R. Koenig

ELISA in the study of homologous and heterologous reactions of plant viruses

Summary Serological relationships between plant viruses within six different taxonomic groups were studied by means of the double antibody sandwich form of enzyme-linked immunosorbent assay (ELISA). This technique was very sensitive not only for the detection of low concentrations of viruses, but also for the differentiation of closely related antigens. With some viruses, e.g. Andean potato latent virus, the specificity of the test was so great that conjugates prepared to one strain failed to detect other serologically closely related strains. With other viruses and with host proteins the cross-reactivity was broader, but distant and even intermediate serological relationships between viruses were usually not detected. The great specificity of ELISA is mainly caused by the behaviour of the conjugated antibodies. The binding of conjugated antibodies was completely inhibited by simultaneously added native antibodies. Antibodies from very early bleedings were not suitable for ELISA.

The trapping of tymovirus particles on electron microscope grids by adsorption and serological binding.

Summary Many purified tymovirus particles are trapped on buffer-treated grids due to non-specific adsorption. Pre-treatment of grids with serum or addition of crude plant sap to virus preparations greatly inhibited non-specific adsorption. Specific serological binding on grids coated with homologous antiserum produced particle counts comparable to those achieved by non-specific adsorption on buffer-treated grids in the absence of plant sap. Specific serological binding was not influenced by crude sap of Nicotiana clevelandii. For both adsorption and serological binding there was a linear relationship between the log virus concentration and log particle counts at virus concentrations below approx. 10 µg/ml; at higher concentrations the grids were saturated. In the presence of excess virus, antisera at dilutions of less than 10-3 yielded lower particle counts than more dilute antisera. Heterologous reactions were detected only with viruses for which very close serological relationships had been found in the agar gel double-diffusion test. Heterologously bound virus was apparently not replaced by subsequently applied homologous virus. However, adsorbed bottom component virus particles could be replaced by top component particles of the same virus and vice versa.

Serological Relationships among Tombusviruses

Summary Serological differentiation indices (SDIs) based on serum titrations in agar-gel double diffusion tests were determined for all ten definitive tombusviruses known at present, namely artichoke mottled crinkle, carnation Italian ringspot, cymbidium ringspot, eggplant mottled crinkle, Moroccan pepper, pelargonium leaf curl, petunia asteroid mosaic, the BS3 and type strains of tomato bushy stunt, and tombus Neckar viruses. More than 2000 titrations using 222 antisera from 36 rabbits were done. The SDIs ranged from 1 to >9, and all intermediate values were found. Simple trigonometry and computer methods were used to classify the results. There was no correlation between the serological relatedness of the particles of these viruses and the homology of their genome nucleic acids previously assessed by hybridization tests. In this respect they resemble the tymoviruses but not the tobamoviruses. The electrophoretic migration of the particles of 12 tombusviruses was studied in 1% agarose containing 20 mm-phosphate buffer pH 7.0. Some serologically indistinguishable tombusvirus strains migrated at different rates.

Nucleic acids in the potato virus X group and in some other plant viruses: comparison of the molecular weights by electrophoresis in acrylamide-agarose composite gels.

The molecular weight of the nucleic acid is a basic property of a virus, and has been shown to be of great value for classifying animal viruses (Bellett, 1967). However, this property is known for few plant viruses. In the potato virus X group, for example, only the RNA of potato virus X (PVX) itself has a known molecular weight. The reported value (2.1 × 106) has been calculated from the total particle weight of the virus and its phosphorus content (Knight, 1963). Recently electrophoresis in acrylamide gels of low concentration has been successfully used to estimate the molecular weights of large RNA molecules (Loening, 1968; 1969). Studies including the RNAs of five plant viruses have been reported by Bishop, Claybrook & Spiegelman (1967), and the RNA of tobacco mosaic virus (TMV) has been used as a marker in the re-examination of the molecular weight of poliovirus RNA (Tannock, Gibbs & Cooper, 1970).

Effect of organic mercury compounds in serological tests on some isometric plant viruses.

Mercury-containing preservatives, such as Merthiolate and Cialit (Koenig, 1969), inhibit the formation of precipitin lines in gel-diffusion tests with several animal and plant viruses (Le Bouvier, 1959; Bancroft, 1962; Cowan, 1966; Koenig & Jankulowa, 1968). With foot-and-mouth disease virus this inhibition is due to a partial degradation into subunits (Cowan, 1966) whereas with belladonna mottle virus (BMV) the diffusion of virus is inhibited (Koenig, 1969). In this paper these studies are extended to other isometric plant viruses. Turnip yellow mosaic (TYMV), eggplant mosaic (EMV), * Andean potato latent (APLV), * viruses and BMV were purified by the method of Paul et al. (1968). A grape-vine isolate of sowbane mosaic virus (SMV) (Bercks & Querfurth, 1969) was purified by the method of Wetter (1960). Rabbits were immunized by two intramuscular injections, given 1 week apart, of virus emulsified in Freunds adjuvant. Complete adjuvant was used for the first injection and incomplete for the second.