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

Nucleotide sequence and genomic organization of an ophiovirus associated with lettuce big-vein disease

The complete nucleotide sequence of an ophiovirus associated with lettuce big-vein disease has been elucidated. The genome consisted of four RNA molecules of approximately 7ò8, 1ò7, 1ò5 and 1ò4 kb. Virus particles were shown to contain nearly equimolar amounts of RNA molecules of both polarities. The 5'- and 3'-terminal ends of the RNA molecules are largely, but not perfectly, complementary to each other. The virus genome contains seven open reading frames. Database searches with the putative viral products revealed homologies with the RNA-dependent RNA polymerases of rhabdoviruses and Ranunculus white mottle virus, and the capsid protein of Citrus psorosis virus. The gene encoding the viral polymerase appears to be located on the RNA segment 1, while the nucleocapsid protein is encoded by the RNA3. No significant sequence similarities were observed with other viral proteins. In spite of the morphological resemblance with species in the genus Tenuivirus, the ophioviruses appear not to be evolutionary closely related to this genus nor any other viral genus

Sequence analysis and genomic organization of Aphid lethal paralysis virus: a new member of the family Dicistroviridae

The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5' ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3' ORF). The deduced amino acid sequences of the 5' ORF and 3' ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genus Cripavirus (family Dicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related to Rhopalosiphum padi virus, and groups in a cluster with Drosophila C virus and Cricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whitefly Trialeurodes vaporariorum, extending its host range to another family of the order Hemipter

Tomato marchitez virus, a new plant picorna-like virus from tomato related to tomato torrado virus

A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as `marchitez disease¿: severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28¿nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898¿nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24¿kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed

Identifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding Potato Leafroll Virus

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain

A new virus infecting Myzus persicae has a genome organization similar to the species of the genus Densovirus

The genomic sequence of a new icosahedral DNA virus infecting Myzus persicae has been determined. Analysis of 5499 nt of the viral genome revealed five open reading frames (ORFs) evenly distributed in the 5' half of both DNA strands. Three ORFs (ORF1-3) share the same strand, while two other ORFs (ORF4 and ORF5) are detected in the complementary sequence. The overall genomic organization is similar to that of species from the genus Densovirus. ORFs 1-3 most likely encode the non-structural proteins, since their putative products contain conserved replication motifs, NTP-binding domains and helicase domains similar to those found in the NS-1 protein of parvoviruses. The deduced amino acid sequences from ORFs 4 and 5 show sequence similarities with the structural proteins of the members of the genus Densovirus. These data indicate that this virus is a new species of the genus Densovirus in the family Parvoviridae. The virus was tentatively named Myzus persicae densovirus. The nucleotide sequence reported in this study appears in the EMBL, GenBank and DDBJ nucleotide sequence databases under accession number AY148187

Voorkomen in plaats van genezen

In een serie artikelen over de verschillende DLP-PO onderzoek programma's wordt in dit artikel ingegeaan op het programma 'Virussen en Viroïden (1998-2001) dat wordt uitgevoerd door Plant Research International B.V., het Praktijkonderzoek Bloembollen en Bolbloemen (PBB) en het Proefstation voor de Bloemisterij en Glasgroenten (PBG). Rubrieken in dit artikel: 1) Achtergrond; 2) Doel; 3) Centrale rol voor karakterisering; 4) Diagnostica; 5) Beheersmaatregelen; 6) Virusresistentie; 7 ) Virussen als vriend; 8) Wat levert het programma op het gebied van identificatie, karakterisering en detectie van plantenvirussen en -viroïden op