Characterization of a viroid associated with avocado sunblotch disease

A viroid has been purified from avocado leaves infected by sunblotch disease and designated the avocado sunblotch viroid. It is a covalently closed circular RNA molecule with a molecular weight lower than that of chrysanthemum stunt viroid and citrus exocortis viroid while hybridization analysis with 32P-labeled complementary DNA indicated that it is a single RNA species. It could be detected as a stainable RNA band on polyacrylamide tube gel electrophoresis of partially purified extracts of only two of four avocado isolates with positive symptoms of sunblotch disease. However, the viroid was detected in all four isolates by hybridization analysis with 32P-complementary DNA; this procedure has potential use for the rapid indexing of sunblotch disease since the viroid was not present in an isolate of healthy avocado. It has yet to be shown that the viroid is the causative agent of sunblotch disease.     

Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization

Summary In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigeninlabelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus.     

Characterization of viroid-like RNAs associated with the citrus exocortis syndrome

Three additional viroid-like RNA species (RNA-I, -II, and -III), smaller than the 371-nucleotide citrus exocortis viroid (CEV), have been identified in citron (Citrus medica) trees which display symptoms of the exocortis disease. The three RNAs migrate on polyacrylamide gels under denaturing conditions in the region between CEV and avocado sunblotch viroid circular molecules, indicating a size range of about 311-335 nucleotide residues in both circular and linear molecular forms. RNA-II is removed from the preparations when chromatographed on CF-11 cellulose. All three RNAs fail to replicate when introduced into Gynura aurantiaca, a herbaceous host of CEV, yet RNA-I and RNA-III can be independently transmitted to citron. Dot-blot hybridization with CEV-cDNA indicates no significant homology between CEV and RNA-I, RNA-II, or RNA-III. The relationship among CEV-RNA and the three viroid-like RNA species in the segregation of expression and intensity of the exocortis disease reaction is discussed.     

Detection of avocado sunblotch viroid by hybridization with synthetic oligonucleotide probes

Two short (20 and 17 nucleotides) DNA hybridization probes, complementary to avocado sunblotch viroid (ASBV) RNA nucleotides 68-87 and 88-104 respectively (Symons, R.H., Nucleic Acid Res. 9, 6527, 1981) were synthesized. The sensitivity and specificity of these radioactively labelled probes for hybridization with RNA of several ASBV isolates are demonstrated.     

Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell-free system from infected avocado leaves.

Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell-free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200 micrograms/ml of the drug, which typically inhibit RNA polymerase II and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polycrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis.     

Studies on encapsidated viroid-like RNA. IV. Requirement for infectivity and specificity of two RNA components from velvet tobacco mottle virus

Neither the virus-like RNA (RNA 1) nor the viroid-like RNA (RNA 2) of velvet tobacco mottle virus (VTMoV) is capable of independent replication in Nicotiana clevelandii. The function of RNA 2 for the RNA 1 replication could not be fulfilled by conventional viroids such as those of chrysanthemum stunt, citrus exocortis, or the viroid-like RNAs associated with avocado sunblotch and cadang-cadang disease of coconut. Similarly, RNA 2 failed to replicate when coinoculated with RNAs of conventional viruses such as those of southern bean mosaic, sowbane mosaic, galinsoga mosaic, red clover necrotic mosaic, and carnation mottle, all of which possess genomes with molecular weights similar to those of VTMoV RNA 1. The interdependence of the two VTMoV RNAs for biological activity was so highly specific that the function of VTMoV RNA 2 could not be replaced by the viroid-like RNA 2 from solanum nodiflorum mottle virus (SNMV), a closely related virus, for the replication of VTMoV RNA 1. Similarly, SNMV RNA 2 could not be replaced by VTMoV RNA 2 for the replication of SNMV RNA 1. These data support the view that RNA 2 cannot be a satellite RNA or an independent viroid. It seems that viruses such as VTMoV may have originated from associations of conventional single-stranded RNA viruses and viroids or, alternatively, may represent a stage in the evolution of viroids from viruses.     

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.     

Oligomeric potato spindle tuber viroid (PSTV) RNA does not process autocatalytically under conditions where other RNAs do

In vitro-synthesized oligomeric linear RNAs representing the replicative intermediates of potato spindle tuber viroid (PSTV) were subjected to a large variety of in vitro conditions where self-splicing of group I introns occurs, and where self-cleavage and self-circularization of the satellite RNA from tobacco ringspot virus particles and self-cleavage of oligomeric forms of avocado sunblotch viroid RNA has been observed. No evidence whatsoever could be obtained that the PSTV RNA oligomers monomerize and subsequently circularize autocatalytically in vitro under these conditions. These results are at variance with the low level of self-cleavage of dimeric PSTV RNA reported by H. D. Robertson et al. Virology 142, 441-447, 1985). A comparable and seemingly autocatalytic in vitro cleavage we have initially observed under certain conditions could be related to an unknown heat-stable RNase contaminating a batch of autoclaved ultrapure ammonium sulfate used in the corresponding incubating mixtures as additive.     

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.     

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

Restriction endonuclease mapping of the DNA of Rous-associated virus O reveals extensive homology in structure and sequence with avian sarcoma virus DNA

We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells.PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA (PvuI andEcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 10⁶M_r and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[Shank, P. R.,et al. (1978b).Cell15, 1383–1395;Hsu, W.,et al. (1978).J. Virol.28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.     

Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.     

Analysis of virus-specific RNA species and proteins in Freon-113 preparations of the Borna disease virus

Treatment of homogenates from Borna disease virus (BDV)-infected brain tissue or cell cultures with Freon-113 yielded infectious particles with a buoyant density of 1.16–1.22 g/ml. Positive- and negative-stranded BDV-specific RNA species as well as three virus-specific proteins, known to be present in BDV-infected cell extracts, were demonstrated in these Freon-treated fractions. When the Freon-purified virus preparations were treated with RNase A prior to RNA extraction, only negative-stranded, genomic RNA was detected in Northern blot hybridizations using sense and antisense RNA probes. These data substantiate that BDV is a negative-stranded RNA virus.     

Class I endochitinase containing a hevein domain is the causative allergen in latex-associated avocado allergy

BACKGROUND: In the medical literature immunoglobulin (Ig)E-mediated sensitization to avocado is rarely reported. On the other hand, more than 50% of subjects having IgE-mediated natural rubber latex allergy are sensitized to avocado fruit as demonstrated by skin-prick testing and/or specific IgE measurements and about 10-20% report hypersensitivity reactions after ingesting avocado. OBJECTIVE: The underlying pathomechanism of latex-associated avocado allergy is still unknown. The conserved hevein domain of the major latex allergen prohevein (Hev b 6.01) is a ubiquitous chitin-binding protein structure that can be found in several plant proteins and may be responsible for the observed cross-reactivity between latex and avocado fruit. METHODS: Chitin-binding avocado proteins (CBAPs) were isolated by affinity-chromatography and their IgE-binding characteristics were studied by immunoblotting using the sera from 15 avocado-sensitized latex patients. Inhibition experiments using isolated hevein and CBAPs as inhibitor solutions were performed to study the immunological cross-reactivity between both protein species and to assess the role of the CBAPs as mediators in latex-associated avocado allergy. RESULTS: In 80% of avocado-sensitized subjects (n = 15), IgE antibodies directed against a 31-kDa allergen were detected by immunoblotting. This IgE-binding protein was identified by protein sequencing to be a class I endochitinase containing a hevein domain at the N-terminus. Purified native and digested (using simulated gastric fluid) endochitinase were able to completely block all avocado-specific IgE antibodies in six out of seven avocado patients. CONCLUSIONS: Sensitization to endochitinase class I containing a hevein domain is the main underlying pathomechanism in latex-mediated avocado allergy.