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     

A non-aphid-transmissible isolate of bean yellow mosaic potyvirus has an altered NAG motif in its coat protein

Summary.  An isolate of Bean yellow mosaic virus (BYMV) not transmitted by aphids (NAT) was compared with the aphid-transmissible isolate (MI) from which it was derived. For each isolate, the sequence of the coat protein and parts of the helper component was determined. A single nucleotide substitution caused a NAG to NAS alteration in the coat protein of the non aphid-transmissible isolate. Loss of aphid transmissibility in isolate BYMV^MI-NAT was most likely caused by this mutation within the NAG motif. Systemic movement and accumulation of the virus in infected plants were not affected by the mutation. Received November 12, 2001; accepted April 30, 2002 Published online July 22, 2002     

Replacement of the coat protein gene of plum pox potyvirus with that of zucchini yellow mosaic potyvirus: characterization of the hybrid potyvirus

Infectious hybrid virus was generated by replacing part of the coat protein gene of plum pox potyvirus with that of the zucchini yellow mosaic potyvirus. This viable hybrid contains 84.5% of zucchini yellow mosaic potyvirus coat protein gene while the rest of the sequence was derived from plum pox potyvirus. Changing the coat protein gene between these two viruses had no effect on the experimental host range. Pathogenicity, stability and replication capacity of the hybrid virus were nearly identical to the parent viruses.     

Aphid transmission of a potyvirus depends on suitability of the helper component and the N terminus of the coat protein

Summary. The present study investigates the specificity of potyviruses for aphid species. Two potyviruses differing in their host range were used: Zucchini yellow mosaic virus (ZYMV) mainly infecting cucurbits and Turnip mosaic virus (TuMV) mainly infecting crucifers. Two sets of aphids species were used as vectors, one polyphagous (Myzus persicae and Aphis gossypii) and the other from crucifers (Brevicoryne brassicae and Lipaphis erysimi). Evidence is provided that the specificity between a vector and a potyvirus depends either on the affinity between the aphid species and the helper component (HC) protein used or on the affinity between the HC and the virions. The difference between the two potyviruses cannot be attributed to the DAG domain which is unaltered in both N termini of the CP. Therefore, a ZYMV full length clone served to exchange a fragment encoding for the N terminus of the ZYMV CP by that of TuMV. This partial exchange in the ZYMV CP, allowed the TuMV HC to transmit the chimeric virus but not the wild type ZYMV. The significance of the N terminus context of the CP in the specificity for the HC is discussed.     

Messenger RNA for the coat protein of southern bean mosaic virus

When RNA from southern bean mosaic virus is fractionated on a sucrose gradient, the resulting absorbance profile shows a major peak corresponding to 1.4 x 10(6) MW together with a considerable amount of slower sedimenting material. The RNA from these gradient fractions was translated in wheat embryo extracts and reticulocyte lysates. Only RNA in the molecular weight range 0.3 - 0.4 x 10(6) was found to induce synthesis of coat protein (designated P3). RNA of molecular weight 1.4 x 10(6) induced synthesis of three proteins, P1, P2, and P4.     

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.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

Summary The sequence of the 3 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M_r of 30 210. The 3 non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3 non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Interaction between coat protein and replication initiation protein of Mung bean yellow mosaic India virus might lead to control of viral DNA replication

In addition to their encapsidation function, viral coat proteins (CP) contribute to viral life cycle in many different ways. The CPs of the geminiviruses are responsible for intra- as well as inter-plant virus transmission and might determine the yield of viral DNA inside the infected tissues by either packaging the viral DNA or interfering with the viral replicative machinery. Since the cognate Rep largely controls the rolling circle replication of geminiviral DNA, the interaction between Rep and CP might be worthwhile to examine for elucidation of CP-mediated control of the viral DNA copy number. Here a reasonably strong interaction between Rep and CP of the geminivirus Mung bean yellow mosaic India virus is reported. The domain of interaction has been mapped to a central region of Rep. The replication initiation activity of Rep, i.e., its nicking and closing function, is down regulated by CP. This report highlights how CP could be important in controlling geminiviral DNA replication.     

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.     

Characterisation of epitopes on barley mild mosaic virus coat protein recognised by a panel of novel monoclonal antibodies

Summary. Barley mild mosaic virus (BaMMV), a member of the family Potyviridae, genus Bymovirus, is involved in the economically important yellow mosaic disease of winter barley in East Asia and Europe. We investigated serological properties of bacterially expressed BaMMV coat protein (CP) of a German isolate. Ten mouse monoclonal antibodies were produced using purified E. coli expressed BaMMV-CP as immunogen. The reactivity of MAbs with different strains of BaMMV was analysed by several immunological methods that are frequently used in diagnostic virology: enzyme-linked immunosorbent assay (ELISA), dot-blot, Western-blotting (WB), direct tissue blotting immunoassay (DTBIA) and immunoelectron microscopy (IEM). The amino acids involved in the formation of epitopes recognised by several MAbs were mapped by using synthetic pin-bound peptides and the localisation of epitopes in assembled virus particles was determined by electron microscope studies. MAbs V29 and M1 decorated the whole virion indicating that their epitopes ⁶PDPI⁹ and ⁹⁶ITDDEK¹⁰¹, respectively, are exposed on the surface. The MAbs V6 and V14 both interacted with ⁴⁴LPEPKM⁴⁹, which seems to be accessible at only one end of the virus particle. The MAbs V6, V14, V29 and M1 detected epitopes common to a wide range of BaMMV isolates and can therefore be used effectively in routine diagnostic tests for BaMMV from barley leaves. We suggest that MAbs M1, V6, V14 and V29 are most suitable for use in TAS-ELISA, V6, V14 and V29 for Western blotting and V29 and M1 for electron microscope serology.     

Strains of Turnip mosaic potyvirus as defined by the molecular analysis of the coat protein gene of the virus

Turnip mosaic virus (TuMV) is a member of the potyvirus genus with a wide host range and highly variable in its biological characteristics. Analysis of the CP gene sequences from databases, combined with the experimental analysis of the CP gene of further isolates, using data derived from sequence or restriction analysis, has allowed the genetic classification of 60 TuMV isolates or sequences. Two main genetic clusters MB (mostly Brassica isolates) and MR (mostly Radish isolates) were found, together with several apparently independent lineages. Isolates in the latter could be grouped as Intermediate between Brassica and Radish clusters (IBR) or outside Brassica and Radish clusters (OBR), according to their genetic distance to the main clusters. The genetic diversity of TuMV isolates deposited in the databases was increased with the sequences of the CP gene of seven selected isolates, mainly belonging to IBR or OBR groups. There was a correlation between the MR genetic cluster and JPN 1 serotype.     

Detection of a 45 kD protein derived from the N terminus of the pea seedborne mosaic potyvirus polyprotein in vivo and in vitro

A 45 kD protein (Pro1) derived from the N terminus of the pea seedborne mosaic potyvirus (PSbMV) polyprotein has been detected in extracts of infected pea plants and among in vitro translation products of PSbMV genomic RNA. The genomic region coding for the first 231 amino acids of the PSbMV polyprotein was cloned and expressed inEscherichia coli as a fusion protein with β-galactosidase. A rabbit antiserum raised against the fusion protein recognized an approximately 45 kD protein in immunoblots of extracts of PSbMV-infected pea leaves that was not present in extracts of healthy leaves. The highest concentration of the 45 kD protein was found in extracts of young leaves, suggesting the protein may be rapidly degraded in vivo. After in vitro translation of PSbMV genomic RNA in a wheat germ extract, the antiserum immunoprecipitated a 45 kD polypeptide as well as some lower molecular weight translation products. On the other hand, an approximately 90 kD polypeptide was immunoprecipitated from in vitro translation products of genomic RNA in a rabbit reticulocyte lysate, corresponding to the combined molecular weights of Prol and the helper component predicted from genomic sequence data.     

Monoclonal antibodies produced to bean yellow mosaic virus,clover yellow vein virus,and pea mosaic virus which cross-react among the three viruses

Summary Monoclonal antibodies prepared against individual potyviruses that infect forage legumes cross-reacted among the viruses. The reaction occurs between capsid subunits and presumably involves epitopes located in the trypsin-resistant core of the coat protein.     

Antigenic properties of the coat of Cucumber mosaic virus using monoclonal antibodies

The coat protein (CP) of Cucumber mosaic virus (CMV) was characterized by antigen-capture-ELISA using a panel of monoclonal antibodies (mAbs) which were produced against Pepo-CMV-CP. Comparative analysis of three mAbs with four different strains by competitive ELISA revealed that the binding affinity of the mAb decreased about 10-fold with both MY17- and Y-CMV than with Pepo-CMV. The CP of these three strains showed high homology (98%) following comparison in the GenBank database. CMV has a negatively charged loop structure, the βH–βI loop, although the amino acid at position 193 is not conserved. In addition, an amino acid residue identified within the variable region spanning amino acids 191–198, specifically at position 194, showed significant changes in Threonine, Alanine, Alanine, and Lysine of the Pepo-, MY17-, Y-, and M2-CMV strains, respectively. Evidence from competitive ELISA and GenBank database amino acid residues, when taken together, provide strong support suggesting that the dominant epitope site of CMV-CP-specific mAbs is the βH–βI loop 191–198. The four mAbs were chosen because they represent distinct, overlapping epitopes within the group-specific determinant located on the CMV-CP and because they all recognize linear epitopes. Knowledge of specific immunoglobulin genes for a common epitope may lead to insight on pathogen–host co-evolution and may help prevent virus infection in plants.     

Selection and performance of monoclonal and polyclonal antibodies in an IgM antibody capture enzyme immunoassay for rubella

Monoclonal anti-human IgM and anti-rubella antibodies were prepared and tested in an IgM capture enzyme immunoassay (MACEIA) for rubella-specific IgM and compared with polyclonal reagents. Assay sensitivity was increased with monoclonal antibodies resulting in the improved discrimination of adult sera with low levels of specific IgM. Despite high IgM binding, interference by IgM anti-Ig was not a major problem. The use of monoclonal antibodies allowed assay simplification by the simultaneous rather than sequential addition of antigen and conjugate. Although comparable results were obtained with 33 test samples in the sequential and simultaneous MACEIA, the specificity and sensitivity of this modification requires further evaluation.     

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.     

Selection of phage-display peptides that bind to cucumber mosaic virus coat protein.

Several discrete peptides that bind specifically to the coat protein of cucumber mosaic virus (CMV) were isolated from a diverse phage library displaying random nonapeptides on the major coat protein VIII. Enrichment was shown by polyclonal phage enzyme linked immunosorbent assay (ELISA) after three rounds of selection. Sequencing of the genes encoding 10 of these peptides revealed an absence of any conserved motifs, although nine of them contained a high proportion of proline residues. Some of the selected peptides were displayed at the N-terminus of thioredoxin and expressed in the cytoplasm of Escherichia coli. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CMV and CMV present in crude leaf extracts from infected plants. By dot blot analysis, a thioredoxin-peptide fusion could readily detect as little as 5 ng of CMV. The peptides did not bind to other plant viruses. These peptides have been shown to be specific and highly sensitive tools in the detection of CMV and, as well as their diagnostic potential, they could form the basis for a novel disease resistance strategy.     

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope

One of the proposed approaches in cancer therapy is to induce and direct the patient’s own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.