A dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral movement encoded by potato virus M.

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Improved serological diagnosis of Poplar mosaic virus with monoclonal antibodies.

Poplar mosaic virus (PopMV) is widespread in all countries where poplar is grown, and causes severe economic losses in terms of quantity and quality of wood production. Control is based on indexing, aimed at the production of healthy propagation material. The currently used diagnostic method is double antibody sandwich (DAS) ELISA with polyclonal antibodies, which is relatively simple and inexpensive and more reliable than visual inspection of symptoms in the nurseries. However, this method also has disadvantages, mainly low sensitivity in relation to low concentration and irregular distribution of the virus in the plant. In this study, a new diagnostic method for PopMV based on production and use of a monoclonal antibody (Mab) in a triple antibody sandwich (TAS) ELISA, is presented. The TAS-ELISA with monoclonal antibodies was optimised by testing a range of reagent combinations and concentrations. PopMV was detected by the optimised TAS-ELISA with sensitivity more than 100 times higher than by DAS-ELISA with polyclonal antibodies. Six PopMV isolates from four European countries were detected with the same efficiency, indicating that no limitations to the practical use of the TAS-ELISA arise due to excessive epitope-specificity of the monoclonal antibody employed.

[Production of monoclonal antibodies to Lily symptomless virus and application in lily virus detection].

Four hybridoma cell lines, 2A2, 5H9, 5H2 and 5E12, secreting monoclonal antibodies (MAbs) against Lily symptomless virus (LSV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the LSV particles. The four MAbs could specifically react with LSV. The titres of ascitic fluids of the four MAbs are up to 10(-6) in ELISA. Isotypes and subclasses of 5H9 and 5E12 belong to IgG1 while those of 2A2 and 5H2 belong to IgG3. Isotypes of light strains of the four MAbs all belong to kappa. The four MAbs were used in antigen-coated plate (ACP)-ELISA for LSV detection, and ACP-ELISA could successfully detect 1.8 ng of purified LSV or virus in plant sap diluted 1:300. The presence of LSV in field lily tissues was investigated with ACP-ELISA.

Development of an immunochromatographic strip test for rapid detection of lily symptomless virus.

A rapid immunochromatographic strip (ICS) test for specific detection of lily symptomless virus (LSV) was developed. The test is based on a double-antibody sandwich format and employs two distinct anti-LSV polyclonal antibodies (IgG1 and IgG2). The first antibody, IgG1 was used as the detection antibody conjugated to colloidal gold and the second antibody, IgG2 was used to as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LSV were tested, the LSV detection limit of the ICS test was 6.0 × 10(-8)mg/mL, which was the same as the quadruplex RT-PCR assay. Relative to quadruplex RT-PCR, the specificity and sensitivity of the ICS were 98.6% and 100%, respectively for field leaf samples. There was significant agreement between the results of the ICS and quadruplex RT-PCR tests (κ = 0.983). Compared with conventional lily virus detection methods, our ICS test has many advantages: simple, fast, low cost, high sensitivity and specificity, and has applications in the laboratory and in the field to detect and control LSV.

Simultaneous detection of five carnation viruses by non-isotopic molecular hybridization.

Several viruses, which in some cases can cause severe losses, affect carnation plants. These viruses include carnation mottle virus, carnation etched ring virus (CERV), carnation vein mottle virus, carnation ringspot virus, carnation Italian ringspot virus and carnation latent virus. A non-isotopic molecular hybridization was developed for the detection of these viruses in host plants and the sensitivity of the technique has been compared with enzyme-linked immunosorbent assay and bioassay methods. A procedure was developed to test simultaneously for the five RNA viruses (except CERV). The conditions established for this simultaneous detection did not include the DNA virus CERV due to the necessity of incorporating an additional step of RNase A treatment in the procedure to eliminate background signals. The sensitivity limits obtained for each virus using this multiple detection assay were identical to those obtained with the individual assays. The relative benefits of using this detection procedure for routine diagnosis of carnation viruses are discussed.

Partial antigenic characterization of potato virus S (Andean strain) by monoclonal antibodies.

Four mouse monoclonal antibodies (MoAbs)) against potato virus S Andean strain (PVSA) were tested. While MoAbs 2 and 3 reacted only with complete virions and were apparently specific for epitopes dependent on quaternary structure, MoAbs 1 and 4 appared to be conformation independent and reacted with exposed regions on native virions as well as on the surface of dissociated coat protein subunits. This seems to be an evidence of metatope existence. The results of competitive binding tests together with reaction patterns of individual MoAbs suggest that the used MoAbs reacted with at least two different epitopes on PVSA particles or polypeptide subunits. Immunoblot analysis of proteolytically cleaved PVSA capsid protein (CP) confirmed close proximity of epitopes recognized by MoAbs 1 and 4. Anti-PVS polyclonal antibody recognized both intact CP and its natural or artificial digest, while the MoAbs bound to intact CP only. These results indicate that the surface virus-specific epitopes are located near the terminus of CP molecule as it is characteristic for potyviruses.

Effect of pH, ions, bovine serum albumin and heterologous antisera on the stability of immunosorbed flexuous potato viruses.

Electron microscopic studies on the stability of immunosorbed (trapped) virions of potato viruses X, S and Y0 (PVX, PVS and PVY0) revealed disintegration and dislodging of PVY0 virions upon incubation with (1) antisera to PVX, PVS, or both diluted in saline, (2) 0.86% NaCl (saline) or 0.1 mol/l CaCl2 but not with 0.1 mol/l CaSO4 or 0.1 mol/l MgSO4. PVX virions, on the other hand, showed partial dislodging upon incubation with an antiserum to PVS diluted in saline, but complete disintegration and dislodging with saline. 0.1 mol/l CaCl2 caused partial dislodging while MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) had no apparent adverse effect. PVS virions were not affected by saline, CaCl2, MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) and were only partially dislodged by antisera to PVX or PVY0. Disintegration and/or dislodging of the PVX and PVY0 virions was prevented when (1) they were fixed with glutaraldehyde prior to incubation or (2) the virus extract contained bovine serum albumin (BSA) or (3) heterologous antisera were diluted in 0.1 mol/l phosphate buffer (PB) before use except the PVS antiserum which still caused disintegration and dislodging of PVY0 virions. Prior fixation of virions prevented their disruption and dislodging by saline only in the case of PVY0 but not PVX. On the other hand, BSA reverted the adverse effect of saline but not that of the PVS antiserum on PVY0 virions. The results presented here suggest (1) a disruptive effect of Cl' on PVX and PVY0 virions particularly when it was associated with Na+ and (2) an interaction between the immunosorbed virions of PVX or PVY0 and the antiserum to PVS.

Development of an indirect ELISA and immunocapture rt-PCR for Lily virus detection.

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

Potato chromosomes IX and XI carry genes for resistance to potato virus M.

Two new loci for resistance to potato virus M (PVM), Gm and Rm, have been mapped in potato. The gene Gm was derived from Solanum gourlayi, whereas, Solanum megistacrolobum is the source of the gene Rm. Gm confers resistance to PVM infection after mechanical inoculation. Rm induces a hypersensitive response in potato plants. Two diploid populations segregating for Gm and Rm, bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), and available potato molecular maps were instrumental for mapping the resistance loci. The novel locus Gm was mapped to a central region on potato chromosome IX. The locus Rm was placed on the short arm of chromosome XI, close to the marker loci GP250 and GP283, where a hotspot for monogenic and polygenic resistance to diverse pathogens is located in the potato and tomato genome.

Molecular characterization of Hop mosaic virus: its serological and molecular relationships to Hop latent virus.

The 3'-terminal sequence of hop mosaic virus (HpMV) genomic RNA was determined. A cDNA of approximately 1.8 kbp was amplified from the HpMV genome by 3' RACE using a degenerate primer, which was designed to anneal to the overlapping region of open reading frames (ORFs) 2 and 3 of eight carlavirus genomes. The sequence contained three ORFs, encoding proteins of 7-, 34-, and 11-kDa, which corresponded to ORFs 4, 5, and 6 of the carlavirus genome, respectively. The amino acid sequence of ORF 5, encoding the coat protein (CP) of HpMV, shows the highest identity (67%) to that of Hop latent virus (HpLV). The HpMV CP N-terminal sequence differs from that of HpLV, but the central and C-terminal sequences of the CP of both viruses are similar. The sequence similarity possibly causes the cross-reaction of heterologous antibodies of HpMV and HpLV. Phylogenetic analyses based on the CP amino acid and 3' non-coding region sequences indicate close relationships among HpMV, HpLV, and Potato virus M. We report here the first molecular characterization of HpMV genomic RNA.

Indian citrus ringspot virus: a proposed new species with some affinities to potex-, carla-, fovea- and allexiviruses.

An isolate of Indian citrus ringspot virus from Kinnow mandarin in northern India had flexuous particles with evident cross-banding and a modal length of 650 nm. It was mechanically transmitted to five herbaceous hosts including Phaseolus vulgaris cv Saxa, in which it became systemic. In thin sections, virus particles were observed in the cytoplasm of parenchyma cells but no specific inclusions were seen. The virus was purified from infected Saxa bean leaves and an antiserum prepared. There was no serological cross-reaction with representative allexi-, capillo-, potex- and trichoviruses, except a faint one-way reaction with Potato virus X. Purified virus yielded a major band, the presumed coat protein (CP), of about 34 kDa, and a single ssRNA of about 7.5 kb, which was infectious. Two ORFs encoding putative proteins of 34 kDa and 23 kDa were located in the 3' part of the RNA. The product of the 34 kDa ORF was confirmed as the CP by expression in E. coli. The derived amino acid sequence of the CP contained some short motifs similar to those of potex-, fovea-, carla- and allexiviruses but otherwise there was no strong similarity to any of these. The 23 kDa ORF contained a zinc finger-like sequence, as in similar ORFs in carla- and allexiviruses but overall amino acid homology with these was low. The virus does not appear to fall into any known genus. A new species is proposed. Serological and molecular diagnostic reagents were prepared.