7.8%) were negative (RH titre less than 4 mm) and an additional 93 (8.7%) gave an RH titre of 4-5 mm, which was regarded as the limit of immunity. Initially, 64 persons of these 177 were vaccinated with the RA27/3 strain of live attenuated rubella virus. Their serum samples were collected at the time of vaccination and at three weeks and three months after vaccination. Altogether 54 vaccinees could be followed for their immune response throughout the study. It became obvious that vaccination generally caused seroconversion only after three months rather than within three weeks. Only one person remained seronegative - even after a booster dose of vaccine. The mean final antibody titres in individuals with pre-existing low level immunity was 6 mm whereas in initially seronegative persons the antibody titre after vaccination was 8.5 mm on average. Thus a pre-existing low level immunity will effectively block the immune response to live rubella virus vaccine and this phenomenon may explain some apparent vaccination failures.

The use of red cells with fused Semliki Forest virus envelope proteins in antibody determinations by hemolysis in gel

Abstract Chicken red blood cells with fused Semliki Forest virus (SFV) proteins on the cell membrane were used in the hemolysis-in-gel (HIG) plates. Optimally the plate contained a 1.5 mm thick gel of 1% agarose with 1% red cells and 1 unit/ml gel of complement. 400 ng of SFV was fused to the red cells in 1 ml of the gel (about 20 virions fused to one red cell). Five μl of inactivated (56°C, 30 min) serum samples were pipetted into wells in the gel. After 20 h of incubation at 37°C the diameters of the hemolytic zones were directly proportional to the logarithm of the serum dilution. This made it possible to calculate the antibody titers for the samples using an experimental formula T = 10 φ k (T is the titer, φ the diameter in mm and k a variable coefficient, which had to be determined for each batch of the plates using a standard serum). Using regression and residual analyses, the formula was shown to fit the experimental results. The fusion-based HIG could be read after as early as 2 h of incubation. The specificity of the test was studied using antisera against Western equine encephalomyelitis, Eastern equine encephalomyelitis, Chikungunya and Uruma viruses, which all gave negative results in the SFV HIG test. Antisera against SFV E1 and E2 proteins were positive, but anti-E3 serum was negative when measured in the SFV HIG test. Chicken red blood cells with fused Semliki Forest virus (SFV) proteins on the cell membrane were used in the hemolysis-in-gel (HIG) plates. Optimally the plate contained a 1.5 mm thick gel of 1% agarose with 1% red cells and 1 unit/ml gel of complement. 400 ng of SFV was fused to the red cells in 1 ml of the gel (about 20 virions fused to one red cell). Five microliters of inactivated (56 degrees C, 30 min) serum samples were pipetted into wells in the gel. After 20 h of incubation at 37 degrees C the diameters of the hemolytic zones were directly proportional to the logarithm of the serum dilution. This made it possible to calculate the antibody titers for the samples using an experimental formula T = 10 phi / kappa (T is the titer, phi the diameter in mm and kappa a variable coefficient, which had to be determined for each batch of the plates using a standard serum). Using regression and residual analyses, the formula was shown to fit the experimental results. The fusion-based HIG could be read after as early as 2 h of incubation. The specificity of the test was studied using antisera against Western equine encephalomyelitis, Eastern equine encephalomyelitis, Chikungunya and Uruma viruses, which all gave negative results in the SVF HIG test. Antisera against SFV E1 and E2 proteins were positive, but anti-E3 serum was negative when measured in the SVF HIG test.