DIG-labeled Probes Research Articles

Development of female specific SCAR marker in spine gourd (Momordica dioica Roxb. Ex. Wild) and its validation

Momordica dioica is a dioecious vegetable crop belonging to the family Cucurbitaceae. Fifty RAPD primers were used to analyze the male and female DNA bulks consisting of six females and six male genotypes of Momordica dioica respectively and 23 of them have differentiated the reproductive DNA bulks. One of the primers, OPB07, amplified a female reproductive bulk specific fragment of 326 bp (OPB07326) and was also present exclusively in all female genotypes used to prepare the reproductive bulk. This unique fragment OPB07326 was cloned and sequenced and was developed into female specific SCAR marker MdFSOPB07255. This SCAR marker has also amplified the unique female-specific fragment of 255 bp in all the six female genotypes used for the preparation of the bulk. The existence of this female-specific fragment in M. dioca genome was also affirmed by southern hybridization of DIG-labeled probe of OPB07326 with EcoRI restricted profile of all the six female genotypes. Further, the MdFSOPB07255 marker was successfully employed to identify eight female genotypes out of ten M. dioica genotypes with unknown reproductive feature. Thus, MdFSOPB07255 could be utilized as an efficient and reliable DNA marker for early differentiation of reproductive type at the sapling stage.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

STUDY OF THE ROLE OF RNA-INTERFERENCE IN THE REGULATION OF DISCRETE FRAGMENTATION OF 18S RRNA IN PLANTS

Under stressful conditions, cells must rapidly reduce the level of total mRNA translation, while maintaining the synthesis of some special proteins that help resist stress. In animals and yeast, the inhibition of protein synthesis is mainly associated with the modification of the translation initiation and elongation factors. In plant cells, many of these regulatory mechanisms are not realized. We hypothesize that plants have an alternative mechanism for suppressing mRNA translation associated with site-specific fragmentation of 18S rRNA molecules within 40S ribosomal subunits. Understanding the molecular mechanisms of plant response to stress is the basis for increasing the efficiency of the breeding process in order to develop stress-resistant varieties of economically important plant species. We found that during a heat shock, discrete RNA molecules accumulate in the cells of various plant species, which are 5'-terminal fragments of 18S rRNA that remain bound in the composition of ribosomal subunits. In this work, the method of nucleic acid hybridization was used to study RNA-interference as a possible mechanism mediating the phenomenon of discrete fragmentation of 18S rRNA in plants in response to heat shock. MicroRNA molecules complementary to the target regions of 18S rRNA, through which its rupture was carried out with the formation of stress-induced 5'-terminal RNA fragments, were not found. The small 5'-terminal fragments of 18S rRNA revealed during the work are proposed to be used as a new type of stress biomarkers in plant cells. The optimized nucleic acid hybridization technique using a DIG-labeled probe complementary to the highly conserved 1-35 5'-end region of plant 18S rRNA can be used to efficiently detect these stress RNA markers.

Novel DNA‐based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D.lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D.lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D.lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D.lepeophtherii spores were significantly associated (p<.05) with the development of D.lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

A Simplified Dot-Blot Hybridization Protocol for Potato spindle tuber viroid Detection in Solanaceae.

A simplified dot-blot hybridization protocol for Potato spindle tuber viroid (PSTVd) detection in Solanaceae species is described here. The protocol uses an RNA DIG-labeled probe and a simplified extraction procedure that avoids the use of hazardous chemicals. PSTVd was detected in composite tomato leaf samples in a ratio of up to 1:15 of PSTVd-infected to non-infected tissue and in composite potato tuber samples in a ratio up to 1:5 of PSTVd-infected to non-infected tissue. In Brugmansia spp., PSTVd was detected solely in the standard sample extract preparation. The method is suitable for a reliable, large-scale sample screening especially where cost is a limiting factor.

Telomere length in dromedary camels (Camelus dromedarius) produced by somatic cell nuclear transfer (SCNT) and their age-matched naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

14 Telomere length in cloned camels produced by somatic cell nuclear transfer is not different from that in their naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, and their age-matched naturally produced counterparts by terminal restriction fragment (TRF) analysis. Genomic DNA was extracted from venous blood collected from 6 cloned animals (aged 3, 12, and 24 months), and their age-matched counterparts by a conventional phenol/chloroform protocol. Denatured and neutralized DNA was blotted onto a positively charged nylon membrane and fixed by baking at 80°C for 3h. After washing in a prewarmed digoxigenin (DIG) easy hybridization solution at 42°C for 1h, DNA hybridization was carried out using a telomere (TTAGGG)n-specific, DIG-labelled hybridization probe (Roche Diagnostics, Germany) at 42°C for 4h. Stringent washes were carried out at the same temperature, followed by chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. Molecular weights of the unknown TRF bands on the gel were calculated using known molecular weight marker provided by Telo TAGGG Telomere Length Assay Kit (Roche Diagnostics). A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated 3 times, and the data, presented as mean±s.e.m., were analysed using a 2-sample t-test (Minitab statistical software). No difference (P&amp;gt;0.05) was found in the mean telomere length of cloned camels compared with their naturally produced age-matched counterparts (21.7±0.3 vs. 22.1±0.3; 21.9±0.3 vs. 22.1±0.3; 22.2±0.5 vs. 22.0±0.1; 20.5±0.5 vs. 22.5±0.7; 20.1±0.1 vs. 22.5±0.7; 21.7±1.1 vs. 22.6±0.2), respectively. In conclusion, this is the first study where telomere length has been reported in naturally produced and cloned dromedary camels produced by SCNT. We found that telomere lengths in cloned camels were similar to those of their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Localisation of probiotic Vibrio midae K811 within the gastrointestinal tract of post-larval abalone, Haliotis midae

An artificial biofilm probiotic-supplemented diet was shown to enhance the growth and survival of post-larval South African abalone, Haliotis midae. To ensure the effectiveness of the probiotic bacterium Vibrio midae K811 in an aquaculture environment, we wished to investigate whether V. midae K811 reaches the abalone intestinal tract where it is expected to exert its probiotic effect. Thus, the aim of this study was to investigate the colonization potential of V. midae K811 and to localise the probiotic within the gastrointestinal tract of post-larval H. midae. The Tn9 cat gene incorporated in V. midae K811 was PCR amplified from total DNA isolated from whole abalone homogenates up to 10 days following the cessation of feeding abalone with the probiotic-supplemented diet, while viable V. midae K811 was detected in abalone homogenates up to 25 days after the post-larval abalone had been switched from an alginate surface supplemented with V. midae K811 to a surface supplemented with Cocconeis sp. Furthermore, in situ hybridization using two Tn9 cat gene-specific DIG-labelled oligonucleotide probes detected V. midae K811 in the intestine of post-larval H. midae, confirming that the probiotic reaches the digestive tract of post-larval abalone placed on an alginate biofilm supplemented with the probiotic, and that the bacterium can persist in the abalone digestive tract for a period of at least 3 weeks.

Early detection and localization of viral haemorrhagic septicemia virus (VHSV) genomic mRNAs in external tissues of juvenile olive flounder (Paralichthys olivaceus) following an immersion challenge

The present study is a continuation of our previous work on in-situ detection and localization of viral haemorrhagic septicemia virus (VHSV) in olive flounder. As VHSV must cross and overcome the external barriers to reach the target organs, the role of external tissues becomes important in early viral uptake. However, investigations in this line are scarce and have not received much attention. Here, we investigated VHSV uptake in olive flounder to decipher the role of external tissues in early viral trafficking using a natural infection model. Juvenile olive flounder were bath (immersion) challenged with live VHSV for 1 h and tissue samples from gill, skin, fin and muscle were aseptically sampled at 15 min, 30 min, 3 h, 12 h, 24 h and 48 h post-challenge. Afterwards, samples were processed for in-situ hybridization (RNA-ISH) to observe spatio-temporal localization of VHSV using our previously designed DIG-labeled antisense RNA probes viz., nucleoprotein (N) and glycoprotein (G) riboprobes. In parallel, a time-dependent quantitation of the viral mRNA transcript (N & G) was done at similar time points using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the viral load. Whereas, VHSV uptake was confirmed in all the tested tissues using qRT-PCR, a weak signal was observed as early as 15 min in the gill using RNA-ISH. The signal intensity gradually increased at the corresponding time points and marked difference was evident at 12 hpc, 24 hpc and 48 hpc in all the tested tissues; particularly in the gill, skin and fin, followed by muscle. Our results suggest that VHSV uptake in flounder possibly occurs via gill immediately after exposure to the virus. Subsequently, virus gains entry through skin and fin base after it crosses the mucosal barriers of the host and then actively replicates in the external tissues. The current findings can be helpful in designing efficient immersion vaccination strategies for VHSV infection in olive flounder.

Expression patterns of ectodysplasin and ectodysplasin receptor during early dental development in zebrafish

This study aims to study the expression patterns of ectodysplasin (EDA) and ectodysplasin receptor (EDAR) during the early development of zebrafish and provide a foundation for further research of the Eda signaling pathway in tooth development. Total RNA was extracted from zebrafish embryos at 48 hours postfertilization (hpf) and then reverse transcribed for cDNA library generation. The corresponding RNA polymerase was selected for the synthesis of the digoxin-labeled antisense mRNA probe of zebrafish pharyngeal tooth specific marker dlx2b and Eda signaling-associated genes eda and edar in vitro. The three sequences were ligated into a pGEMT vector with a TA cloning kit, and polymerase chain reaction (PCR) was applied to linearize the plasmid. The resultant PCR sequences were used as templates for synthesizing Dig-labeled mRNA probe dlx2b, eda, and edar. Zebrafish embryos were collected at 36, 48, 56, 60, 72, and 84 hpf, then whole mount in situ hybridization was performed for the detection of eda and edar expression patterns. Then, their expression patterns at 72 hpf were compared with the expression pattern of dlx2b. The mRNA antisense probes of dlx2b, eda, and edar were successfully obtained. The positive signals of eda and edar were observed in zebrafish pharyngeal tooth region at 48-72 hpf and thus conform to the signals of dlx2b in the positive regions. The ligand eda and edar, which are associated with the Eda signaling pathway, are strongly expressed only at the pharyngeal tooth region in zebrafish from tooth initiation to the morphogenesis stage. Thus, the Eda signaling pathway may be involved in the regulation of the early development of zebrafish pharyngeal teeth.

A novel species of RNA virus associated with root lesion nematode Pratylenchus penetrans.

The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of plants. While analysing transcriptomic datasets of P. penetrans, we have identified a full-length genome of an unknown positive-sense single-stranded RNA virus, provisionally named root lesion nematode virus 1 (RLNV1). The 8614-nucleotide genome sequence encodes a single large polyprotein with conserved domains characteristic for the families Picornaviridae, Iflaviridae and Secoviridae of the order Picornavirales. Phylogenetic, BLAST and domain search analyses showed that RLNV1 is a novel species, most closely related to the recently identified sugar beet cyst nematode virus 1 and potato cyst nematode picorna-like virus. In situ hybridization with a DIG-labelled DNA probe confirmed the presence of the virus within the nematodes. A negative-strand-specific RT-PCR assay detected RLNV1 RNA in nematode total RNA samples, thus indicating that viral replication occurs in P. penetrans. To the best of our knowledge, RLNV1 is the first virus identified in Pratylenchus spp.

Comparative performance of TCBS and TSA for the enumeration of trh+ Vibrio parahaemolyticus by direct colony hybridization

Vibrio parahaemolyticus is one of the important foodborne pathogens is of public health concern due to the emergence of pandemic strains causing disease outbreaks worldwide. We evaluated the DNA based colony hybridization technique for the detection and enumeration of total and pathogenic V. parahaemolyticus from the bivalve shellfish, clam using non-radioactive, enzyme-labeled probe targeting the tlh and trh genes, respectively. The digoxigenin (DIG) labeled probes designed in this study showed 100% specificity by dot blot assay. Colony hybridization using DIG probes was performed using both non-selective, trypticase soy agar (TSA) and the selective medium, thiosulfate citrate bile salts sucrose (TCBS) agar. Of 32 clam samples analyzed, 71.88% had>10,000 V. parahaemolyticus cells/g in TSA whereas it was 18.75% in case of TCBS. All the samples showed the presence of total V. parahaemolyticus in TSA and 97% in the case of TCBS. Interestingly, results of the trh+V. parahaemolyticus samples were quite high while using TCBS plates (62.5%) as compared to TSA (43.75%). However, the cell numbers obtained from TSA were higher than from TCBS. Several yellow colonies on TCBS turned out to be V. parahaemolyticus using colony hybridization, which was further confirmed by PCR and sucrose utilization test. Colony hybridization using DIG-labeled probe was found to be highly sensitive and could differentiate and enumerate pathogenic and non-pathogenic strains of V. parahaemolyticus. Since traditional methods are not only labor-intensive and time-consuming but also less sensitive, colony hybridization using DIG-labeled probes would be a useful alternative for the enumeration of V. parahaemolyticus in naturally contaminated seafood.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Whole Mount in situ Localization of miRNAs and mRNAs During Somatic Embryogenesis in Arabidopsis.

Somatic embryogenesis (SE) results from the transition of differentiated plant somatic cells into embryogenic cells that requires the extensive reprogramming of the somatic cell transcriptome. Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount in situ hybridization (WISH) method (Bleckmann and Dresselhaus, 2016; Dastidar et al., 2016) in order to analyze the spatiotemporal localization of miRNAs (miR156, miR166, miR390, miR167) and mRNAs such as WOX5 and PHABULOSA-target of miR165/166 during the SE that is induced in Arabidopsis explants. This study presents a detailed step-by-step description of the WISH procedure in which DIG-labeled LNA and RNA probes were used to detect miRNAs and mRNAs, respectively. The usefulness of the WISH in the functional analysis of the SE-involved regulatory pathways is demonstrated and the advantages of this method are highlighted: (i) the ability to analyze intact non-sectioned plant tissue; (ii) the specificity of transcript detection; (iii) the detection of miRNA; and (iv) a semi-quantitative assessment of the RNA abundance.

Non-isotopic RNA In Situ Hybridization for Functional Analyses in Medicago truncatula.

Different strategies have been developed and implemented during the last decades aiming to decipher the function of particular genes. Among the different techniques, in situ hybridization of mRNA remains an essential experiment to fully understand gene function. Here, we describe a protocol for the in situ localization of gene transcripts in plants. It is optimized for use of paraffin-embedded tissues and DIG-labeled probes and has successfully applied to floral bud tissues from Medicago truncatula. Using this protocol, we have analyzed the expression of MADS-box transcription factors where some of them have been preserved as duplicates in the genome. When duplicated genes are analyzed, the tissue and cellular location of the transcripts is the only technique that accounts for small variations in the pattern of gene expression that occurred after duplication and diversification. The use of a well-standardized in situ hybridization protocol is vital for the systematic analysis of the function of genes in Medicago truncatula.

FIRST REPORT OF TOMATO INFECTIOUS CHLOROSIS VIRUS IN GLOBE ARTICHOKE ECOTYPES IN APULIA, SOUTHERN ITALY

Apulia (southern Italy) hosts a rich variety of globe artichoke ecotypes of high interest for breeding programs and industrial production. Most of the ecotypes are at risk of genetic erosion because of the high incidence of plant pathogens, which include infections from taxonomically different viruses. In the early autumn of 2014 and 2016 five samples were collected from each of the early-flowering ecotypes, Brindisino, Carciofo di Lucera, Francesina di Brindisi, Francesina di Trinitapoli, Locale di Mola, Nero di Ostuni, Tricasino, Troianella, Verde di Putignano, Violetto di Putignano and the late-flowering ecotypes Bianco di Taranto and Locale di Mola tardivo, and tested for viral infections by dot blot hybridization with Dig-labeled DNA probes and RT-PCR (Minutillo et al., 2012). In the five samples of the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella the hybridization signal revealed the presence of tomato infectious chlorosis virus (TICV) that in the ecotype Troianella was in mixed infection with artichoke Italian latent virus and turnip mosaic virus. TICV infection was confirmed by RT-PCR with TICV-specific primer pair for 5'- TCAGTGCGTACGTTAATGGG-3' and rev 5'-CACAGTATACAGCAGCGGCAG-3' (Minutillo et al. 2012). Amplicons from the ecotypes Verde di Putignano and Troianella were sequenced. Nucleotide sequence analyses revealed 99% identity with a TICV isolate from USA (accession No. FJ542306). Previous surveys detected TICV in globe artichoke in Liguria, Sardinia, Latium, Campania and Sicily but this is the first report of TICV in artichoke in Apulia and in the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella. Artichoke plants infected by TICV did not show symptoms and thus may act as perennial TICV reservoirs.

Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3’ poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3’ proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659.

Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection

The present study reports outbreaks of tilapia lake virus (TiLV), an emerging pathogen causing syncytial hepatitis of tilapia (SHT), in farmed tilapia in Thailand. Occurrence of the virus was confirmed by RT-PCR and nucleotide sequence homology to the TiLV from Israel. Diseased fish exhibited typical histopathological features of syncytial giant cells in the liver examined through H&E and semi-thin sections. Presence of intracytoplasmic viral particles was revealed by TEM. In situ hybridization using a specific DIG-labeled probe derived from a partial genome segment 3 of TiLV genome revealed multiple tissues tropism of the virus including liver, kidney, brain, spleen, gills and connective tissue of muscle. An alternative semi-nested RT-PCR protocol has been developed in this study for disease diagnosis. Additionally, comparative genetic analysis revealed genetic variations of Thailand-originated virus to the Israel TiLV strains, sharing 96.28 to 97.52% nucleotide identity and 97.35 to 98.84% amino acid identity. Outbreaks of TiLV in different continents might signal a serious threat to tilapia aquaculture globally.

Investigating the Molecular Basis of PPCD3: Characterization of ZEB1 Regulation of COL4A3 Expression.

PurposeTo investigate the role of the zinc finger e-box binding homeobox 1 (ZEB1) transcription factor in posterior polymorphous corneal dystrophy 3 by demonstrating its ability to regulate type IV collagen gene transcription via binding to putative E2 box motifs.MethodsPutative E2 box motifs were identified by in silico analysis within the promoter region of collagen, type IV, alpha3 (COL4A3) and collagen, type IV, alpha4 (COL4A4). To test the ability of ZEB1 to bind to each identified E2 box, electrophoretic mobility shift assays were performed by incubating ZEB1-enriched nuclear extracts with DIG-labeled probes containing one of each of the identified E2 box motifs. Dual-luciferase reporter assays were performed to test the effects of ZEB1 on the luciferase activity of COL4A3 and cadherin 1 (CDH1) promoter constructs, and to determine the effect of a ZEB1 truncating mutation on CDH1 promoter activity.ResultsZEB1 exhibited binding to six of the nine COL4A3 E2 box probes, whereas no binding was observed for either of the two COL4A4 E2 box probes. ZEB1 overexpression resulted in reduced activity of the COL4A3 promoter construct containing all identified E2 box motifs, whereas a truncating ZEB1 mutation led to the loss of ZEB1-dependent repression of the CDH1 promoter.ConclusionsCOL4A3 gene expression is negatively regulated by ZEB1 binding to E2 box motifs in the COL4A3 promoter region. Therefore, the altered expression of type IV collagens, particularly COL4A3, in the corneal endothelium in individuals with PPCD3 is likely due to reduced transcriptional repression in the setting of a single functional ZEB1 allele.