Development and application of ELISA assays for the detection of two members of the family Luteoviridae infecting legumes: Pea enation mosaic virus (genus Enamovirus) and Bean leafroll virus (genus Luteovirus)

An antigen‐coated plate enzyme‐linked immunosorbent assay (ACP‐ELISA) method was developed and validated for the detection of Bean leafroll virus (BLRV) and Pea enation mosaic virus (PEMV), two of the important viral pathogens of several legume crops. The coat protein (CP) gene of each of the viruses was bacterially expressed as a fusion protein containing an N‐terminal hexa‐histidine tag and used as an antigen to produce antisera in rabbits. The antiserum to BLRV could detect the virus in leaf samples in up to 1:1000 dilution, and the PEMV antiserum detected the homologous virus in leaf samples of dilutions up to 1:6400. No serological cross‐reactivity was observed between anti‐BLRV and anti‐PEMV sera. The ACP‐ELISA assays were then used for estimating the prevalence of these two viruses in alfalfa, pea and vetch over a three‐state area in the US Pacific Northwest over a 2‐year period and virus incidence was mapped. Availability of rapid and sensitive ELISA assays facilitate virus disease mapping efforts and screening germplasm for virus resistance.     

Diversity among Grapevine Leafroll-associated Virus 2 Isolates Detected by Heteroduplex Mobility Assay

Grapevine leafroll‐associated virus 2 (GLRaV‐2) was detected by serological and molecular analyses in several grapevine accessions of different varieties from Italian, Greek, French and Brazilian vineyards in a 2001–2002 survey. In order to study the genetic variability among GLRaV‐2 isolates in the open reading frame (ORF) coding the coat protein (CP), heteroduplex mobility assays were performed on 17 isolates and six strains used as reference. Eight diverse GLRaV‐2 variants were identified among the infected grapevines tested. The most common variant was found in the majority of the samples characterized; it was indistinguishable from the reference strains from the Semillon and Pinot noir 95 accessions. GLRaV‐2 variants found in Italian cvs Negro amaro and Vermentino were identical to the reference strain from cv. Muscat de Samos (Greece). Three other GLRaV‐2 variants from Southern and Central Italy were different from all the reference strains. A grapevine accession from Tuscany was found to contain two diverse GLRaV‐2 variants. None of the variants tested sample identical to the American strain H4 or the reference strains from cvs Chasselas 8386 (Switzerland) or Alphonse Lavallée 224 (France); the latter three accessions were different from one another. The estimated nucleotide homology in CP gene among 23 GLRaV‐2 isolates was in some cases <88%.     

Grapevine Viruses' Detection and Sanitary Selection in Grapevine Germplasm of Calabria (Southern Italy)

A survey of grapevine viruses present in the region of Calabria (southern Italy) was carried out, and the sanitary selection was conducted on various indigenous varieties. Serological (ELISA) and molecular (multiplex RT‐PCR) tests were used to detect the viruses included in the Italian certification programme: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1 (GLRaV‐1), Grapevine leafroll associated virus 2 (GLRaV‐2), Grapevine leafroll associated virus 3 (GLRaV‐3), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine fleck virus (GFkV). The frequency with which the above viruses have been detected was 37.4, 32.6, 12.8, 7.7, 7.3, 1.9 and 0.3%, respectively, for GVA, GLRaV‐3, GFLV, GFKV, GLRaV‐1, GLRaV‐2 and GVB. ArMV was never found. The sanitary selection allowed for the detection of 6 putative clones of ‘Arvino’, 2 of ‘Magliocco dolce’ and 2 of the rootstock ‘17–37’ free of the above‐mentioned viruses. The necessary process for the commercialization of these clones as ‘certified’ propagation material was accomplished, and their official approval by the Italian Ministry of Agriculture is currently in progress.     

First Report of the Occurrence of Grapevine leafroll‐associated virus‐5 in Turkish Vineyards

Surveys were made in the main grape growing region (Southeast Anatolia) of Turkey for the occurrence of Grapevine leafroll‐associated virus‐5 (GLRaV‐5). Plant samples with typical leafroll symptoms and mealybugs, Planococcus ficus (Signoret) were used for assessing the occurrence of GLRaV‐5 by RT‐PCR. A 272 bp band representing GLRaV‐5 infection was successfully detected in plants and mealybugs in some vineyards of the Southeast Anatolia region and the virus is the first time reported in Turkish vineyards.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Occurrence and Distribution of Grapevine Leafroll-associated Viruses 1, 2, 3 and 7 in Turkey

Grapevines in central Anatolia region of Turkey were surveyed for the prevalence of grapevine leafroll viruses. The field study and collection of samples were conducted in nine major grapevine‐growing areas. Samples collected from 622 vines were tested for Grapevine leafroll‐associated virus 1, 2, 3 and 7 (GLRaV‐1, ‐2, ‐3 and ‐7). According to diagnostic tests and surveys, 27 of 41 cultivars and 95 of 622 samples (15.27%) were found to be infected at least one virus. GLRaV‐1 (8.36%) was found to be the most frequently encountered virus associated with leafroll disease of grapes, followed by GLRaV‐3 (5.78%), GLRAV‐7 (3.86%) and GLRAV‐2 (2.41%).     

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

The coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).     

Complete Nucleotide Sequence of the RNA 3 of Bacopa chlorosis virus and Production of Polyclonal Antibodies to a Recombinant Coat Protein

The complete sequence of the RNA 3 of a virus causing chlorosis in Impatiens in Germany was determined and identified as an isolate of Bacopa chlorosis virus (BaCV, genus Ilarvirus). BaCV has previously only been reported from bacopa in the USA, but no coat protein (CP) sequence has been previously available. Both RNA 3 encoded proteins, CP and movement protein, showed highest sequence identity to Parietaria mottle virus, a subgroup 1 ilarvirus. Attempts to purify BaCV failed, so an antiserum was raised against a recombinant CP. The polyclonal antiserum so produced allowed specific detection of BaCV but showed no serological cross‐reaction with other ilarviruses and was unsuitable for immunoelectron microscopy. The host range includes many important flowering plant species, highlighting the potential threat BaCV might pose for the horticultural industry. This is the first report of BaCV occurring in Germany and outside the US.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.     

Transmission of Grapevine virus A and Grapevine leafroll‐associated viruses 1 and 3 by Planococcus ficus and Planococcus citri fed on mixed‐infected plants

The Grapevine virus A (GVA) and Grapevine leafroll‐associated viruses 1 and 3 (GLRaV‐1 and GLRaV‐3) are associated with grapevine diseases that induce severe reductions in yield and berry quality. These three viruses are known to coexist in both grapevine and insect vectors, but their cotransmission has been poorly characterised so far. This study investigates the acquisition and transmission of GLRaV‐1, GLRaV‐3 and GVA by Planococcus ficus and Planococcus citri (Hemiptera: Pseudococcidae) following feeding on multiple‐infected plants. The retention and load of the three viruses in the two insect species were analysed. After feeding onto GVA, GLRaV‐1 and GLRaV‐3 mixed‐infected grapevines, nymphs of P. ficus and P. citri showed similar virus acquisition rates and retained low quantities of viruses until the third post‐acquisition day. Despite the similar acquisition patterns, the two vectors differed in transmission efficiency: P. ficus showed a higher efficiency in transmitting GVA and GLRaV‐3, whereas P. citri transmitted GLRaV‐1 more efficiently. When focusing on the virus cotransmission, it appears that GVA could be transmitted to grapevine without GLRaV‐1 and/or GLRaV‐3 and that the transmission of both GLRaVs could take place in the absence of GVA. This comparative study involving different viruses and vector species improves the current knowledge of the semi‐persistent transmission of these three viruses and contributes to the understanding of grapevine virus epidemiology.     

Effects of Grapevine leafroll‐associated virus 3 on the physiology in asymptomatic plants of Vitis vinifera

Grapevine leafroll disease is one of the most important viral diseases of grapevine (Vitis vinifera) worldwide. Grapevine leafroll‐associated virus 3 (GLRaV‐3) is the most predominant virus species causing this disease. Therefore, it is important to identify GLRaV‐3 effects, especially in plants which do not systematically show visual symptoms. In this study, effects of GLRaV‐3 on grapevine physiology were evaluated in asymptomatic plants of Malvasía de Banyalbufar and Cabernet Sauvignon cvs. Absolute virus quantification was performed in order to determine the level of infection of the treatment. The net carbon dioxide (CO₂) assimilation (A_N) and electron transport rate (J_flux) were the main parameters affected by the virus. The A_N reduction in infected plants was attributed to restrictions in CO₂ diffusion caused by anatomical leaf changes and a reduction of Rubisco activity. Those effects were more evident in Malvasia de Banyalbufar plants. The reduction of A_N leads to a decrease in the total oxygen uptake rate by the activity of the cytochrome oxidase pathway, producing slight differences in plant growth. Therefore, even though no symptoms were expressed in the plants, the effects of the virus compromised the plant vital processes, showing the importance of early detection of the virus in order to fight against the infection.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and their Effectiveness for Virus Detection

Citrus tristeza virus (CTV) is distributed worldwide and causes the most economically important virus diseases of citrus. Enzyme‐linked immunosorbent assay (ELISA) and/or immunoprinting have become an indispensable tools for large‐scale diagnosis of CTV worldwide. Several CTV detection kits are commercially available, based on either polyclonal or monoclonal antibodies developed against purified virus preparations. We have developed polyclonal antibodies to recombinant p25 CTV coat proteins (rCP) and determined their effectiveness for both trapping and as the intermediate antibody in double‐antibody sandwich indirect (DASI) ELISA. The p25 coat protein gene of three CTV isolates was amplified by RT‐PCR and further cloned and expressed in Escherichia coli cells. The rCP was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV‐specific antibodies reacted positively with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extracts of CTV‐infected citrus tissue, but not with extracts from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in DASI‐ELISA, discriminating healthy and CTV‐infected citrus, with optical density (OD₄₀₅) values in the range of 0.151–2.415 for CTV‐infected samples and less than 0.100 for healthy tissue. Commercially available anti‐CTV antibodies were used as a reference. Previous reports indicate that antibodies developed to recombinant antigens, including those of CTV, may not be functional for trapping the target antigens under non‐denaturing conditions. Our results showed the feasibility of CTV antibodies developed to the rCP for use as both trapping and intermediate antibodies in DASI‐ELISA, when the recombinant antigen was fractioned with polyacrylamide electrophoresis gel and further extensively dialysed against phosphate buffer saline prior to its use as immunogen.     

Impacts of leafroll‐associated viruses (GLRaV‐1 and ‐3) on the physiology of the Portuguese grapevine cultivar ‘Touriga Nacional’ growing under field conditions

The impact of mixed infection of grapevine leafroll‐associated virus 1 and 3 (GLRaV‐1&‐3) on physiological performance of the Portuguese grapevine variety ‘Touriga Nacional’ was evaluated during 3 years with the main purpose of understanding the drastic reduction in yield. Overall, gas exchange was negatively affected in leaves with these leafroll virus infections. Particularly at ripeness stage, the reduction in stomatal conductance (g_s) was higher than in net CO₂ assimilation rate (A), leading to higher intrinsic water use efficiency (A/g_s) in infected leaves. However, the decrease in g_s and A were not a consequence of the decrease in bulk water potential, as the water index/normalised difference vegetation index ratio suggested similar magnitude for both treatments. The maximum quantum efficiency of photosystem II was unaffected by GLRaV‐1&‐3, whereas quantum effective efficiency of PSII, apparent electron transport rate and photochemical quenching significantly decreased in infected leaves and these was paralleled by a significant increase of non‐photochemical quenching. Relative to carbon metabolism, the analyses of the net CO₂ assimilation rate/photosynthetic photon flux density (A/PPFD) and net CO₂ assimilation rate/internal CO₂ concentration (A/C_i) curves revealed that virus infection had a negative effect on light saturated rate of CO₂ fixation at high irradiances and carboxylation efficiency but, in contrast, apparent quantum yield of CO₂ fixation was significantly higher. Meanwhile, the presence of GLRaV‐1&‐3 resulted in a marked decrease in photosynthetic pigments, soluble sugars and soluble proteins contents, while starch and anthocyanins were significantly improved. N, P, Ca, S and Fe leaf concentrations significantly decreased, while K, Mg, B, Cu, Zn and Mn were unaffected by these two leafroll virus species. Infected plants showed a significant decrease in yield, mainly due to a lower cluster weight. These results emphasised the important role of GLRaV‐1&‐3 as a biotic stress for the grapevine physiology and consequently to yield attributes.     

Detection and Sequence Analysis of Grapevine Leafroll‐Associated Virus 2 Isolates from China

Several grapevine leafroll‐associated viruses (GLRaVs) have been found frequently in grapevines behaving GLD. Among them, GLRaV‐2 is the only one belonging to Closterovirus, and mainly induces leafroll symptoms and graft incompatibility. In this study, new degenerate primer pairs designed against the HSP70 gene were applied in polymerase chain reaction (PCR) and nested PCR (nPCR) to detect GLRaV‐2 in 132 samples collected from 14 provinces and regions of China. Of the samples, 51.5% were infected with GLRaV‐2, and most did not exhibit GLD symptoms. Some popular grape cultivars had a high incidence of GLRaV‐2 infection, such as Cabernet Sauvignon (92.3%), Chardonnay (80%), Red Globe (75%) and Italian Riesling (73.7%). ‘Beta’ rootstocks, previously identified as negative samples, were also found to be highly infected with GLRaV‐2 (50%). GLRaV‐2 isolates obtained in this study showed identities ranging from 68.9% to 100% and 76.47% to 100.0% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the HSP70 gene showed that all GLRaV‐2 isolates in China belong to three of five reported phylogenetic groups. Different variants belonging to the PN and RG groups were present in a single isolate. The results showed that the new degenerate primer pairs could detect more GLRaV‐2 isolates than the previously reported primers. This is the first detailed report on the prevalence and gene diversity of GLRaV‐2 in China and also provides an nPCR method to improve the sensitivity of PCR as an alternative method when no real‐time PCR device is available.     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.