J.F.J.M. van den Heuvel

Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae

In order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots. In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins. The one most readily detected has an M(r) of 63K, and was identified as symbionin. This is the predominant protein synthesized by the bacterial endosymbiont of the aphid and is released into the haemolymph. Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin. Inhibition of prokaryotic protein synthesis by feeding M. persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus transmission by more than 70%. The major coat protein of the virus was found to be degraded in the antibiotic-treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss of infectivity. For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction.

Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.

Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.

Expression of the Potato Leafroll Virus ORF0 Induces Viral-Disease-like Symptoms in Transgenic Potato Plants

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.

Development of a multiplex AmpliDet RNA assay for simultaneous detection and typing of potato virus Y isolates

A multiplex AmpliDet RNA assay was developed for the specific detection of potato virus Y (PVY), and for the differentiation of the PVY(N), PVY(O/C) strains and the tuber necrotic isolates (PVY(NTN)). The assay is based on the generic amplification of a region within the coat protein coding region of all known PVY isolates by nucleic acid sequence-based amplification (NASBA) and strain-specific detection by molecular beacons. PVY(NTN)-specific diagnosis is achieved by detecting PVY(N) and PVY(O)-specific sequences flanking a recombination site that is associated with the tuber necrotic pathotype. The assay exhibited good specificity toward the various PVY strains in both single and mixed infections. The technique was validated by the use of 47 PVY isolates originating from six countries. The results of the AmpliDet RNA assay were identical or consistent with those of biological characterisation in the decisive majority of cases.

Characterization of a new densovirus infecting the green peach aphid Myzus persicae

A new icosahedral DNA virus was isolated from aphids (Myzus persicae) that showed abnormal growth and development. The purified virus particles have a diameter of 20 nm and contain a single-stranded DNA molecule of approximately 5.7 kb. The viral particles are composed of five structural proteins (92, 85, 68, 64, and 57 kDa). As the main biophysical properties of this virus are similar to those of the members of the genus Densovirus it was tentatively named Myzus persicae densovirus (MpDNV). A PCR-based detection method and a polyclonal antiserum raised against MpDNV allowed the detection of the virus in a single-infected aphid. MpDNV is immunologically related to Junonia coenia densovirus, but not to other members of the subfamily Densovirinae. Biological assays showed that MpDNV could be both transmitted transovarially and horizontally via honeydew and saliva. MpDNV was able to infect whiteflies but not other aphid species tested.

The use of molecular beacons combined with NASBA for the sensitive detection of Sugarcane yellow leaf virus

Sugarcane yellow leaf virus (ScYLV) is widely distributed in Brazil and other sugarcane producing countries causing significant yield losses. Due to the high incidence of the aphid vector, the virus is widespread in the field and in parental clones used in sugarcane breeding programmes. Aiming to present a sensitive and reliable detection of ScYLV, we have adapted an AmpliDet RNA system, compared it with the currently available detection methods and discussed its applicability for routine diagnosis. AmpliDet RNA consists of nucleic acid sequence-based amplification (NASBA) of the target RNA with specific primers and simultaneous real-time detection of the amplification products with molecular beacons. The results showed that the system produced a detection level of at least 100fg of purified virus. Virus was readily detected in plant tissues with low levels of infection (without the need of previous RNA extraction) and in the hemolymph of aphids. The method showed to be virus-specific, testing negative for other species of the Luteoviridae. In conclusion, the system has potential to become a diagnostic method for the detection of sugarcane viruses.

Mechanical transmission of poleroviruses

Previously, transmission of poleroviruses has relied solely on the use of their aphid vectors. Biolistic inoculation allowed for the first time the mechanical transmission of Beet western yellows virus (BWYV) and Potato leafroll virus (PLRV) to several host plants. Inoculation with purified preparations and viral RNA extracts of PLRV resulted in 30-50% systemically infected Nicotiana occidentalis P1 plants and 15-30% infected Nicotiana clevelandii plants. Particle bombardment was also used successfully to infect N. clevelandii plants with in vitro RNA transcripts of full-length cDNA of BWYV.

The Genome-linked Protein (VPg) of Southern Bean Mosaic Virus is Encoded by the ORF2

The sequence of the 20 N-terminal amino acids of the viral protein (VPg) which is covalently attached to the genomic RNA of the bean strain of Southern bean mosaic virus (SBMV-B) has been determined. The obtained VPg sequence mapped to position 327 to 346 of the SBMV-B ORF2 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Thus indicating that the sobemovirus genomic arrangement is similar to that of subgroup II luteoviruses. Comparison with other viral sequences revealed a high similarity with the sequence of the ORF2-product of the cowpea strain of SBMV (SBMV-C). No significant similarities were detected with amino acid sequences derived of other sobemoviruses or non-related viruses.

Expression of potyvirus proteins in insect cells infected with a recombinant baculovirus

The N-terminal portion (P1-HC-Pro-P3) of the tobacco vein mottling virus (TVMV) polyprotein was expressed in insect cells and larvae by a recombinant baculovirus. The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature P1, HC-Pro and P3 proteins were detected by specific antisera in Western blots. Antisera to P1, HC-Pro and P3 also recognized polypeptides with apparent M(r) values predicted for the intermediate processing products of the polyprotein fragment. The results of this study indicate that the autocatalytic processing of TVMV HC-Pro from the polyprotein is supported by insect cells. Helper component activity in extracts of cells infected with recombinant baculovirus was not detected by aphid transmission assay.