Acquisition and transmission of potato leafroll virus by Myzus persicae : quantitative aspects

Studying the transmission of potato leafroll virus (PLRV) by Myzus persicae from infected Physalis floridana plants, revealed that the ability of aphids to transmit the virus differed widely among individuals and strongly depended on the biotype of the vector aphid and the age of the virus source. Virus transmission was more frequent by M. persicae from top leaves that hardly showed any disease symptom than from bottom leaves with pronounced symptoms. The percentage of successful PLRV transmission decreased with the infection age. Furthermore, the median latency period (LP 50 ) of the virus was significantly shorter in aphids that fed on top leaves than on bottom leaves. The differences in virus transmission from bottom and top leaves could not be explained by the amount of viral antigen in the sources, since a higher concentration of viral antigen was detected in bottom leaves than in top leaves. The feeding behaviour of aphids on the virus sources also did not account for the observed differences; the honeydew excretion rate of M.persicae nymphs feeding on bottom leaves was higher than on top leaves of infected plants (Chapter 1).To investigate to what extent the amount of virus acquired by M. persicae contributed to differences in virus transmission, the sensitivity of the double antibody sandwich (DAS) variant of the enzyme-linked immunosorbent assay (ELISA) had to be increased. To this end, the procedure was modified by incubating sample and conjugate simultaneously (cocktail-ELISA), and by amplifying the enzyme reaction in which a dephosphorylated substrate catalytically triggers an enzyme-mediated redox cycle. Cocktail-ELISA preceding enzyme amplification was 10- to 15-fold more sensitive than DAS-ELISA and could easily be applied to detect viral antigen in individual nymphs which had been feeding for only a short period of time on infected P. floridana plants (Chapter 2).Nymphs that had fed on bottom leaves with pronounced symptoms of P. floridana plants infected with PLRV acquired considerably less virus in the same acquisition access period than nymphs feeding on top leaves. The observed dissimilarity between the viral antigen content in the virus sources and in the aphids feeding on them suggests that the availability of the virus for acquisition by aphids is considerably lower in bottom than in top leaves (Chapter 3). Changes in availability of virus for acquisition were also noticed on eight potato genotypes with different levels of field resistance to PLRV. Early in the growing season, a lower virus titer in the secondarily-infected potato plants resulted in a lower capacity of the plants to act as a virus source. As plants aged, and symptoms became apparent, the virus was still readily detectable in the plants but virus acquisition by M. persicae was impaired and did not correlate anymore with the amount of viral antigen in the source (Chapter 4). PLRV acquisition and transmission by M. persicae from artificial diets containing purified PLRV demonstrated that the amount of viral antigen in the nymphs, and the percentage of viruliferous nymphs were linearly related to the log 10 transformed virus concentration in the diet. Therefore, the amount of viral antigen present in aphids is a more reliable parameter in deducing the potential of a plant to act as a virus source, than the viral antigen concentration of the plant itself.Artificial diet studies furthermore demonstrated that the LP 50 of the virus in M. persicae was not influenced by the virus concentration in the diet, but by interactions between the virus and its vector. Four-day old M. persicae nymphs displayed a longer LP 50 than one-day old nymphs. PLRV purified from top leaves of infected P.floridana was transmitted with a significantly shorter LP 50 than virus purified from bottom leaves. This finding shows that the previously observed differences in virus transmission from intact top and bottom leaves of P.floridana can not be solely explained by differences in the virus concentration available for acquisition. As similar amounts of purified virus from top and bottom leaves in artificial diet were fed to the aphids, it is likely that intrinsic properties of the virus also determine the transmissibility of the virus occurring in top and bottom leaves. This may concern changes at the surface of the viral capsid (Chapter 3).To study possible relationships between the transmissibility of PLRV and protein structures at the surface of the viral capsid, monoclonal antibodies (MAbs) were generated to PLRV. After two fusion experiments, nine different MAbs to PLRV were selected, and the topological relationships and the nature of the epitopes, to which they were directed, determined (Chapter 5). PLRV isolates which differed in their transmissibility by M. persicae, were tested in a triple antibody sandwich ELISA with this panel of MAbs. It was shown that four MAbs reacted significantly stronger with isolates which were readily transmitted than with the poorly transmitted isolates. Moreover, when mixtures of PLRV and MAbs suspensions were fed to M.persicae, these four MAbs reduced the probability of virus transmission and significantly increased the latency period of the virus in its vector. Hence, two lines of evidence indicate that the epitopes to which these MAbs were directed might be functionally involved in virus transmission by M.persicae (Chapter 6). The four MAbs reacted with conformation-dependent epitopes which are supposedly dependent on the tertiary protein structures. Competitive binding assays indicated that these epitopes strongly overlap which each other (Chapter 5). Anti-idiotypic antibodies (AiAbs) were raised to the four MAbs in rabbits. They may be considered as blueprints of the epitopes to which the MAbs are directed. When suspensions containing these AiAbs were fed to M.persicae prior to virus acquisition, PLRV transmission was reduced by up to 71% (Chapter 6). AiAbs may have blocked specific sites in the aphid which have a function in transcellular transport of virus particles.