High-throughput sequencing detected a virus-viroid complex in a single pokeweed plant.

In this study, total RNA high-throughput sequencing (HTS) of a single symptomatic Phytolacca americana plant enabled the obtention of a nearly complete genome of two new isolates of turnip yellows virus (TuYV), named TuYV-ITA1 and TuYV-ITA2, and revealed a mixed infection with a new variant of citrus exocortis viroid (CEVd), named CEVd-ITA1. The TuYV-ITA2 isolate diverged from the known virus isolates of TuYV and showed variability in the P0 and P5 readthrough domain. Recombination analysis revealed its recombinant nature between TuYV and an unidentified polerovirus. The putative recombination event was identified in the P5 readthrough domain of the TuYMV-ITA2 isolate. Our results thus represent the first report of TuYV in Italy and some molecular evidence for the possible natural co-existence of TuYV and CEVd in a new natural host for both infectious entities. This study is adding further knowledge about the role of weed plants as virus reservoirs, and thus additional biological and impact studies would be desirable to determine in particular the role of P. americana in the spread of TuYV and if this virus should be considered a new threat for the susceptible Italian crops.

Loquat (Eriobotrya japonica) Is a New Natural Host of Tomato Mosaic Virus and Citrus Exocortis Viroid.

Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic virus (ToMV) and citrus exocortis viroid (CEVd) were identified in the transcriptome data. The complete genome sequence of ToMV and CEVd were obtained from the loquat leaves. The full-length genome of the ToMV-loquat is 6376 nt and comprises four open reading frames (ORFs) encoding 183 kDa protein, RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), respectively. A pairwise identity analysis showed that the complete sequence of the ToMV-loquat had a nucleotide identity between 98.5 and 99.3% with other ToMV isolates. A phylogenetic analysis indicated that ToMV-loquat was more closely related to ToMV-IFA9 (GenBank No. ON156781). A CEVd sequence with 361 nt in length was amplified based on the HTS contigs, sequence alignment indicated CEVd-loquat had the highest identity with the strain of CEVd-Balad (GenBank No. PP869624), phylogenetic analysis showed that CEVd-loquat was more closely related to CEVd-lettuce (GenBank No. ON993891). This significant discovery marks the first documentation and characterization of ToMV and CEVd infecting loquat plants, shedding light on potential threats to loquat cultivation and providing insights for disease management strategies.

Effects of RNA silencing during antagonism between citrus exocortis viroid and citrus bark cracking viroid in Etrog citron (Citrus medica).

Citrus exocortis viroid (CEVd) and citrus bark cracking viroid (CBCVd) are two important viroids that infect citrus plants and frequently occur as mixed infections in orchards. However, the mechanism of antagonism between the two viroids in mixed infections remains unclear. The CEVd/CBCVd-citron system and small RNA sequencing (sRNA-seq) were used to study the antagonism. When CBCVd was inoculated before CEVd, the CEVd titre was significantly reduced and the symptoms were attenuated. Viroid-derived sRNAs (vd-sRNAs) from CEVd and CBCVd were predominantly 21-nucleotide (nt) and 22-nt in length and had similar 5' base biases. Homologous sequences of the two viroids in the terminal right (TR) region are rich in vd-sRNAs, and the high frequency vd-sRNAs selected from the CBCVd TR region can be used to degrade the transcripts of CEVd in vivo directly. These results suggest that RNA silencing may play an important role in the antagonism of the two viroids, thus deepening our understanding of the molecular interaction of long noncoding RNAs in woody plants.

Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids.

The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study.

Detection of Viroids Using RT-qPCR.

Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.

Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure.

Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid-host interactions.

Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).

Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points (H52R, A277G) compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified.

The Gender-Based Differences in Vulnerability to Ambient Air Pollution and Cerebrovascular Disease Mortality: Evidences Based on 26781 Deaths.

The gender-based differences in the vulnerability to ambient air pollution have not been widely explored. This study aimed to investigate vulnerability differences to the short-term effects of PM2.5, PM10 and O3 between cerebrovascular diseases (CEVD) deaths of men and women. The general additive models (GAMs) and distributed lag non-linear models (DLNMs) were adopted, and both single-pollutant and two-pollutant models were performed to analyze the associations between ambient air pollution and daily CEVD deaths. Both models indicated that O3 was the most suspicious pollutant that could induce excess CEVD deaths, and women tended to be more vulnerable to O3. These results were confirmed by seasonal analysis, in which we also found both genders were more vulnerable to O3 in winter. The exposure-response relationships revealed that women were usually more vulnerable to ambient air pollution than men, and the exposure-response curves differed significantly between genders. Our findings suggested that more attention should be paid on the adverse effects of ambient O3, and the protection of women CEVD population against air pollution should be emphasized.

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

Global Transcriptomic Analysis Reveals Insights into the Response of 'Etrog' Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected 'Etrog' citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.

Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid-Tomato Pathosystem.

Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested.

Serum Hepatocyte Growth Factor Is Associated with Small Vessel Disease in Alzheimer's Dementia.

Background: While hepatocyte growth factor (HGF) is known to exert cell growth, migration and morphogenic effects in various organs, recent studies suggest that HGF may also play a role in synaptic maintenance and cerebrovascular integrity. Although increased levels of HGF have been reported in brain and cerebrospinal fluid (CSF) samples of patients with Alzheimer's disease (AD), it is unclear whether peripheral HGF may be associated with cerebrovascular disease (CeVD) and dementia. In this study, we examined the association of baseline serum HGF with neuroimaging markers of CeVD in a cohort of pre-dementia (cognitive impaired no dementia, CIND) and AD patients. Methods: Serum samples from aged, Non-cognitively impaired (NCI) controls, CIND and AD subjects were measured for HGF levels. CeVD (cortical infarcts, microinfarcts, lacunes, white matter hyperintensities (WMH) and microbleeds) were assessed by magnetic resonance imaging (MRI). Results: After controlling for covariates, higher levels of HGF were associated with both CIND and AD. Among the different CeVD MRI markers in CIND and AD, only small vessel disease, but not large vessel disease markers were associated with higher HGF levels. Conclusion: Serum HGF may be a useful peripheral biomarker for small vessel disease in subjects with cognitive impairment and AD.

Emergence of carp edema virus (CEV) and its significance to European common carp and koi Cyprinus carpio.

Carp edema virus disease (CEVD), also known as koi sleepy disease, is caused by a poxvirus associated with outbreaks of clinical disease in koi and common carp Cyprinus carpio. Originally characterised in Japan in the 1970s, international trade in koi has led to the spread of CEV, although the first recognised outbreak of the disease outside of Japan was not reported until 1996 in the USA. In Europe, the disease was first recognised in 2009 and, as detection and diagnosis have improved, more EU member states have reported CEV associated with disease outbreaks. Although the structure of the CEV genome is not yet elucidated, molecular epidemiology studies have suggested distinct geographical populations of CEV infecting both koi and common carp. Detection and identification of cases of CEVD in common carp were unreliable using the original PCR primers. New primers for conventional and quantitative PCR (qPCR) have been designed that improve detection, and their sequences are provided in this paper. The qPCR primers have successfully detected CEV DNA in archive material from investigations of unexplained carp mortalities conducted >15 yr ago. Improvement in disease management and control is possible, and the principles of biosecurity, good health management and disease surveillance, applied to koi herpesvirus disease, can be equally applied to CEVD. However, further research studies are needed to fill the knowledge gaps in the disease pathogenesis and epidemiology that, currently, prevent an accurate assessment of the likely impact of CEVD on European koi and common carp aquaculture and on wild carp stocks.

Survey and analysis of simple sequence repeats (SSRs) present in the genomes of plant viroids.

Extensive simple sequence repeat (SSR) surveys have been performed for eukaryotic prokaryotic and viral genomes, but information regarding SSRs in viroids is limited. We undertook a survey to examine the presence of SSRs in viroid genomes. Our results show that the distribution of SSRs in viroids may influence secondary structure, and that SSRs could play a role in generating genetic diversity. We also discuss the potential evolutionary role of repeated sequences in the viroid genome. This is the first report of SSR loci in viroids, and our study could be helpful in understanding the structure and evolution of viroid genomes.

Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.