Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Serological Detection of Grapevine leafroll virus 2 Using an Antiserum Developed against the Recombinant Coat Protein†

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL‐c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP‐CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti‐MBP‐CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti‐MBP‐CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate‐trapped antigen enzyme‐linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins.     

Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis

Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.     

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).     

Immunodiagnosis of Parietaria mottle virus in Tomato Crops Using a Polyclonal Antiserum against its Coat Protein Expressed in a Bacterial System

Recombinant DNA technology was used to raise a polyclonal antiserum against the coat protein (CP) of Parietaria mottle virus (PMoV). The CP gene was expressed in Escherichia coli as a fusion to a 6xHis tag and purified by affinity chromatography. Recombinant purified protein was used as antigen to raise a polyclonal antiserum. This polyclonal antiserum consistently detected PMoV specifically infected tomato plants from different commercial tomato crops by indirect enzyme-linked immunosorbent assay (I-ELISA) and direct tissue-printing immunoassay (DTBIA).     

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.     

应用斑点免疫结合法检测植物病毒

用改进的斑点免疫结合法,检测了烟草花叶病毒、黄瓜花叶病毒、豇豆花叶病毒、芋花叶病毒、柑桔速衰病毒和柑桔顽固病螺原体。无论用单克隆抗体还是多克隆抗体,全血清还是提纯的IgG,均获得满意的结果。直接法与间接法结果相同。在测定豇豆花叶病毒和芋花叶病毒时,与酶联免疫吸附试验和免疫电镜进行比较。表明无论是对纯化病毒还是感病植物汁液,其测定的敏感性均优于后二法,豇豆花叶病毒的可测感度是0.35ng,芋花叶病毒为0.83ng。以含0.5％吐温20的Tris缓冲液封闭硝基纤维素薄膜固相载体的未结合部位,其效果与牛血清白蛋白相同。应用碱性磷酸酶标记抗体以及羟基吲哚磷酸盐和氮蓝四唑为底物较合适,这种底物可在室温下长期保存,且反应产物为不褪色的紫色。易于观察和保存。     

Differentiation of Dasheen Mosaic Potyvirus Isolates Based on Variability in the Apparent Size of the Capsid Protein in Western Blots

The capsid protein (CP) sizes of seven dasheen mosaic virus (DsMV) isolates and one isolate of vanilla mosaic virus were estimated to be 38–47 kDa and 47 kDa, respectively, based on Western blot analyses using DsMV polyclonal antiserum. The CP sizes of 12 other potyviruses were estimated to be 31–36 kDa. Apparent CP sizes of the DsMV isolates extracted from their original hosts were 47 kDa ( Xanthosoma caracu ), 45 kDa ( Colocasia esculenta , Zantedeschia aethiopica ), and 38–46 kDa ( Caladium hortulanum ). Propagation in seedlings of Philodendron selloum did not affect the CP sizes of any of the individual DsMV isolates. The same characteristic CP sizes were also detected in Western blot analyses of these isolates, using polyclonal antisera of eight other potyviruses, or using Agdia Poty 1 monoclonal antiserum, and using three monoclonal antisera of papaya ringspot virus type W. The apparent CP size and pattern of apparent breakdown products as revealed by Western blots of extracts from infected aroids may be used in the characterization and differentiation of DsMV isolates.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

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.     

Production of Polyclonal Antibodies Using Recombinant Coat Protein of Papaya ringspot virus and their Use in Immunodiagnosis

Production of polyclonal antibodies requires large amount of purified virus that can be avoided by the use of recombinant coat protein (CP). Recombinant CP of Papaya ringspot virus (PRSV) was thus used for the production of polyclonal antibodies as the virus purification from papaya tissues provides low virus yields. CP was expressed as a fusion protein (～72 kD) containing a fragment of E. coli maltose binding protein. Polyclonal antibodies from rabbits immunized with the fusion protein, successfully detected natural infection of PRSV in papaya and cucurbits samples collected from different locations at 1:4000 dilution in direct antigen-coated enzyme-linked immunosorbent assay.     

An Unusual Feature at the N-terminal end of the Coat Protein of Maize dwarf mosaic virus isolated in Hungary

Maize dwarf mosaic is the most widespread virus disease affecting corn production in Hungary. In attempts to identify the causal virus by test plant reactions, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), only Maize dwarf mosaic virus (MDMV) was detected. To further characterize Hungarian isolates of MDMV, one isolate from each of the sweet corn varieties Dallas, Royalty and GH23‐85 was selected for sequence analysis of its coat protein (CP) gene. The three Hungarian isolates shared CP amino acid sequence similarities of 95–98% not only with one another but also with MDMV isolates from other countries. However, the N‐terminus of the CP of the ‘Dallas’ isolate was unusual in containing a stretch of 13 additional amino acids. This is the first report of variation in the size of the N‐terminus of the MDMV CP.     

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.     

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.