High throughput sequencing from Angolan citrus accessions discloses the presence of emerging CTV strains.

BACKGROUND: Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use. METHODS: Double-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3). RESULTS: From the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs. CONCLUSION: Phylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV.

Ultrasensitive immunoassay for detection of Citrus tristeza virus in citrus sample using disposable microfluidic electrochemical device.

Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DµFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DµFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95-10.0 × 103 fg mL-1 and ultralow detection limit of 0.3 fg mL-1. The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.

Codon Usage Bias Analysis of Citrus tristeza Virus: Higher Codon Adaptation to Citrus reticulata Host.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host⁻virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations.

Aphid transmission is a major factor in the formation of citrus tristeza virus (CTV) populations. Here, we examined the effect of population interaction on aphid transmissibility of different CTV genotypes. We found that there was no correlation between the proportion of viral genotypes in the source population and what was transmitted. We next examined the transmission of a poorly transmitted infectious cDNA clone (T36) in mixture with other CTV genotypes. T36 transmission increased from 0.5% alone, to up to 35.7%, depending on the coinfecting genotype. These results suggest that interaction between CTV genotypes affects the transmission of this virus.

Identification of rehmannia virus 1, a novel putative member of the genus Closterovirus, from Rehmannia glutinosa.

Transcriptome sequencing analysis of a symptomatic Rehmannia glutinosa plant revealed a virome containing two known RNA viruses and one novel virus. In this study, we examined the molecular and biological characteristics of the novel virus. The complete genome of the novel virus is composed of monopartite single-stranded RNA of 15,322 nucleotides with 69% nucleotide sequence identity (with 68% coverage) to tobacco virus 1. Its genome organization is typical of the members of the genus Closterovirus, containing nine putative open reading frames. Molecular and phylogenetic analyses of the genome and encoded protein sequences strongly support that the identified virus is a new species of the genus Closterovirus in the family Closteroviridae. The name rehmannia virus 1 (ReV1) is proposed for this novel virus.

Molecular characterization and detection of a new closterovirus identified from blackcurrant by high-throughput sequencing.

Two large contigs with high sequence similarities to several closteroviruses were identified by high-throughput sequencing from a blackcurrant plant. The complete genome of this new virus was determined to be 17,320 nucleotides. Its genome contains ten open reading frames (ORF) that include, in the 5'-3' direction, a large ORF encoding a putative viral polyprotein (ORF 1a) and nine ORFs that encode RNA-dependent RNA polymerase (RdRp, ORF 1b), p6 (ORF 2), heat shock protein 70-like protein (Hsp70h, ORF 3), Hsp-90-like protein (p61, ORF 4), CP minor (ORF 5), CP (ORF 6), p17 (ORF 7), p11 (ORF 8), and p26 (ORF 9), respectively. BCCV-1 shares nucleotide sequence identities of 43-45% with other 9 closteroviruses at genome sequences. The amino acid sequence identities between BCCV-1 and the closteroviruses were 49-55% (RdRp), 37-41% (Hsp70h), 19-33% (p61), 26-38% (CPm), and 19-28% (CP), respectively. Phylogenetic analysis of Hsp70h sequences placed the new virus with members of genus Closterovirus in the same group. The results indicate that this new virus, which is provisionally named as Blackcurrant closterovirus 1, should represent a new species of the genus Closterovirus. A RT-PCR was developed and used to detect BCCV-1 in more germplasm accessions of Ribes spp.

Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis.

BACKGROUND: The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS: The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS: Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS: The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.

Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza).

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

Population structure and diversity of citrus tristeza virus (CTV) isolates in Hunan province, China.

Stem-pitting (SP) is the main type of citrus tristeza virus (CTV) that causes severe damage to citrus trees, especially those of sweet orange, in Hunan province, China. Understanding the local CTV population structure should provide clues for effective mild strain cross-protection (MSCP) of the SP strain of CTV. In this study, markers for the p23 gene, multiple molecular markers (MMMs), and sequence analysis of the three silencing suppressor genes (p20, p23 and p25) were employed to analyze the genetic diversity and genotype composition of the CTV population based on 51 CTV-positive samples collected from 14 citrus orchards scattered around six major citrus-growing areas of Hunan. The results indicated that the CTV population structure was extremely complex and that infection was highly mixed. In total, p23 gene markers resulted in six profiles, and MMMs demonstrated 25 profiles. The severe VT and T3 types appeared to be predominantly associated with SP, while the mild T30 and RB types were related to asymptomatic samples. Based on phylogenetic analysis of the amino acid sequences of p20, p23 and p25, 19 representative CTV samples were classified into seven recently established CTV groups and a potentially novel one. A high level of genetic diversity, as well as potential recombination, was revealed among different CTV isolates. Five pure SP severe and two pure mild strains were identified by genotype composition analysis. Taken together, the results update the genetic diversity of CTV in Hunan with the detection of one possible novel strain, and this information might be applicable for the selection of appropriate mild CTV strains for controlling citrus SP disease through cross-protection.

Detection of Citrus tristeza virus by using fluorescence resonance energy transfer-based biosensor.

Due to the low titer or uneven distribution of Citrus tristeza virus (CTV) in field samples, detection of CTV by using conventional detection techniques may be difficult. Therefore, in the present work, the cadmium-telluride quantum dots (QDs) was conjugated with a specific antibody against coat protein (CP) of CTV, and the CP were immobilized on the surface of gold nanoparticles (AuNPs) to develop a specific and sensitive fluorescence resonance energy transfer (FRET)-based nanobiosensor for detecting CTV. The maximum FRET efficiency for the developed nano-biosensor was observed at 60% in AuNPs-CP/QDs-Ab ratio of 1:8.5. The designed system showed higher sensitivity and specificity over enzyme linked immunosorbent assay (ELISA) with a limit of detection of 0.13µgmL(-1) and 93% and 94% sensitivity and specificity, respectively. As designed sensor is rapid, sensitive, specific and efficient in detecting CTV, this could be envisioned for diagnostic applications, surveillance and plant certification program.

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves.

Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10(-6) to 10(-7) in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees.

Citrus trees harbor a large number of viral and bacterial pathogens. Citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), Citrus yellow mosaic virus (CYMV), Citrus tristeza virus (CTV) and a bacterium, Candidatus Liberibacter asiaticus (CLa) associated with huanglongbing (HLB) disease, the most prevalent pathogens in citrus orchards of different regions in India and are responsible for debilitating citriculture. For detection of these viral and bacterial pathogens a quick, sensitive and cost effective detection method is required. With this objective a multiplex polymerase chain reaction (mPCR) assay was developed for simultaneous detection of four viruses and a bacterium in citrus. Several sets of primers were designed for each virus based on the retrieved reference sequences from the GenBank. A primer pair published previously was used for greening bacterium. Each pair of primers was evaluated for their sensitivity and differentiation by simplex and mPCR. The constant amplified products were identified on the basis of molecular size in mPCR and were compared with standard PCR. The amplicons were cloned and results were confirmed with sequencing analysis. The mPCR assay was validated using naturally infected field samples for one or more citrus viruses and the huanglongbing bacterium. The mPCR assay developed here will aid in the production of virus free planting materials and rapid indexing for certification of citrus budwood programme.