- Volume 58, Issue 2, 1982

An indirect ELISA is described in which (i) virus is trapped by F(ab′)2 fragments of specific IgG immobilized on a solid phase support, (ii) trapped virus is detected by intact IgG (from the same or a different antiserum) and (iii) positive reactions are identified using an enzyme conjugate specific for the Fc portion of IgG. Pepsin digestion of the Fc portion of the trapping antibody permits the use of a general purpose enzyme conjugate to discriminate between trapping and detecting antibody. Consequently, the assay requires only a single virus-specific antiserum which is often all that is available to the plant virologist. The assay was at least as sensitive for detecting small amounts of antigen as the standard double-antibody sandwich procedure and, for some viruses, two- to fourfold more sensitive. The improvement in performance resulted largely from lower and more consistent background reactions. Both assays were equally effective in revealing heterologous reactions when optimized for detecting homologous antigen. However, increased cross-reactions were obtained in the F(ab′)2 procedure by the use of higher concentrations of detecting antibody. The assay is considered particularly suited for comparing antisera from different sources or of different bleeds from the same source, and for investigations involving so few tests that the effort or expense of preparing individual virus-specific conjugates is not justified.

The kinetics of infection of barley protoplasts with barley stripe mosaic virus (BSMV) was followed by enzyme-linked immunosorbent assay (ELISA) using the double-sandwich procedure. Infected protoplasts were identified by an immunoperoxidase method taking precautions to inhibit endogenous peroxidase. About 70% of the protoplasts were infected with BSMV when the inoculum contained 50 µg/ml purified BSMV in 10 mm-potassium citrate buffer pH 4.7 containing 0.65 m-mannitol and 5 µg/ml poly-l-ornithine. It was estimated by ELISA that about 1400 to 1600 virus particles were adsorbed per protoplast. Following an apparent latent period, virus increased linearly between about 14 and 48 h after inoculation. After 48 h the virus concentration reached between 1.62 × 106 and 2.0 × 106 particles per protoplast.

RNA of tomato spotted wilt virus (TSWV) was analysed by gel electrophoresis on methylmercury-agarose gels. Three segments were detected with mol. wt. of 2.7 × 106, 1.7 × 106 and 1.1 × 106. The behaviour on oligo(dT)-cellulose columns indicated that TSWV RNA does not contain poly(A) sequences. TSWV RNA directed the synthesis of several proteins in a wheatgerm cell-free system and a messenger-dependent rabbit reticulocyte lysate. Analysis of the translation products by gel electrophoresis revealed that two major polypeptides were synthesized in both systems. One could be identified by immunoprecipitation as the nucleocapsid protein; the identity of the other, having a mol. wt. of 60000, is unknown. The above results demonstrate that TSWV contains a positive-stranded RNA and this conclusion was confirmed by other experiments. TSWV RNA labelled with 125I did not hybridize with polyribosomal RNA from infected leaves and transcriptase activity could not be detected in purified preparations of TSWV or in nucleocapsid extracts from infected leaves. This study supports the view that TSWV is a unique membrane-bounded plant virus whose genome consists of three RNA segments which act as mRNA.

In hybridization experiments using complementary DNA copies, no homology was detected between RNA-3 and either of the genome RNA species of tomato black ring virus. It is estimated that sequences amounting to 160 nucleotide residues shared by RNA-3 and RNA-1, or to 120 nucleotide residues shared by RNA-3 and RNA-2 would have been detected. It is concluded that RNA-3 is a true satellite RNA.