Development of a Bio-PCR Protocol for the Detection of Xanthomonas arboricola pv. pruni.

A real-time SYBR Green I assay was developed and evaluated as a biological and enzymatic polymerase chain reaction (Bio-PCR) protocol for the detection of Xanthomonas arboricola pv. pruni. Suppression subtractive hybridization was used to generate a X. arboricola pv. pruni-specific subtracted DNA library, using X. arboricola pv. corylina as the driver strain. Primer pair 29F/R, designed from cloned sequence, showed no homology to GenBank sequences and amplified a 344-bp product in all X. arboricola pv. pruni isolates. Compared with other published X. arboricola pv. pruni primers, this primer pair was shown to be the only one capable of differentiating X. arboricola pv. pruni from all other X. arboricola pathovars. A real-time assay was developed and shown to be capable of detecting less than 10 CFU and 0.1 pg of DNA. Epiphytic bacteria isolated from plum tissue was used to further evaluate the specificity of the assay. A Bio-PCR protocol, developed for field evaluation, confirmed X. arboricola pv. pruni isolation from asymptomatic and symptomatic plum tissue over a 9-week period between host flowering and the first appearance of leaf and fruit symptoms in an orchard. Dilution plating enabled X. arboricola pv. pruni numbers to be quantified, providing supportive evidence for the usefulness of the Bio-PCR protocol in plant pathology and quarantine surveillance.

Drosophila melanogaster mounts a unique immune response to the Rhabdovirus sigma virus.

Rhabdoviruses are important pathogens of humans, livestock, and plants that are often vectored by insects. Rhabdovirus particles have a characteristic bullet shape with a lipid envelope and surface-exposed transmembrane glycoproteins. Sigma virus (SIGMAV) is a member of the Rhabdoviridae and is a naturally occurring disease agent of Drosophila melanogaster. The infection is maintained in Drosophila populations through vertical transmission via germ cells. We report here the nature of the Drosophila innate immune response to SIGMAV infection as revealed by quantitative reverse transcription-PCR analysis of differentially expressed genes identified by microarray analysis. We have also compared and contrasted the immune response of the host with respect to two nonenveloped viruses, Drosophila C virus (DCV) and Drosophila X virus (DXV). We determined that SIGMAV infection upregulates expression of the peptidoglycan receptor protein genes PGRP-SB1 and PGRP-SD and the antimicrobial peptide (AMP) genes Diptericin-A, Attacin-A, Attacin-B, Cecropin-A1, and Drosocin. SIGMAV infection did not induce PGRP-SA and the AMP genes Drosomycin-B, Metchnikowin, and Defensin that are upregulated in DCV and/or DXV infections. Expression levels of the Toll and Imd signaling cascade genes are not significantly altered by SIGMAV infection. These results highlight shared and unique aspects of the Drosophila immune response to the three viruses and may shed light on the nature of the interaction with the host and the evolution of these associations.

Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian 'blotch' strain of peanut stripe potyviruses.

We have analysed the coat protein gene sequences of two potyviruses infecting peanut. The 3' terminal 1247 nucleotides (nt) of an Australian strain of peanut mottle virus (PeMoV-AU) and the 3' terminal 1388 nt of an Indonesian 'blotch' strain of peanut stripe virus (PStV-Ib) were cloned and sequenced. Those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3' non-coding sequences. Comparison of the nucleotide sequences of PeMoV-AU and PStV-Ib revealed a sequence similarity of 64.4% for the coat protein gene and 34.6% for the 3' non-coding region. The deduced amino acid sequences of PeMoV-AU and PStV-Ib coat proteins are 66.7% identical. These results provide further evidence that PeMoV and PStV are distinct viruses. Comparisons of the 3' terminal sequences of PeMoV-AU and PStV-Ib with those of the genomic RNA of other strains of PeMoV and PStV and with other potyviruses are discussed.

Reducing agents interfere with the detection of lettuce necrotic yellows virus in infected plants by immunoblotting with monoclonal antibodies.

A procedure is described for the detection of lettuce necrotic yellows virus (LNYV) nucleocapsid protein (N) or envelope glycoprotein (G) by immuno-blotting with their respective monoclonal antibodies. The antigens can be detected in 1-10 mg of fresh tissue from systemically infected Nicotiana glutinosa leaves showing prominent symptoms. The composition of the extraction buffer played a crucial role in the recovery of antigenically active proteins. Procedures involving tissue extraction in the presence of the reducing agents 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) failed to detect either the N or G antigen. For optimum detection of the N or G antigens, leaf tissue was ground with 10 mM phosphate buffer, pH 7.6 (PB), containing 3% (w/v) sodium dodecyl sulphate (SDS) and 1 mM of the protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK). The extract was then mixed with an equal volume of dissociation buffer containing 2-ME before electrophoresis and immunoblotting.

Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization.

A simple, single-step plant tissue preparation protocol suitable for the detection of viruses by the polymerase chain reaction and reverse transcription-polymerase chain reaction is described. The effect of buffer components and pH, and the incubation temperature for the release of virus from plant material was evaluated. A small amount of plant tissue was heated in a solution containing 100 mM Tris-HCl, pH 7.4 or 8.4, 1 M KCl and 10 mM EDTA for 10 min at 95 degrees C and the supernatant used for enzymatic amplification. This protocol was suitable for the detection of both DNA and RNA viruses in a variety of plant species and tissues and reduced plant inhibitory factors which may interfere with PCR. The application of this method was demonstrated for the detection of banana bunchy top virus in banana leaves, root and corn, zucchini yellow mosaic potyvirus in squash leaves and lettuce necrotic yellows rhabdovirus in lettuce and Nicotiana glutinosa leaves.

Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELISA.

Nonspecific binding of immunoglobulins to coat protein of cucumber mosaic virus and several other plant viruses was observed in Western immunoblots, and to a much lesser extent in enzyme-linked immunosorbent assays. The binding appears to occur between immunoglobulin molecules and the basic domains of viral coat proteins which bind to RNA during encapsidation. In all cases tested, the nonspecific reactions could be prevented by addition of 5 U/ml of heparin to the incubation buffers.

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana.

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Identification of Zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability.

A reverse transcription-polymerase chain reaction (RT-PCR) method was used to identify Zucchini yellow mosaic virus (ZYMV) in leaves of infected cucurbits. Oligonucleotide primers which annealed to regions in the nuclear inclusion body (NIb) and the coat protein (CP) genes, generated a 300-bp product from ZYMV and also from the closely related watermelon mosaic virus type 2 (WMV-2). However, no product was obtained from papaya ringspot potyvirus which also infects cucurbits. ZYMV and WMV-2 were differentiated using a third primer which was complementary to a sequence in the 3'-untranslated region; a 1186-bp amplified product was obtained for ZYMV only. Nucleotide sequence analysis of the 300-bp fragments of Australian ZYMV and WMV-2 strains revealed 93.7-100% sequence identity between ZYMV strains. Multiple sequence alignments indicated that the nucleotide sequence which codes for the N-terminus of the CP was 74-100% identical for different isolates of ZYMV. The Australian isolate of WMV-2 was 43-46% identical to all isolates of ZYMV and was 84.6% identical to a Florida isolate of WMV-2.

Genetic Diversity Among Banana streak virus Isolates from Australia.

ABSTRACT Banana streak virus (BSV) is an important pathogen of bananas and plantains (Musa spp.) throughout the world. We have cloned and sequenced part of the genomes of four isolates of BSV from Australia, designated BSV-RD, BSV-Cav, BSV-Mys, and BSV-GF. These isolates originated from banana cvs. Red Dacca, Williams, Mysore, and Goldfinger, respectively. All clones contained a sequence covering part of open reading frame III and the intergenic region of the badnavirus genome. The sequences were compared with those of other badnaviruses, including BSV-Onne, a previously characterized isolate from Nigeria. The BSV-RD sequence was virtually identical to that of BSV-Onne, differing by only two nucleotides over 1,292 bp. However, BSV-Cav, -Mys, and -GF were divergent in nucleotide sequence. Phylogenetic analyses using conserved sequences in the ribonuclease H domain revealed that all BSV isolates were more closely related to each other than to any other badnavirus. BSV-Cav was most closely related to BSV-Onne, and there was 95.1% identity between the two amino acid sequences. Other relationships between the BSV isolates were less similar, with sequence identities ranging from 66.4 to 78.2%, which is a magnitude comparable to the distance between some of the recognized badnavirus species. Immunocapture-polymerase chain reaction assays have been developed, allowing specific detection and differentiation of the four isolates of BSV.

The rhabdoviruses: biodiversity, phylogenetics, and evolution.

Rhabdoviruses (family Rhabdoviridae) include a diversity of important pathogens of animals and plants. They share morphology and genome organization. The understanding of rhabdovirus phylogeny, ecology and evolution has progressed greatly during the last 30 years, due to enhanced surveillance and improved methodologies of molecular characterization. Along with six established genera, several phylogenetic groups at different levels were described within the Rhabdoviridae. However, comparative relationships between viral phylogeny and taxonomy remains incomplete, with multiple representatives awaiting further genetic characterization. The same is true for rhabdovirus evolution. To date, rather simplistic molecular clock models only partially describe the evolutionary dynamics of postulated viral lineages. Ongoing progress in viral evolutionary and ecological investigations will provide the platform for future studies of this diverse family.

Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus.

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

Characterization of antigenic structures on arabis mosaic virus with monoclonal antibodies.

Seven different epitopes on arabis mosaic virus (ArMV) were discerned. Neo-, crypto-, and epitopes exposed on the virion and isolated coat protein were differentiated by their reactivity with monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. The monoclonal antibodies, produced in vitro and in vivo, were of IgGl, IgM and IgA isotype. No epitope exclusively specific for one isolate was found. One epitope was specific for ArMV isolates only. With the common epitopes an operational antigenic map was devised. An immunological relationship between nepoviruses of different serological subgroups was demonstrated. Grapevine fanleaf virus, a member of the ArMV subgroup could not be shown to expose crossreactive epitopes. For serotyping ArMV isolates were discriminated by comparing the reactivity of two or more monoclonal antibodies specific for different epitopes.

Immunological properties and biological function of monoclonal antibodies to tobacco mosaic virus.

Monoclonal antibodies to TMV vulgare produced in hybridoma cultures as well as in ascitic fluid were characterized according to their reactivity with the virion and/or the coat protein monomer thus revealing specificity for epitopes, cryptotopes or neotopes. Different patterns of crossreactivity of these monoclonal antibodies with TMV strains dahlemense and Holmes' Ribgrass occurred. Some monoclonal antibodies showed stronger reactivity with these strains than with the immunizing strain. The monoclonal antibodies were TMV-specific as they did not react with ArMV and PLRV and proteins of healthy tobacco plants. The monoclonal antibodies were of the IgG2a or IgM isotype. The specific activity (Ext405nm/hour/100 micrograms MCA) with the immunizing virus and its coat protein monomers was determined as characteristic property of each monoclonal antibody. A monoclonal antibody specific for the C-terminal epitope of TMV coat protein was selected by means of the corresponding chemically synthesized tetrapeptide. With this monoclonal antibody infectivity of TMV was neutralized.

Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-biphosphate carboxylase share a common antigenic determinant.

An immunological relationship was detected between the coat protein of the common (U1) strain of tobacco mosaic virus (TMV) and the large subunit of the ubiquitous CO2-fixing host enzyme, ribulose-1,5-biphosphate carboxylase (RuBisCo). When assayed by Western immunoblotting or indirect ELISA, polyclonal antisera to TMV coat protein and to RuBisCo reacted with both antigens. In addition, a monoclonal antibody specific for the C-terminal antigenic determinant of TMV coat protein reacted with RuBisCo. Conversely, several monoclonal antibodies generated to the large subunit of RuBisCo reacted with TMV coat protein. This cross-reactivity was verified by an examination of the amino acid sequences of both proteins. A region of homology was found between the carboxy proximal portion of coat protein and the sequence 60-73 residues from the amino terminus of RuBisCo large subunit. This homology was not mirrored at the nucleic acid level because of different codon usages for the two proteins.

Alleged common antigenic determinant of tobacco mosaic virus coat protein and the host protein ribulose-1,5-bisphosphate carboxylase is an artifact of indirect ELISA and western blotting.

We have reported the detection of an antigenic determinant shared by the tobacco mosaic virus coat protein and the large subunit of ribulose-1,5-bisphosphate carboxylase, a host protein (R.G. Dietzgen and M. Zaitlin Virology 155, 262-266, 1986). This conclusion was questioned by D. Zimmermann and M.H.V. Van Regenmortel (Arch. Virol. 106, 15-22, 1989). Thus we have reinvestigated this unexpected serological cross-reaction in Western immunoblotting and indirect ELISA. We found that when skimmed milk instead of bovine serum albumin was used as a blocking agent and as a diluent for antibodies and alkaline phosphatase conjugates, the alleged cross-reaction was abolished. In light of these findings, we retract our previous conclusions.

Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia.

Isometric virus-like particles, 18 nm in diameter, have been isolated from banana (Musa spp.) affected by bunchy top disease in Australia. Banana bunchy top disease-associated virus-like particles (BBTV) banded as a single component with buoyant density of 1.28 to 1.29 g/ml in Cs2SO4 and sedimented at about 46S in isokinetic sucrose density gradients. The A260/A280 of purified preparations was about 1.33. A single coat protein of Mr 20,500 was identified with antibodies to BBTV particles from Australia. Single-stranded DNA of about 1 kb as well as ssRNA smaller than 0.45 kb was also associated with the particles. A polyclonal antiserum to BBTV, suitable for use in ELISA, was prepared. Stability and antigenicity of purified BBTV was impaired by storage at pH greater than or equal to 8.5 and freezing at -20 degrees C without protectants. BBTV was detected by double antibody sandwich-ELISA with monoclonal and polyclonal antibodies, in field-infected banana plants, single aphids from an infective colony, and in experimentally aphid-inoculated banana plants. After transmission of BBTV particles by aphids from a banana bunchy top disease-affected to an uninfected banana plant, the disease was induced and BBTV was detected by ELISA in symptomatic leaves only. BBTV isolates from Australia, Taiwan, People's Republic of China, Tonga, Western Samoa and Hawaii were found to be serologically related, which suggests a common aetiology for the disease.

Monoclonal antibodies against a plant virus.

Eight stable hybridoma clones derived from fusion of spleen cells of Tobacco Mosaic Virus (TMV) immunized mice with murine myeloma cells were grown in mass culture. They were obtained by 2 subsequent limiting dilution cloning cycles and subcultivation. Five of these clones secreted monoclonal antibodies which reacted with TMV nucleocapsid but not with capsid monomers and the monoclonal antibodies secreted by 3 other clones reacted with the TMV capsid monomer but not with the nucleocapsid. The specificity was determined by Enzyme Linked Immunosorbent Assay (ELISA) adjusted for the detection of antibodies in hybridoma culture supernatants.