- Volume 48, Issue 2, 1980

Measles virus nucleocapsids were labelled with 3H-amino acids and 32P-orthophosphate, and purified from the cytoplasm of persistently infected human amnion cells (AV+). When analysed by SDS-PAGE, the two major capsid-associated polypeptides (P, mol. wt. 69000, and NP, mol. wt. 60000) were shown to be phosphorylated. Subsequent characterization of the phosphorylated polypeptides by acid hydrolysis and high voltage paper electrophoresis showed that serine and threonine were the major phosphorylated amino acid species. The similarities between the peptide phosphorylation patterns obtained in these studies and those reported earlier for the virus phosphoproteins produced in acute infections (Robbins & Bussell, 1979) indicate that major phosphorylative modifications of the capsid proteins are not involved in measles virus persistence in AV3 cells.

Vaccinia and pseudorabies viruses are resistant to ACV [Acyclovir or 9-(2-hydroxyethoxymethyl)guanine] in normal cells. However, both viruses are sensitive in thymidine kinase (TK)-transformed cells in which the resident HSV-specific TK is able to phosphorylate the drug. This demonstrates the sensitivity of these viruses to phosphorylated ACV and suggests a wider antiviral activity for the phosphorylated drug.

A simple and highly sensitive radioimmunosorbent assay (RISA) for the detection of plant viruses is described. The RISA procedure is a microplate method based on the principle of ‘double-antibody sandwich’ and follows essentially the protocol of the enzyme-linked immunosorbent assay (ELISA) (Clark & Adams, 1977), with the exception that 125I-labelled γ-globulin is substituted for the γ-globulin enzyme conjugate; the bound 125I-γ-globulin is dissociated by acidification from the double-antibody sandwich. The radioactivity is proportional to virus concentration, and cauliflower mosaic virus (CaMV) and lettuce mosaic virus (LMV) could be detected at concentrations as low as 5 and 2 ng/ml, respectively. Direct evidence of the adverse effects of conjugation with enzyme on the binding abilities of antibodies is presented. The RISA procedure should prove valuable with viruses for which the ELISA values are too low to be dependable.

Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-l-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 90% of the protoplasts were infected by CPMV when the inoculation medium contained 0.4 m-sorbitol, 0.5 µg virus/ml, 1.5 µg PLO/ml, 10 mm-potassium phosphate buffer, pH 6.3, and 0.5 mm-CaCl2, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaCl2 was omitted. Inoculation at temperatures below 10 °C also decreased infection substantially. With SBMV a maximum of 30 to 35% of the protoplasts were infected when 0.4 m-sorbitol, 2 to 2.5 µg virus/ml, 2 µg PLO/ml, 10 mm-tris-HCl buffer, pH 8.0, 1 mm-MgSO4 and 0.5 mm-CaCl2 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.

Infection of turnip protoplasts with cauliflower mosaic virus (CaMV) was investigated using an immunofluorescent technique. Up to 80% of protoplasts were infected when they were inoculated with 10 µg/ml CaMV, together with 1 µg/ml poly-l-ornithine (PLO) in 20 mm-citrate buffer, pH 5.0. Infection was first detected by fluorescent antibody staining 24 h after inoculation and the CaMV inclusion bodies were observed by electron microscopy of the cytoplasm 72 h after inoculation.