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.     

The bipartite genome of red clover necrotic mosaic virus

Purified preparations of red clover necrotic mosaic virus isolated in Australia have been shown to contain three RNA components whose electrophoretic mobilities in polyacrylamide gel electrophoresis indicate molecular weights of 1.5 x 106 (RNA 1), 0.5 x 10(6) (RNA 2), and 0.14 x 10(6) (RNA 3). Comparisons of the RNAs by hybridization analysis with 3H-labeled complementary DNAs synthesized in vitro have established that RNAs 1 and 2 are unique RNA molecular species with little or no sequence homology between them. However, RNA 3 appears to be a complex mixture of breakdown fragments of both RNA 1 and RNA 2. Infectivity experiments with highly purified preparations of RNAs 1 and 2 have demonstrated that both molecules are essential for infectivity.     

Light microscopic cytochemistry and ultrastructure of red clover vein mosaic virus-induced inclusions.

Characteristic crystalline inclusions were observed in the cytoplasm of epidermal strips from stems, petioles, and leaves of Pisum sativum, Trifolium pratense, and Vicia faba infected with red clover vein mosaic virus (RCVMV). These inclusions vary in their shapes and sizes, from regular hexagonal prisms (up to 30 × 16 μm) to globular structures with irregular borders. The chemical nature of these inclusions was determined by cytochemical tests. The inclusions were ninhydrin positive, stained red with pyronin Y-methyl green, stained blue to purple with azure B, and fluoresced flame red with dilute acridine orange. Inclusions treated with RNase or hydrolyzed with perchloric acid did not fluoresce red with acridine orange or stain with pyronin, whereas treatment with DNase had no effect on the staining properties of these inclusions. These results are consistent with the presence of protein and a single-stranded ribonucleic acid in the inclusions. Electron microscopy of thin sections, obtained from sectioning an inclusion at different angles, showed that these inclusions were composed of polyhedral particles (approximately 10 nm in diameter) arranged in a hexagonal pattern. The polyhedral nature of these particles and their arrangement in a cubic symmetry was confirmed by viewing thick sections of the inclusions with a high voltage electron microscope equipped with an axis center tilting stage. The inclusions were not aggregates of RCVMV rods.     

Serotypes of red clover necrotic mosaic virus. I. Characterization of three serotypes

Three serotypes (A, B and C) were distinguished based on serological differences between isolates of red clover necrotic mosaic virus (RCNMV). Isolate TpM34, representative of serotype A, induced the formation of antibody only against homologous antigen. By contrast, isolate TpM48, representative of serotype B, induced the formation of 3 groups of antibody; the group of type-specific antibody was present in a higher titre than the other two antibody groups. Isolate 63/70, representing serotype C, also induced the production of type-specific antibody in a higher titre as compared with antibodies reacting with type A and B antigens. The distinct behaviour of the 3 serotypes was also manifested on immunoelectrophoresis in agarose gel. In anionic barbital buffer, serotypes A and C showed a higher mobility than representatives of serotype B, but in cationic environment serotype A showed a higher mobility than serotypes B and C.     

Determination of serotypes of red clover necrotic mosaic virus by enzyme-linked immunosorbent assay.

The direct double antibody sandwich (DAS) type of enzyme-linked immunosorbent assay (ELISA) was used to determine the degree of serological and antigenic differences, among the three serotypes (A, B, C) of red clover necrotic mosaic virus (RCNMV). Homologous and heterologous antibody titres in the used IgGs to isolates TpM34 (serotype A), TpM48 (serotype B) and isolate No. 6 (serotype C) as determined by ELISA were 100- to 200-fold higher than by ring precipitation test. Intensity of homologous and heterologous reactions in ELISA depended on the concentration of antigen, of the IgG used for coating and of the labelled IgG. The IgG preparations used contained 50 to 100 times higher concentration of homologous (serotype-specific) than heterologous (interserotype-specific) antibodies. Such a great difference between the two antibody types accounts for a comparatively high degree of selectivity of the homologous reactions.     

Serotypes of red clover necrotic mosaic virus. II. Typing of 34 isolates

Examination of 34 isolates of red clover necrotic mosaic virus (RCNMV) from Czechoslovakia in agar gel double diffusion precipitation tests and immunoelectrophoresis in agarose gel showed that 16 isolates belonged to serotype B, 6 isolates to serotype C and 4 isolates to serotype A; 3, 1 and 4 isolates represented mixtures of serotypes A + B, A + C and B + C, respectively. The distribution of the individual RCNMV serotypes in Czechoslovakia is not bound to definite geographic areas; two serotypes were even involved in mixed infections of single plants. Isolates of a given serotype were serologically and electrophoretically identical with the respective type isolate, i.e. there was a correlation between antigenic properties and electrophoretic mobility.     

The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1

The complete nucleotide sequence of red clover necrotic mosaic virus (RCNMV) RNA-1 has been determined. RNA-1 is 3889 nucleotides in length with a 5' terminal m7GpppA cap. The RNA contains three large open reading frames (ORFs): the 5' proximal ORF, encoding a 27-kDa polypeptide; the internal ORF, coding for a 57-kDa polypeptide; and the 3' terminal ORF, encoding the 37-kDa capsid protein. The sequence results confirm in vitro translation of 27-, 50-, and 37-kDa products but do not account for the observed 90-kDa product. A translational frameshift event from the 27- to the 57-kDa ORFs is proposed to explain the synthesis of the observed 90-kDa in vitro product. The putative translational frameshift region is structurally similar to several retrovirus frameshift regions and the putative barley yellow dwarf virus (BYDV) frameshift regions. Extensive amino acid homology was observed in the 57-kDa downstream ORF with the downstream domains of the carnation mottle virus (CarMV), turnip crinkle virus (TCV), maize chlorotic mottle virus (MCMV) readthrough, and BYDV fusion proteins. The 57-kDa ORF contained the conserved "GDD" motif. A significant alignment between the capsid proteins of RCNMV, CarMV, and TCV was also observed. Given the extensive amino acid sequence similarity of RCNMV, CarMV, and TCV polymerase and capsid proteins, we speculate that they are closely related, evolutionarily.     

Serotypes of red clover necrotic mosaic virus. III. Immunoelectrophoresis under different conditions

The effects of 4 buffers at different pH and of different molarities was tested on the immunoelectrophoresis of representatives of three serotypes and their natural mixtures of red clover necrotic mosaic virus (RCNMV). No substantial differences were observed with buffers at pH 7.2, 8.0 and 8.6. The representatives of the three RCNMV serotypes showed different mobilities in 0.1 and 0.01 mol/l ionic buffers in the agarose gel from cathode to anode. In nonionic 0.01-0.1 mol/l N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES) buffer, the RCNMV isolates moved from the anode to the cathode. Isolate TpM48 (serotype B) moved the most rapidly, while isolates No. 6 (serotype C) and TpM34 (serotype A) showed lower mobilities distinct from one another. This made possible a reliable differentiation of serotype B from serotypes A and C as well as of serotype A from serotype C. Agarose gels in 0.001 mol/l buffers proved to be unsuitable for the immunoelectrophoresis of RCNMV.     

Cassava vein mosaic virus (CsVMV), type species for a new genus of plant double stranded DNA viruses?

Summary.  The complete sequence of 8159 nucleotides of the double stranded DNA genome of cassava vein mosaic virus (CsVMV) was determined (# U59751) and revealed a significant difference in genome organization when compared with a previous report (# U20341). When transferred to cassava plants by microbombardment, the full length CsVMV clone was infectious, confirming the genome organization here described. Sequence comparisons between CsVMV and members of the genera Caulimovirus and Badnavirus revealed high homologies between consensus sequences of several proteins that are indispensable for virus replication, including a potential transactivator factor not reported previously. The presence of a sequence complementary to a plant Met tRNA confirms that CsVMV is a plant pararetrovirus and is most closely related to members of the genus Caulimovirus as previously assessed. However, differences in genome organization, number and size of the ORFs, in addition to sequence comparisons with other plant pararetroviruses, shows that either the genetic variability of caulimoviruses is much greater than previously thought, or that CsVMV is the unique representative of a new genus within the Caulimoviridae family. On the basis of this study, it is proposed to upgrade the floating genus Caulimovirus to the family level and to divide the Caulimoviridae family into at least three genera with CsVMV being the type member of a new genus. Accepted December 16, 1997 Received July 31, 1997     

Electron microscopy of leaves infected with sowbane mosaic virus and other small polyhedral viruses

Leaves of Chenopodium amaranticolor were infected with sowbane mosaic virus (SMV), sectioned and examined by electron microscopy. Leaves of Brassica pekinensis and C. amaranticolor were infected with turnip yellow mosaic virus and cowpea mosaic virus, respectively, and similarly studied. With all three viruses it was difficult, in sections, to distinguish the small isometric virus particles from ribosomes though sometimes this was possible, especially when the viruses crystallized. Pretreatment of tissue with permanganate or EDTA appeared to destroy the ribosomes but resulted in excessive disorganization of the tissue. Although SMV did not normally crystallize, wilting the infected leaves caused it to do so. All three viruses were found free in the cytoplasm and were absent from nuclei, chloroplasts, and mitochondria. Some abnormal structures found in the infected tissues are described.     

Enzyme-linked immunosorbent assay for detection of red clover necrotic mosaic virus in the host plants.

Detection of three isolates of red clover necrotic mosaic virus (RCNMV) representing A, B, and C serotypes was experimentally proved in 18 host plant species by enzyme-linked immunosorbent assay (ELISA). In all plant species tested, the homologous serotype reactions showed high selectivity. Individual virus serotypes could be reliably detected in the extracts of infected plants only with the homologous IgG fraction. Group specific detection of RCNMV without serotype determination was possible using the mixture of IgG directed to all virus serotypes occurring in the region of investigation. Intensity of positive reaction of optimally diluted IgG with the extracts from infected plants differed markedly from that of negative reaction and from the reaction background. The latter depended on the quality of serum used for the IgG preparation. For detection of small amounts of RCNMV, virus infectivity test on indicator plants was more sensitive than ELISA.     

The assembly of clover yellow mosaic virus and its protein

Clover yellow mosaic virus has been reconstituted near neutrality at low ionic strength. Reconstitution is stopped by low temperatures or by 0.1 M KCl or 10(-3) M MgCl2. Assembly is not confined to homologous RNA. Protein assembles into stacked-ring tubes near pH 8.0 or helical structures of two diameters at pH 5.0 to 5.5.     

The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses

Summary. The complete nucleotide sequence of a Beijing isolate of Wisteria vein mosaic virus was determined to be 9695 nucleotides in length excluding the poly(A) tail. Sequence analysis predicted a single large open reading frame of 9279 nucleotides potentially encodes a polyprotein of 3092 amino acids. Phylogenetic analysis based on the genomic and deduced amino acid sequences support the current status of Wisteria vein mosaic virus (WVMV) as a distinct virus of the genus Potyvirus and a member of the Bean common mosaic virus (BCMV) subgroup. Sequence comparisons of WVMV and other members of the BCMV subgroup showed that WVMV is most closely related to both soybean mosaic virus and watermelon mosaic virus.     

A comparative study of the cowpea and bean strains of southern bean mosaic virus

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.     

A comparative study of the alkaline disassembly of two strains of tobacco mosaic virus

Exposure of tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) to dilute alkaline solutions (pH > 8.0) at 0 degrees results in disassembly of the virus particles. Over the range of pH and NaCl concentration studied, Tm-TMV was more sensitive to alkaline conditions than was Tb-TMV. Kinetic analysis demonstrates that both isolates disassemble in a stepwise manner and that each produces six major intermediate-size particles.