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Two viral proteins, the helper component-protease and the coat protein, are required for the non-persistent aphid transmission of potyviruses. In the potyvirus coat protein, the tripeptide aspartate-alanine-glycine (DAG) has often been shown to be involved. A monoclonal antibody, raised against a synthetic decapeptide containing the DAG tripeptide, reacted with the peptide as well as with isolates of soybean mosaic, tobacco etch and tobacco vein mottling potyviruses. Experiments indicate that the monoclonal antibody recognizes a conformational rather than a sequential epitope. The data support the suggestion that the DAG region plays a structural role to determine a coat protein-helper component-protease conformation that influences aphid transmission.

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY(O)). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY(O) whilst failing to detect three isolates of PVY(N) and 12 isolates of PVY(NTN). Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vlambda1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

A sap transmissible virus, causing mosaic and leaf curl disease of Celosia argentea, was isolated at vegetable farms in Amuwo Odofin, Tejuoso, and Abule Ado, Lagos, Nigeria. The virus had a restricted host range confined to a few species of the Amaranthaceae, Chenopodiaceae and Solanaceae families. It failed to infect several other species of the Aizoaceae, Brassicaceae, Cucurbitaceae, Fabaceae, Lamiaceae, Malvaceae, Poaceae and Tiliaceae families. The virus was transmitted in a non-persistent manner by Aphis spiraecola and Toxoptera citricidus but not by eight other aphid species tested. There was no evidence of transmission by seeds of C. argentae varieties. The viral coat protein had a relative molecular mass (M(r)) of about 30.2 K. Electron microscopy of purified virus preparations revealed flexuous rod shaped particles of about 750 nm in length. Serological studies were performed using the enzyme-linked immunosorbent assay (ELISA), immunosorbent electron microscopy (ISEM) and Western blot analysis. The virus reacted positively with an universal potyvirus group monoclonal antibody (MoAb) and MoAb P-3-3H8 raised against peanut stripe potyvirus. It also reacted with polyclonal antibodies raised against several potyviruses including asparagus virus-1 (AV-1), turnip mosaic virus (TuMV), maize dwarf mosaic virus (MDMV), watermelon mosaic virus (WMV-2), plum pox virus (PPV), soybean mosaic virus (SoyMV), lettuce mosaic virus (LMV), bean common mosaic virus (BCMV) and beet mosaic virus (BMV) in at least one of the serological assays used. On the basis of host range, mode of transmission, and available literature data, the celosia virus seems to be different from potyviruses previously reported to infect vegetables in Nigeria. The name celosia mosaic virus (CIMV) has been proposed for this virus.

Serological differences between two zucchini yellow mosaic virus (ZYMV) isolates (ZYMV-169 and ZYMV-M) obtained from two distinct geographical locations in Japan were determined by mapping epitopes on the coat proteins (CPs) of the two isolates. A total of 45 monoclonal antibodies (MAbs) against the two isolates were produced and the epitopes on the CPs were delineated by reacting these MAbs with trypsin-treated ZYMV particles and Escherichia coli-expressed ZYMV CP fragments. Six MAbs of groups I-a and I-b, specific for ZYMV-169, recognised two epitopes in the N-terminal region at amino acids (aa) 1-28 and 6-41 of ZYMV-169 CP. Fourteen MAbs of group II, specific for ZYMV-M, recognised epitopes in the N-terminal region of ZYMV-M CP. Twenty-one MAbs of groups III-a, III-b(i), III-b(ii), and III-b(iii), reacting with both isolates, recognised four epitopes; one epitope was located in the N-terminal region at aa 6-28 and the remaining three epitopes were located in the core region at aa 42-95, 171-227 and 228-259 of ZYMV CPs.

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

The potato tuber ringspot necrosis isolate of potato virus Y (PVY(NTN)) is a recently recognized and highly aggressive isolate of the PVY(N) group of strains. In order to screen specifically sources of resistance to PVY(NTN) a method to separate PVY(NTN) from PVY(N) is needed. To achieve this, 61 isolates from 13 imported and locally developed potato cultivars in Slovenia were studied. On the basis of the reactions in indicator plants Nicotiana tabacum cv. Samsun and Solanum brachycarpum and with a PVY(N) specific monoclonal antibody (4E7), all Slovenian isolates (Sl-NTN) were identified as PVY(N). Using two primer pairs from the P1 gene of a Hungarian isolate of PVY(NTN) by a conventional single primer pair, reverse transcription polymerase chain reaction (RT-PCR) both PVY(NTN) and PVY(N) were amplified similarly. However, specific amplification of PVY(NTN) was achieved by a nested-PCR at an annealing temperature of 63 degrees C. A simplified form of the nested-PCR, termed 3-primer PCR was developed, which is applicable for large-scale testing of samples. Using the 3-primer PCR at annealing temperature of 63 degrees C, known mixtures of PVY(NTN) and PVY(N) were correctly separated. PVY(NTN) was detected in dormant tubers and leaves from all Sl-NTN isolates. The 3-primer PCR was specific to PVY(NTN) and did not react with nine isolates of PVY(N), 13 isolates of PVY(o), one isolate of PVY(C), six commonly occurring potato viruses and a viroid.

The P1 protein is liberated from the N-terminal region of the potyviral polyprotein by cleavage depending on its own autoproteolytic activity. Existence of the 32-kDa P1 protein in tobacco plants infected with potato virus Y ordinary strain (PVY-O) was detected by an antiserum against a recombinant PVY-O P1 protein. In vivo analysis using tobacco protoplasts confirmed that the Phe284-Ser285 was the cleavage site separating the P1 protein from the PVY-O polyprotein. Phe284 was indispensable for proteolysis and Ser285 was needed for optimal cleavage susceptibility.

Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections. Antiserum to CP reacted with virions of seven out of 18 other potyviruses. CP was distributed throughout the cytoplasm of infected cells. Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses. NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions. Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein. Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP. An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus. Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated. Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI. Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments. The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP.

The coat protein of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed using a recombinant vaccinia virus (VV) system. Ultra-thin section electron microscopy demonstrated that the coat protein assembled into potyvirus-like particles (PVLPs) in recombinant VV infected cells. Infection of cells with two additional VV recombinants expressing coat protein plus N-terminal and N- and C-terminal extensions also resulted in the formation of PVLPs. These results suggest that the ability of VV to express the potyvirus coat protein at sufficient levels to allow PVLP formation in vitro, could make VV a suitable vector for the delivery of PVLPs displaying vaccine antigens in vivo without the need for particle purification and/or inclusion of adjuvant. Use of such a vaccine strategy would also benefit from the proven advantages of poxviruses as vaccines such as stability in a freeze dried form, resistance to environmental factors and the potential for oral administration.

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.

Potato plants carrying the Ry(sto) gene from Solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. The resistance may be due to Ry(sto) or to a closely linked gene. In this investigation, we used potato virus Y (PVY) and a tobacco etch virus construct that encodes beta-glucuronidase (TEV-GUS) to monitor virus infections of potato plants. Systemic spread of either virus in resistant potato plants was not detectable by serology, RT-PCR, GUS assay or bioassay although each replicated in the initially infected cells of leaves from resistant potato cultivars and was transported into neighbouring cells. However, 3 days post-inoculation (p.i.) a necrotic reaction set in that stopped movement and accumulation of both viruses by 7 days p.i. The resistance reaction (probably a hypersensitive reaction) became visible as necrotic streaks on veins on the lower leaflet surfaces of some potato cultivars carrying the Ry(sto) gene and may be elicited by a common potyviral gene product.

An immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay has been developed for the detection of yam-infecting potyviruses. Based upon the same format two distinct simple tests have been developed, which allow the reliable diagnosis of yam mosaic virus and the tentatively named yam mild mosaic virus. By using immunocapture and a single-buffer RT-PCR reaction, the test can be performed in a single tube. The tests described have been shown to exhibit a thousand-fold increase in detection sensitivity compared to existing ELISA assays.

Comparison of the predicted coat protein amino acid sequence of the 'sweet cherry' strain of plum pox potyvirus (PPV-SwC) with the corresponding regions of several other PPV strains indicated that the main differences are in the N-terminal region. Polyclonal antibodies were produced against a synthetic peptide corresponding to the 1-14 sequence of the N-terminal region of PPV-SwC coat protein. They specifically detected PPV-SwC in different immunochemical tests.

DNA-based immunization is a promising new technique for generating antibodies in laboratory animals for diagnostic purposes in biological science. The main advantages are the elimination of time and labor and the technically demanding steps of antigen purification. The DNA sequence of the protein of interest, cloned in a suitable in vivo expression vector that is administered intramuscularly or intradermally, is sufficient to induce an immune response in animals. We report the induction of antibodies to tobacco mosaic virus (TMV) coat protein (CP) as a highly immunogenic structural protein and potato virus Y (PVY) P1 protein (P1) as a nonstructural protein. The appropriate nucleotide sequences were introduced in a mammalian expression vector (pSG5) and injected intramuscularly into New Zealand White rabbits (Oryctolagus cuniculus). By 10 days post-injection (dpi) a specific immune response was detected against TMV-CP, while it took about 5 weeks for a response to PVY P1. In both cases the antibody titers were significantly above the corresponding pre-immune serum, however, they were considerably below the titer of the matching conventionally produced antiserum. To our knowledge, this is the first report of DNA-based immunization in order to generate antibodies to plant viral proteins, but further improvements are necessary to increase antibody titers before this promising new technique can be introduced broadly in plant science for diagnostic purposes.

The cost protein (CP) genes of two potato virus Y necrotic isolates (N27 and a mutant strain N27-92), which differed in their reactivity to a monoclonal antibody (mab), were characterized. Both isolates could be detected by mab 4E7, but mab VN295.5 selectively reacted to N27 and not to N27-92. The CP genes of both isolates coded for 267 amino acids with approximately 99.0% identity at both the nucleotide and the amino acid levels. nucleotide sequence comparison indicated five substitutions in N27-92 compared with N27. Three of these changes resulted in substitution of amino acids. Two transitions (A-->G) in N27-92 changed threonine to alanine and lysine to arginine at positions 7 and 55, respectively, whereas a A-->T transversion changed asparagine to isoleucine at positions 27. The surface probability curves of both the isolates could almost be superimposed, except at amino acid positions 7 and 27. Since amino acid substitution at position 55 is conservative, changes from polar to hydrophobic amino acids (threonine-->alanine and asparagine-->isoleucine) at positions 7 and 27 might have changed the epitope(s) of N27-92, abolishing its detection by mab VN295.5.

Partial genomic sequences from an unknown garlic potyvirus and from an onion isolate of the onion yellow dwarf potyvirus (OYDV) were obtained. Comparison of the deduced amino acid sequences showed a similarity of 88% between the respective viral coat proteins. The garlic potyvirus coat protein was expressed in E. coli cells, purified, and subjected to Western blot analysis using antibodies raised against different garlic-infecting viruses. The expression protein was consistently recognised by anti-OYDV antibodies and did not react with antibodies specific for leek yellow stripe potyvirus (LYSV), garlic common latent carlavirus (GCLV) and shallot latent carlavirus (SLV). Besides, the garlic potyvirus coat protein was obtained as a fusion protein and used as antigen to produce polyclonal antibodies. These antibodies reacted with purified OYDV virions, but failed to recognise LYSV particles. In the light of this evidence the garlic potyvirus was identified as the garlic strain of OYDV.

Potato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I, Eco RV, Hinf I, Rsa I and Taq I). Genetic distances between the different PVY "restrictotypes" were calculated and used to define the PVY genetic strains. Three main clusters were found: PVYO, PVYN, and non-potato PVY (PVYNP), in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed.

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1-14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Banana bract mosaic (BBMV) is a relatively new, non-persistently aphid transmitted disease of bananas in the Philippines. Partially purified preparations from infected plants contained low numbers of flexuous virions, 660 to 760 nm in length, and a 38kDa protein, possibly the coat protein, which reacted with a general potyvirus antiserum in western blots. There were insufficient virions for conventional antiserum production or cDNA synthesis. Therefore, DNA was amplified using potyvirus-specific degenerate primers and reverse transcriptase PCR. The PCR products were cloned, sequenced and analysed and contained a 5' open reading frame of up to 150 amino acids and a 3' untranslated region of up to 190 nucleotides which were clearly related to the C-terminal half of the coat proteins and the 3' untranslated regions, respectively of potyviruses. The BBMV open reading frame amino acid sequence was most similar to the C-terminal half of the maize dwarf mosaic potyvirus coat protein (71.3% similarity) and the BBMV 3' untranslated region was most similar to that of ornithogalum mosaic potyvirus (39.6% similarity). Our results show that BBMV is a distinct potyvirus and also demonstrate the application of virus group specific primers in the characterisation of previously undescribed viruses.

An isolate of potato virus Y, named PVY-36, reacted with polyclonal antibody against PVY-O (an ordinary strain), but not with any of eight monoclonal antibodies (MAbs) specific to PVY-O or with two MAbs specific to PVY-T (a necrotic strain). From its host range and symptomatology, PVY-36 belongs to the PVYO group. The nucleotide sequence of the coat protein (CP) coding region of the PVY-36 genome was determined and the amino acid sequence was predicted. Based on the CP amino acid sequence, PVY-36 is more closely related to PVY-O than to PVY-T. There were eight amino acid differences between the CPs of PVY-36 and PVY-O in the N-terminal 30 amino acids. It is suggested that amino acids 8-15 and/or 26-30 from the N-terminus may determine a PVY-O-specific epitope (or epitopes).

Effect of titer and pH of decorating antiserum, and the virus-source host species on the virion morphology upon decoration of potato viruses X, S and Y was studied. There was good decoration without any apparent adverse effect in the case of PVX and PVS with exception of pH 6.0 and antiserum titer 1:0.5, which caused decoration of only a small proportion of the virions. On the other hand, the PVYo virion morphology showed only slight to extensive disorganization depending on the pH and titer of the antiserum and the virus-source host species. Virion structure was, however, preserved when either PVYo (o strain) and its antiserum were made to react in liquid phase, or virions were fixed with 3% glutaraldehyde before decoration.

Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (ClYVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of ClYVV a total of ten MAbs were obtained which reacted strongly with ClYVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five ClYVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with ClYVV, form the BYMV subgroup of potyvirses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The ClYVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five ClYVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the ClYVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of ClYVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the ClYVV coat protein. Comparison of published coat protein sequences of BYMV and ClYVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of ClYVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of ClYVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1-25 and 4-30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Two hybridomas stably secreting monoclonal antibodies (McAb) against sweet potato feathery mottle virus (SPFMV) were constructed by fusing mouse myeloma (Sp2/0-Ag14) with mouse splenocytes immunized with SPFMV-LF. The McAb secreted by the two hybridomas specifically reacted with both SPFMV-LF (China strain) and SPFMV-RC (American strain) in dot-blot-ELISA. A large amount of McAb was produced with BALB/c mice. The titer of hybridoma cultured supernatant was 1:256, and that of the ascitic fluids was 1:1 x 10(4); and the immunoglobulins of McAb were IgG2a and IgG3 respectively. The detection of SPFMV-infected sweet potato plants with McAb confirmed that the McAb obtained in this test were desirable for large-scale routine surveys of SPFMV. This study on construction of hybridomas and detection of SPFMV using McAb is the first report in the world.

Monoclonal antibodies (McAb) of hybridomas derived from the fusing of the mouse splenocytes immunized by TuMV with BALB/c mouse myeloma cells (SP2/0-Ag14). Five kinds of hybridoma cell line were produced by indirect-ELISA screening and cloning three times with limiting dilution. Four kinds of hybridoma produced antibodies respectively reactive to TuMV C1, C3, C4 and C5. One kind was reactive to all five strains of TuMV. In indirect-ELISA and sandwich-ELISA tests, TuMV specific monoclonal antibodies did not react with CaMV, CMV, TMV, PVX, and PVY. Antibody titers of ascitic fluids were about 1:256,000 to 2,048,000 in indirect-ELISA. The biological, physical, and chemical properties of the hybridoma cell lines and McAb were identified. The identification of TuMV strains, the specificity and stability of McAb, the coat proteins, and the antigenic site of TuMV were discussed and analyzed with SDS-PAGE and western-blotting.