Selection of polyclonal antibodies to the N terminus of bean yellow mosaic potyvirus coat protein by induction of tolerance with monoclonal antibody

Polyclonal antisera to potyviruses contain virus-specific as well as cross-reacting antibodies. The virus-specific antibodies are directed to the surface-located, N-terminal region of the coat protein, whereas cross-reacting antibodies are produced against multiple epitopes within the core region of the coat protein (minus N and C termini), which displays extensive sequence homology among distinct potyviruses. In the present study, immunological tolerance was induced in mice against the cross-reactive central core region of bean yellow mosaic virus (BYMV) using a rat monoclonal antibody (mAb) to the L3T4 molecule (the mouse equivalent of CD4). Generation of specific antisera reactive to the N terminus of BYMV was attained in tolerized mice by secondary immunization with whole viral coat protein from BYMV. This approach appears to be ideally suited to potyviruses where a two-third of the coat protein molecule contains immunogenic epitopes which can result in cross-reacting antibodies.     

The use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein.

Whilst monoclonal antibodies (Mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. In the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. Mice were immunized with coat protein from either one potyvirus (potato virus Y, PVY-D) or a mixture of five distinct potyviruses. Two well-defined peptides were used for selective electrofusions. Peptide-1 was selected from the highly specific N terminal region of PVY-D and peptide-2 from the highly conserved N terminal/core junction region of Johnson grass mosaic virus (JGMV). Conventional PEG-mediated fusions using mice immunized with these peptides did not result in hybridoma formation. On the other hand, electrofusions using biotin-streptavidin to bridge peptide-specific B cells to myeloma cells produced hybridomas secreting antibodies either highly specific to PVY-D or cross-reactive with all potyviruses, depending on the peptide used.     

Towards a system for the identification and classification of potyviruses: I. Serology and amino acid composition of six distinct viruses

Antigenic relationships of six distinct potyviruses were studied by immunodiffusion tests using highly purified sonicated virus preparations and anti-intact virus sera devoid of detectable antibodies to host-plant antigens. Three variants of bean yellow mosaic virus (BYMV) including BYMV sensu stricto and two variants of pea mosaic virus (PMV and SPMV) were shown to be antigenically very similar and also relatively closely related to lettuce mosaic virus (LMV). Distant antigenic relationships were detected between the BYMV variants and bean common mosaic virus (BCMV); between BCMV and passionfruit woodiness virus (PWV); and between PWV and potato virus Y (PVY). No antigenic relationships were detected between any of these viruses and sugarcane mosaic virus (SCMV). Antibodies in anti-viral sera were very poor in recognizing coat proteins dissociated with LiCl from homologous viruses and failed altogether to recognise those dissociated with pyrrolidine. Attempts to prepare antisera in mice against isolated viral coat proteins dissociated with either LiCl or pyrrolidine were unsuccessful due to poor immunogenicity of the preparations.Electrophoretic mobilities of the viral coat proteins relative to marker proteins in the presence of sodium dodecyl sulphate suggest that the protein subunits of all the viruses studied have molecular weights of about 33,000. However, the coat proteins were prone to partial degradation. The amino acid compositions of the antigenically closely related viruses were very similar, but similarities of those distantly related were no greater than those of the apparently unrelated viruses.The problems in the use of serological and amino acid composition data obtainable with currently available techniques for the classification of potyviruses are discussed.     

Possible members of the potyvirus group transmitted by mites or whiteflies share epitopes with aphid-transmitted definitive members of the group

Summary There are at least ten viruses identified in the literature that resemble definitive potyviruses in having flexuous filamentous particles and inducing the formation of pinwheel cytoplasmic inclusions in infected cells but that are transmitted by eriophyid mites, whiteflies or soil fungi and not by aphids, the vectors of the definitive potyviruses. The taxonomic status of these viruses is uncertain at present. Using a broadly cross-reactive antiserum raised against the dissociated coat protein core (residues 68–285) of a definitive potyvirus (Johnsongrass mosaic virus), we have shown that wheat streak mosaic virus which is transmitted by mite and sweet potato mild mottle virus which is transmitted by whitefly have coat proteins that share epitopes with definitive potyviruses. This finding further supports their classification as definitive members of the potyvirus group. The cross-reactive antiserum used here had been shown previously to react with coat proteins of fifteen different definitive potyviruses. The antiserum did not react with coat proteins of potexviruses and tobamoviruses.     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

Coat protein of potyviruses 7. Amino acid sequence of peanut stripe virus

Summary The amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47–57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70–76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.     

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

Summary.  Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 A1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups. Received May 18, 1998 Accepted October 26, 1998     

Identification of potyvirus isolates from faba bean (Vicia faba L.), and the relationships between bean yellow mosaic virus and clover yellow vein virus

Summary Clover yellow vein virus (CYVV) isolate H and the related potyvirus isolates E178, E197, and E242 could be distinguished from bean yellow mosaic (BYMV) isolates by their wider host range among non-legume test plant species, the peculiar enlargement of the nucleolus in infected plants, and the larger size of their coat protein as evidenced by slower migration in SDS-PAGE. Serologically, they are qualitatively indistinguishable in electro-blot immunoassay (EBIA) also with antibodies specific to the N-terminal part of BYMV-B25 coat protein, implying therefore that CYVV and BYMV coat proteins contain identical amino acid sequences in the N-terminal region.The faba-bean virus isolates from Sudan, Syria, and The Netherlands could be identified as BYMV isolates especially adapted to faba bean. All of them were weakly pathogenic toPhaseolus bean with the exception of SV205, assuming an intermediate position betweenPhaseolus-bean isolates, with low pathogenicity to faba bean, and faba-bean isolates, usually having low pathogenicity toPhaseolus bean. Strains of BYMV are thus hard to delimit.     

Nucleotide sequence of the 3'-terminal region of potato virus A RNA.

The sequence of the 3'-terminal region of the genome of the potato virus A (PVA) was obtained from two independent cDNA clones. This sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon TAA and followed by untranslated region of 205 nucleotides. Since the N-terminal amino acid of the coat protein of PVA was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus sequences and by the analogy to other potyviruses. The resulting coat protein is 269 amino acids long and has a calculated MW of 30257. Two independently sequenced cDNA clones show sequence heterogeneity at four nucleotide positions: C422/A422, G432/A432, G446/A446 and T706/C706. Three first nucleotide differences are located at the PVA coat protein N-terminal region and led to the change of the amino acid. The coat protein of PVA displayed significant (73-78%) sequence homology to the coat proteins of six other potyviruses: papaya ringspot virus (PRV), pepper mottle virus (PeMV), plum pox virus (PPV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and tobacco vein mottling virus (TVMV). Even higher sequence homology (82%) was detected with a coat protein of a seventh potyvirus, tobacco etch virus (TEV). Major differences among the coat protein of PVA and of other potyviruses are located at the N-terminal region of the coat protein.     

The complete nucleotide sequences of the coat protein cistron and the 3′ non-coding region of a newly-identified potyvirus infecting sweetpotato,as compared to those of sweetpotato feathery mottle virus

Summary Complementary DNA representing 728 nucleotides of the 3 end of the genomic RNA of sweetpotato virus G (SPV-G), a newly-identified potyvirus infecting sweetpotato, was cloned and sequenced. This sequence was combined with that previously determined for the 5 terminal part of the coat protein cistron of the virus. The whole sequence contained a single open reading frame (ORF) of 1065 nucleotides, with the capacity to encode a coat protein of 355 amino acids, significantly larger than that of other potyviruses. The ORF was followed by an untranslated region of 222 nucleotides and a poly (A) tail. The coat protein of SPV-G was only distantly related to that of known potyviruses, with the exception of sweetpotato feathery mottle virus (SPFMV). Indeed, sequence identity in the C-terminal three quarters of the coat protein (more than 80%) and in the 3 untranslated region (more than 70%) indicate that SPV-G should be considered as closely related to, though distinct from SPFMV. This subset relationship is similar to that previously reported for members of the bean yellow mosaic virus subgroup or the bean common mosaic virus subgroup.     

Characterization of epitopes recognized by monoclonal antibodies to aphid transmissible and non-transmissible strains of turnip mosaic virus

Summary Fourteen monoclonal antibodies (MAbs) were prepared against two strains of turnip mosaic virus (TuMV) differing in aphid transmissibility. Serological specificity of fourteen MAbs against the two strains was tested by indirect ELISA. Three MAbs were able to distinguish aphid transmissible TuMV strain 1 from non-aphid transmissible strain 31 while four MAbs reacted only with strain 31. No cross-reactivity between the two strains was found using these specific MAbs. Based upon the ability of Mab to inhibit the reaction of other MAbs, antibody competition test indicated that fourteen MAbs recognized six different epitopes on the virus particle; MAbs specific to strain 1 recognized two epitopes while MAbs specific to strain 31 also recognized two epitopes. The remaining two epitopes are common. Since the six amino acid differences between the coat proteins of the two strains were found at the N-terminal regions, MAbs specific to strain 1 or 31 bound to the different epitopes on the N-terminal regions in coat proteins of the two strains.     

The potyviruses of Australia

Many potyviruses have been found in Australia. We analyzed a selected region of the coat protein genes of 37 of them to determine their relationships, and found that they fall into two groups. Half were isolated from cultivated plants and crops, and are also found in other parts of the world. Sequence comparisons show that the Australian populations of these viruses are closely related to, but less variable than, those in other parts of the world, and they represent many different potyvirus lineages. The other half of the potyviruses have only been found in Australia, and most were isolated from native plants. The sequences of these potyviruses, which are probably endemic, are on average five times more variable than those of the crop potyviruses, but surprisingly, most of the endemic potyviruses belong to one potyvirus lineage, the bean common mosaic virus lineage. We conclude that the crop potyviruses entered Australia after agriculture was established by European migrants two centuries ago, whereas the endemic plant potyviruses probably entered Australia before the Europeans. Australia, like the U.K., seems recently to have had c. one incursion of a significant crop potyvirus every decade. Our analysis suggests it is likely that potyviruses are transmitted in seed more frequently than experimental evidence indicates, and shows that understanding the sources of emerging pathogens and the frequency with which they 'emerge' is essential for proper national biosecurity planning.     

Complete nucleotide sequence and analysis of the putative polyprotein of maize dwarf mosaic virus genomic RNA (Bulgarian isolate)

Summary.  The complete nucleotide sequence of maize dwarf mosaic virus Bulgarian isolate (MDMV-Bg) was determined. The viral genome was 9515 nt and contained an open reading frame encoding 3042 amino acids, flanked by 3′- and 5′-UTRs of 139 and 250 nucleotides, respectively. MDMV-Bg was more conserved in the coding region (52.9%) than in the UTRs (45.8%) when compared to the 15 other potyviruses. Of ten putative gene products of MDMV-Bg, the P1 was the most variable protein (24.9%) while the NIb was the most conserved protein (67.3%). Several sequence variations were observed between MDMV-Bg and Johnson grass mosaic virus (JGMV), and more between MDMV-Bg and the dicot potyviruses. Phylogenetic analysis suggested that MDMV-Bg was the most closely related to JGMV. Received January 15, 1998 Accepted April 29, 1998     

Monoclonal antibodies define genus-specific, species-specific, and cross-reactive epitopes of the chlamydial 60-kilodalton heat shock protein (hsp60): specific immunodetection and purification of chlamydial hsp60.

Ocular and urogenital tract infections with Chlamydia trachomatis can progress to chronic inflammatory diseases that produce blindness and tubal infertility. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the chlamydial 60-kDa heat shock protein (hsp60) has been implicated as a major target antigen that stimulates the immunopathological response. The lack of chlamydial hsp60 antibodies and purified hsp60 has severely restricted studies to define more thoroughly the role of this protein in the immunopathogenesis of chlamydial disease. We produced a panel of antichlamydial hsp60 monoclonal antibodies (MAbs) and defined their specificities by immunoblotting against lysates of C. trachomatis, C. psittaci, and six other genera of bacteria. Three patterns of anti-hsp60 immunoreactivity were observed: chlamydial species specific, chlamydial genus specific, and cross-reactive. The epitopes recognized by these MAbs were localized within the primary amino acid sequence of hsp60 by immunoblotting against recombinant amino-terminal truncated hsp60 fusion polypeptides and then precisely mapped by use of overlapping synthetic peptides. The majority of the MAbs mapped to either the amino or the carboxyl termini of hsp60. Epitopes defining all three MAb reactivities mapped within amino-terminal residues 6 to 16. Genus-specific hsp60 MAbs mapped to epitopes located within this region and to residues 17 to 28 and 177 to 189. Antichlamydial hsp60 MAbs stained inclusions as effectively as MAbs specific for the major outer membrane protein. Homogeneous preparations of full-length recombinant chlamydial hsp60 and amino-terminal truncated recombinant hsp60 polypeptides were obtained by immunoabsorption chromatography with an hsp60 MAb reactive to the carboxyl terminus of the protein. Thus, the antichlamydial MAbs described here should be extremely useful for the specific immunodetection of hsp60 in tissues from individuals having different disease manifestations and for the purification of hsp60 or truncated hsp60 polypeptides for use in serologic and lymphocyte proliferation assays. The availability of these MAbs will facilitate studies to define more precisely the role of hsp60 in the immunopathogenesis of chlamydial disease.     

Immunochemical studies of tobacco mosaic virus—VII. Use of comparative surface accessibility of residues in antigenically related viruses for delineating epitopes recognized by monoclonal antibodies

The binding properties of 18 monoclonal antibodies (MAbs) directed against the tobacco mosaic virus (TMV) coat protein were studied with five related tobamoviruses and seven mutant viruses, as well as with the dissociated coat proteins of these variants. Ten of the antibodies bound to both TMV and TMV protein, but these were able to discriminate between different mutants only when whole virus particles were compared in the immunoassay. Three MAbs reacted with TMV but not with dissociated viral subunits and these recognized the same residue exchanges. Five MAbs recognized synthetic peptides of 13–28 residues corresponding to parts of the protein. By comparing the common accessible surface between TMV and antigenically related viruses, it was possible to narrow down the region recognized by some of these MAbs. The linear peptide sequence recognized by these antibodies did not represent the entire epitope and residue exchanges around this region were able to affect the binding of MAbs.     

Watermelon mosaic virus-Morocco is a distinct potyvirus

Summary The relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV 2, soybean mosaic virus (a virus closely related to WMV 2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV 1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61–68% identity to equivalent sequences in PRSV-W, WMV 2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.     

Sequence diversity in the coat protein coding region of the genome RNA of <Emphasis Type="Italic">Johnsongrass mosaic virus</Emphasis> in Australia

Summary. Sequence diversity in the coat protein coding region of Australian strains of Johnsongrass mosaic virus (JGMV) was investigated. Field isolates were sampled during a seven year period from Johnsongrass, sorghum and corn across the northern grain growing region. The 23 isolates were found to have greater than 94% nucleotide and amino acid sequence identity. The Australian isolates and two strains from the U.S.A. had about 90% nucleotide sequence identity and were between 19 and 30% different in the N-terminus of the coat protein. Two amino acid residues were found in the core region of the coat protein in isolates obtained from sorghum having the Krish gene for JGMV resistance that differed from those found in isolates from other hosts which did not have this single dominant resistance gene. These amino acid changes may have been responsible for overcoming the resistance conferred by the Krish gene for JGMV resistance in sorghum. The identification of these variable regions was essential for the development of durable pathogen-derived resistance to JGMV in sorghum.     

Cross-reactive and major virus-specific epitopes are located at the N-terminal halves of the cylindrical inclusion proteins of turnip mosaic and zucchini yellow mosaic potyviruses

Summary.  To investigate the antigenic nature of cylindrical inclusion proteins (CIPs) of the potyviruses Turnip mosaic virus (TuMV) and Zucchini yellow mosaic virus (ZYMV), monoclonal antibodies (MAbs) against the two CIPs were produced and epitopes on the CIPs were localized using Escherichia coli-expressed CIP fragments in Western blot analysis. All 23 MAbs against ZYMV CIP reacted only with ZYMV CIP. In contrast out of the 18 MAbs produced against TuMV CIP, 14 MAbs were TuMV CIP-specific while the remaining four MAbs cross-reacted with both CIPs. The four cross-reactive MAbs recognized two distinct epitopes in the N-terminal half of TuMV CIP corresponding to amino acid residues 103–119 and 224–237. Thirteen out of 14 TuMV CIP-specific MAbs recognized two distinct epitopes within residues 1–102 and 120–214, while the other one recognized an epitope within residues 301–644. On the other hand, 21 out of 23 ZYMV CIP-specific MAbs recognized epitopes within residues 1–118, while the remaining two recognized epitopes within residues 301–522. These results suggest that cross-reactive and major virus-specific epitopes are located at the N-terminal half of the respective CIPs. Received August 10, 1999 Accepted March 2, 2000