Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms.

Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV.

5'-coterminal subgenomic RNAs in citrus tristeza virus-infected cells.

Three unusual 5' coterminal positive-stranded subgenomic (sg) RNAs, two of about 0.8 kb and one of 10 kb (designated LMT1, LMT2, and LaMT, respectively), from Citrus spp. plants and Nicotiana benthamiana protoplasts infected with Citrus tristeza virus (CTV) were characterized. The 5' termini of the LMT RNAs were mapped by runoff reverse transcription and found to correspond with the 5' terminus of the genomic RNA. The LMT 5'-coterminal sgRNAs consisted of two modal lengths of 744--746 and 842--854 nts. The 3' of the LaMT RNAs terminated near the junction of ORF 1b and ORF 2 (p33). None of the 5' sgRNAs had detectable amounts of corresponding negative-sense RNAs, as occurs with the genomic and 3' coterminal subgenomic RNAs of CTV. The abundance of the short and long 5' sgRNAs differed considerably in infected cells. The LMT RNAs were considerably more abundant than the genomic RNAs, while the larger LaMT RNA accumulated to much lower levels. The kinetics of accumulation of LMT1 and LMT2 in synchronously infected protoplasts differed. The larger RNA, LMT1, accumulated earlier with a strong hybridization signal at 2 days postinfection, a time when only traces of genomic and 3' sgRNAs were detected. The lack of corresponding RNAs, that could be 3' cleavage products corresponding to the 5' coterminal sgRNAs and the lack of complementary negative strands, suggest that these sgRNAs were produced by termination during the synthesis of the genomic positive strands.

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies.

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic RNA molecules.

Plants infected by citrus tristeza virus (CTV) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. The CTV particles and RNA extracts from purified CTV preparations were fractionated by sucrose gradient centrifugation and the RNA molecules from different fractions translated in a reticulocyte translation system. Fractions containing predominantly an RNA band of approximately 3.2 kb directed the synthesis of CTV coat protein (CP), which in SDS-PAGE had an estimated molecular mass of 28 kDa. Three additional polypeptides, with estimated sizes of 21 kDa, 23 kDa and 27 kDa, were translated from a range of RNA molecules smaller than 3.2 kb. Hybridization with cDNA to the CP gene (CTV-CPG) and with a 350 base clone complementary to the 3' and 5' termini of the genes for CTV p20 and p23.5, respectively, indicated that preparations of CTV particles contain, in addition to the genomic (20 kb) RNA, two sub-genomic RNA molecules of 3.2 kb and 2.4 kb and probably also two smaller molecules of 1.6 kb and 0.9 kb. Only the 3.2 kb RNA, its corresponding dsRNA molecule and populations of larger RNAs, including the 20 kb genomic RNA, hybridized with a CTV-CPG probe, thus conflicting with our previous assignment of the CTV-CPG to the 0.8 kbp dsRNA. Based on these results we propose that distinct populations of CTV particles encapsidate smaller RNAs which were formed as a nested set of subgenomic RNAs. Sequence analysis of 2540 nucleotides downstream to CTV-CPG of strain VT revealed four open reading frames (ORFs) potentially encoding in the 5' to 3' direction, 18 kDa (p18), 13 kDa (p13), 20 kDa (p20) and 23.5 kDa (p23.5) proteins. The CTV-VT ORFs showed variable but usually close levels of homology with the corresponding ORFs of CTV-T36 from Florida.

Rapid isolation of double stranded RNA segments from disulphide crosslinked polyacrylamide gels.

A simple method was developed to isolate dsRNA segments from disulphide crosslinked polyacrylamide gels. The dsRNA preparations from the P4 killer virus strain 77 of Ustilago maydis containing 7 genomic segments with molecular sizes ranging from 0.36 to 6.7 kbp, the 20 kbp dsRNA associated with the '447' cytoplasmic male sterility in Vicia faba and the 23 kbp genomic dsRNA of citrus tristeza virus (CTV) were separated on disulphide crosslinked polyacrylamide gels. After UV visualisation, the dsRNA bands were excised from the gel and dissolved using 2-mercaptoethanol. The dsRNA were then purified from the solubilized fractions by specific adsorption on microgranular cellulose and elution with a small volume of water. The method is rapid, simple and convenient for the isolation of all the tested dsRNAs segments.

A simple procedure for the extraction of double-stranded RNA from virus-infected plants.

A simple procedure for the isolation of double-stranded (ds) RNA from virus-infected plants is described. The method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds RNA by phenol extraction and precipitation with 30% ethanol. The presence of both negative and positive virus RNA strands in RNA fractionated in agarose gels was verified by Northern blot hybridization with polynucleotide kinase labelled genomic RNA or complementary DNA (cDNA) probes. The procedure enabled detection of three major ds RNA species (MWs 4.2, 1.05 and 0.48 X 10(6)) and at least 4 minor bands with estimated MWs of 3.5, 2.5, 2.2 and 2.0 X 10(6) in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV). Cucumber mosaic virus (CMV)-infected Pachystachys coccinea plants contained 2 minor bands of MWs 0.49 and 0.35 X 10(6) in addition to the previously described 4 major ds RNAs and ds CARNA 5 (MW 0.22 X 10(6)). The patterns of ds RNA are useful for diagnosing natural infections of CMV and TMV in N. glauca plants and of citrus tristeza virus in Citrus spp.

Translational competition between related virus RNA species in cell-free systems.

The competition between the RNAs from two tobacco mosaic virus (TMV) strains for translation in rabbit reticulocyte lysate and wheat germ cell-free systems was investigated. The virus translational products were distinguished by the size difference of the I2 polypeptides. The RNAs of the two strains were translated simultaneously if added to the translation system together at the beginning of the reaction. Each of the TMV RNAs could inhibit translation of the other when the reaction was initiated by the RNA of one strain and that of the other strain was added after active translation had commenced. In contrast, translation of alfalfa mosaic virus (AMV) RNA was not appreciably inhibited when its RNA was added after the start of translation of TMV RNA; the addition of TMV RNA to an AMV RNA-initiated reaction also resulted in independent synthesis of the products of both virus RNAs.

Hen egg yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): a comparison of two plant viruses.

A method of raising antibodies against plant viruses in hen egg yolk is described. Laying hens were immunized with citrus tristeza virus (CTV) or tobacco mosaic virus-avocado isolate (TMV-A). Anti-viral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (HADAS-ELISA). Antibodies first appeared in yolk 7 days after injection and peak levels were attained on day 9-11; these levels persisted for about 6-12 days. Non-specific yolk antibodies were removed by absorption with an extract of uninfected plant tissue. Using the HADAS-ELISA technique we found that yolk titres were equal to, or higher than those in serum. The benefits of using laying hens over conventional laboratory animals as a source of antiviral antibody are discussed.

Initiation factor eIF-4B (IF-M3)-dependent recognition and translation of capped versus uncapped eukaryotic mRNAs.

Translation of capped and uncapped eukaryotic mRNAs is stimulated by addition of eIF-4B to an mRNA-dependent reticulocyte lysate system. m7G5 ppp inhibits translation of capped but not uncapped mRNAs and reduces translation of capped vaccinia mRNA to the level obtained with uncapped vaccinia mRNA. Exogenous eIF-4B but no other initiation factor reverses inhibition of protein synthesis by m7G5'ppp. Both capped and uncapped mRNAs interact directly with eIF-4B to form a stable complex, which can be detected by a simple nitrocellulose filter binding assay. However, addition of a 5'-cap to beta-eliminated globin mRNA or satellite tobacco necrosis virus RNA (normally uncapped) increased binding affinity of these mRNAs for eIF-4B and causes binding of these mRNAs to become sensitive to inhibition by m7G5'ppp. These results indicate that the role of the mRNA 5'-cap in translation is related specifically to the function of eIF-4B in forming a complex with mRNA (prior to association of mRNA with the 40 S ribosomal subunit) and that both cap and non-cap sequences participate in this process.