Nuredin Habili

Comparative studies on tomato aspermy and cucumber mosaic viruses: I. Physical and chemical properties

Abstract Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 10 6 ,1.10 × 10 6 , 0.90 × 10 6 , and 0.43 × 10 6 daltons, and CMV-RNA, species of 1.26 × 10 6 , 1.10 × 10 6 , 0.77 × 10 6 , and 0.34 × 10 6 daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.

Comparative studies on tomato aspermy and cucumber mosaic viruses: III. Further studies on relationship and construction of a virus from parts of the two viral genomes

Although the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) do not show any detectable serological relationship or nucleotide base sequence homology, it was possible to construct a pseudo-recombinant virus from parts of their RNA genomes. The pseudo-recombinant from the two largest RNA molecules of TAV (T1 and T2) when combined with the third largest molecule of CMV (C3) formed a stable virus whose coat protein was like that of CMV, but it induced symptoms on several plants indistinguishable from those produced by TAV. It is concluded that TAV and CMV have functionally divided genomes and that the coat protein cistron is located on RNA component 3, whereas the cistron or cistrons determining host reactions are located on either RNA component 1 or 2, or on both 1 and 2.

Stabilization of capsid structure and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by formaldehyde

Abstract Treatment of the Q strain of cucumber mosaic virus (QCMV) with 0.2% formaldehyde for 24 hr at 4 ° in 0.005 M borate buffer, pH 9, stabilizes capsid structure and enhances immunogenicity in mice. Treated virus remained in suspension on mild heating in the presence of electrolyte whereas normal virus precipitated under similar conditions. Protein shells of formaldehyde-treated virus retained their structure during digestion of the viral RNA with pancreatic ribonuclease while those of normal virus degraded rapidly. Formaldehyde-treated capsids retained their structure on exposure to phosphotungstic acid, pH 6.8, during preparation for electron microscopy under conditions which degraded normal virus. It is concluded that the observed changes in the physical properties of QCMV by formaldehyde treatment are the results of cross linking of reactive sites on the adjacent subunits of the protein shells rendering the structural integrity of the capsids less dependent on bonding between the viral RNA and protein. The enhanced immunogenicity of formaldehyde-treated QCMV may be due to ability of the antigen to retain its structural integrity in the tissues of the animal.

Comparative studies on tomato aspermy and cucumber mosaic viruses: II. Virus stability

Abstract Although tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) are structurally similar in many respects, the conditions required for their stability differ considerably. TAV is more resistant than CMV to degradation by pancreatic RNase and to precipitation by NaCl. Whereas TAV is stabilized by Mg 2+ , CMV precipitates in the presence of the cation. CMV is stabilized, but TAV is degraded by EDTA. Both viruses are dissociated by low concentrations of sodium dodecyl sulphate indicating that their capsid structures depend on RNA-protein interactions.

Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus

Photobiotin was used to prepare biotinylated, nonradioactive nucleic acid probes for the detection of the RNA of barley yellow dwarf virus (BYDV) in plant extracts. A 1.7-kb cDNA of the PAV isolate of BYDV in the plasmid pUC8 vector was biotinylated and then used intact or as sonicated double-stranded DNA fragments. Simple methods were developed for the preparation of partially purified nucleic acid extracts of cereals and their spotting, after formaldehyde treatment, onto nitrocellulose membranes. After hybridization, biotin-labelled DNA bound to BYDV RNA on the nitrocellulose was detected with an avidin-alkaline phosphatase conjugate. BYDV RNA was readily detected with a sensitivity similar to that found with the same probe labelled with 32P by nick translation. Healthy plant extracts gave colourless spots.

Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts

Avocado sunblotch viroid (ASBV), coconut cadang cadang viroid (CCCV), chrysanthemum stunt viroid (CSV) and potato spindle tuber viroid (PSTV) were detected in plant extracts by dot-blot hybridization using nonradioactive photobiotin-labelled nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were biotinylated with photobiotin and used as sonicated double-stranded DNA fragments. Using fresh leaf material, a general method (suitably modified for avocado tissue) was developed for the rapid preparation of purified nucleic acid extracts. Plant extracts from a range of field samples were spotted onto nitrocellulose, subjected to hybridization and the biotin-labelled DNA bound to the target nucleic acid was detected with an avidin-alkaline phosphatase conjugate. Under the stated hybridization and washing conditions, each individual viroid probe was specific. Each viroid was readily detected with a sensitivity similar to that obtained with the same (or a like) probe labelled with 32P. Healthy plant extracts gave colourless spots.

Extensive Variation of Sequence within Isolates of Grapevine Virus B

Four regions covering 1247 nucleotides of the RNA genome of 20 isolates of a Vitivirus, Grapevine virus B (GVB), from three countries were analyzed. All the regions in these isolates varied in sequence as compared to the published GVB sequence. Of these, the intergenic region varied the most, with 73.2% nucleotide sequence homology, while ORF4 encoding coat protein varied the least when compared both at nucleotide sequence (80.3% homology) and at amino acid sequence levels (90.6% homology). The variations were scattered along each region length and were higher at the nucleotide level than at the amino acid level, but none resulted in a frame shift or stop codon. These results indicate that GVB may exist as a heterogeneous population, possibly resulting from mixing different strains by grafting practices or by RNA-RNA recombination in the grapevine, the only known natural host of this virus. Although it has been reported that GVB is associated with corky bark disease, no corky bark symptoms were observed in any of the GVB positive grapevine sample collected from Australia.

Comparative studies on tomato aspermy and cucumber mosaic viruses. IV. Immunogenic and serological properties.

Abstract Immunogenicity of TAV was not significantly different from that of CMV when compared by intraperitoneal injections of purified virus preparations into mice in doses ranging from 5 to 320 μg of antigen per injection per mouse. It is concluded that both viruses are poor immunogens. However, their immunogenicities could be enhanced by fixation with formaldehyde. Anti-TAV sera always contained antibodies specific to viral capsids and sometimes also to viral coat protein subunits, whereas anti-CMV sera invariably contained antibodies only to intact capsids. Both capsid-specific and protein coat-subunit-specific antibodies were shown to be immunoglobulins of the IgG class. Preparations of protein coat subunits from CMV failed to elicit antibody production in mice. However, those from TAV were weakly immunogenic producing low-titered antisera containing antibodies specific to TAV coat protein subunits but not to intact capsids. TAV capsid-specific and TAV protein coat subunit-specific antibodies were shown to be antigenically distinct. No antigenic relationship could be demonstrated between TAV and CMV.

Three group 16SrII phytoplasma variants detected in co-located pigeonpea, lucerne and tree medic in South Australia

Legume-infecting phytoplasmas identified in symptomatic tree medic (Medicago arborea), lucerne (M. sativa) and pigeonpea (Cajanus cajan) growing at a single trial site at Urrbrae, South Australia, were all shown to belong to the 16SrII phytoplasma taxonomic group, but each host species was found to be infected with a different genotype. Within one species (M. sativa) the genotypes were more uniform as demonstrated by comparing five isolates from lucerne plots. These lucerne genotypes differed from the Australian lucerne yellows phytoplasma genotype previously described in New South Wales, and the pigeonpea phytoplasma isolate was more similar to the non-legume infecting Bonamia little leaf (BoLL) than to pigeonpea little leaf phytoplasma. This diversity supports the view that these legumes were infected from different reservoir phytoplasma hosts rather than from a single perennial host such as tree medic.

First detection and molecular characterization of new Apple scar skin viroid variants from apple and pear in Iran

Apple scar skin viroid (ASSVd) is one of the most destructive viroids of pome fruit. We report 12 new sequence variants of ASSVd from apple and pear trees in north-eastern Iran. The Iranian variants ranged in size from 329 to 334 nucleotides. They formed a cluster distinct from other reported ASSVd isolates, and showed sequence variations in specific regions of the viroid RNA. This is the first report of the identification and molecular characterization of ASSVd in Iran.