A. Hadidi

Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification

DNA primers specific for grapevine virus A (GVA), grapevine virus B (GVB) or grapevine leafroll-associated virus III (GLRaV-III) were constructed based on the nucleotide sequence of a segment of each viral genome. DNA primers were utilized for cDNA synthesis and polymerase chain reaction (PCR) amplification of a 430 bp fragment from extracts of GVA-infected grapevine tissue or viruliferous mealybugs and 450 bp and 340 bp DNA fragments from extracts of GVB and GLRaV-III-infected grapevine tissues, respectively. The amplified DNA fragment of each virus was identified by Southern hybridization analysis with a cRNA probe of cloned viral genome. Reverse transcription (RT)-PCR, immunocapture (IC)-RT-PCR and/or multiplex (M)-RT-PCR assays were developed for the detection of GVA, GVB, and/or GLRaV-III in extracts of infected grapevine leaves, dormant cuttings and/or in viruliferous mealybugs. Viral specific DNA was absent from amplified extracts of uninfected grapevine tissue or nonviruliferious mealybugs. IC-RT-PCR was easier to perform than RT-PCR for the detection of GVA from viruliferous mealybugs. M-RT-PCR was easier and faster than IC-RT-PCR for the detection of GLRaV-III from infected grapevine tissue and it allows the sensitive detection of GVB, for which a high titer antiserum is not yet available.

Development of a plant-derived subunit vaccine candidate against hepatitis C virus.

Summary. Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 “mimotopes” previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines.

Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

DETECTION OF POME FRUIT VIROIDS BY ENZYMATIC CDNA AMPLIFICATION

We have studied the detection of apple scar skin, dapple apple, and pear rusty skin viroids in nucleic acid extracts of infected pome fruit tissues by reverse transcription-polymerase chain reaction (RT-PCR) with viroid cDNA-specific primers. Analysis of RT-PCR-amplified products by gel electrophoresis or by Southern blot hybridization indicated that the new procedure is more sensitive than existing detection methods and provides information about viroid detection without requiring large samples or using molecular hybridization. Our results also suggest the potential utility of the PCR technology in detecting other viroids and possibly other plant pathogens.

Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization.

A chemiluminescent molecular hybridization protocol was compared to 32P autoradiography for detecting potato spindle tuber viroid (PSTVd) and apple scar skin group viroids (ASSVd). Labeled cRNA probes for PSTVd and ASSVd were synthesized by SP6 RNA polymerase transcription using digoxigenin-11-UTP or alpha-[32P]UTP. Dot blot hybridization of purified viroids and sap extracts from infected plants showed that chemiluminescent detection using digoxigenin-labeled probes was as sensitive as autoradiography using 32P probes. A minimum of 2.0-2.5 pg purified viroid was detected. ASSVd could be detected in as little as 0.4 ng of total nucleic acid extract from infected tissue or in sap extracts diluted to 10(-3) with healthy extracts. Tissue blots of PSTVd-infected potato tubers and tomato roots, stems and leaves and ASSVd-infected apple fruit, stems and petioles, gave positive reactions when hybridized with the digoxigenin probe. No reaction with similar tissues from healthy plants was observed.

Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid, and MLO nucleic acids

A rapid, simple method for preparing plant tissues infected with viruses, viroids, or MLOs using a commercial product known as Gene Releaser is described. The Gene Releaser polymeric matrix method produced plant extracts suitable for PCR amplification without the use of organic solvents, ethanol precipitation, or additional nucleic acid purification techniques. Modification of maceration methods and/or extraction buffers resulted in the PCR amplification of potato spindle tuber, apple scar skin, and dapple apple viroids, as well as, genomic segments of plum pox potyvirus, grapevine virus B, grapevine leafroll-associated virus III, and elm yellows MLO. These pathogens were amplified from tissue of woody and herbaceous hosts such as peach, apricot, apple, grapevine, elm, periwinkle and potato. The application of this product for use with intractable tissue avoids lengthy and laborious extraction procedures. In our hands, about 20 samples could be prepared for PCR or RT-PCR in 1-2 h versus 1-3 days.

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera : Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

Scar skin and dapple apple viroids are seed-borne and persistent in infected apple trees

The closely related apple scar skin viroid (ASSV) and dapple apple viroid (DAV) were identified in whole seeds from infected pome fruits by hybridization of extracted nucleic acids with a 32P-labelled ASSV cRNA probe. Viroid amounts were greater in seed coats and subcoats than in seed cotyledons and embryos. ASSV or DAV was also detected in nucleic acid extracts from infected seeds, cotyledons and embryos by reverse transcription/polymerase chain reaction with viroid-cDNA-specific primers followed by Southern blot hybridization analysis of the amplified products with an ASSV cRNA probe. These results indicate that ASSV and DAV are seed-borne. ASSV and DAV were also found in the anthers, petals, receptacles, leaves, bark and roots of infected trees. The results suggest that viroid-infected trees constitute potential sources of the viroid in field spread. ASSV and DAV infections have been observed sporadically in commercial orchards in the United States and Canada and the infected trees have been eliminated. The use of viroid-free sources of seeds, seedlings, rootstocks and budwood should greatly reduce the risk of the future spread of the viroid.

Next-Generation Sequencing and Genome Editing in Plant Virology

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization

A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was developed. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was digoxigenin (DIG) -labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100-fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. The cRNA probe was several fold more sensitive than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue.