Clematis vitalba Is a Natural Host of the Novel Ilarvirus, Prunus Virus I.

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.

Structural hierarchy of mechanical extensibility in human von Willebrand factor multimers.

The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C1-6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A2 domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.

Quantitative and Qualitative Assessment of Fluorescence in Aesthetic Direct Restorations.

Currently available direct restoration materials have been developed to have improved optical properties to interact with light in the same manner as the natural tooth. The objective of this study was to investigate the fluorescence of different enamel resin composites. In the present study, nine brands of enamel composites were tested in vitro, some of which are cited by manufacturers as having color adjustment potential. Fluorescence spectra of the composite specimens and the human natural enamel were measured with a fluorescence spectrophotometer immediately after preparation and after 6 months. Qualitative data of the specimens were also collected. Statistical analyses were conducted by Kruskal−Wallis and Mann−Whitney U nonparametric tests (p < 0.05). Almost all tested resin composites presented a significant decrease in the fluorescence values after a period of 6 months. There was no significant decrease in fluorescence in the case of Harmonize™ resin composite samples, which presented the lowest initial fluorescence values. The highest value in the reduction of the initial fluorescence intensity after 6 months (22.95%) was observed for the Charisma® specimens. Composites with a color adjustment did not perform significantly better than other composites in terms of reduction in fluorescence intensity.

Isolation and molecular characterization of a tomato brown rugose fruit virus mutant breaking the tobamovirus resistance found in wild Solanum species.

A new tobamovirus named tomato brown rugose fruit virus (ToBRFV) overcomes the effect of the Tm-1, Tm-2, and Tm-22 resistance genes introgressed from wild Solanum species into cultivated tomato (Solanum lycopersicum). Here, we report the isolation and molecular characterization of a spontaneous mutant of ToBRFV that breaks resistance in an unknown genetic background, demonstrated recently in Solanum habrochaites and Solanum peruvianum. The wild isolate ToBRFV-Tom2-Jo and the mutant ToBRFV-Tom2M-Jo were fully sequenced and compared to each other and to other ToBRFV sequences available in the NCBI GenBank database. Sequence analysis revealed five nucleotide substitutions in the ToBRFV-Tom2M-Jo genome compared to ToBRFV-Tom2-Jo. Two substitutions were located in the movement protein (MP) gene and resulted in amino acid changes in the 30-kDa MP (Phe22 → Asn and Tyr82 → Lys). These substitutions were not present in any of the previously described ToBRFV isolates. No amino acid changes were found in the 126-kDa and 183-kDa replicase proteins or the 17.5-kDa coat protein. Our data strongly suggest that breaking the newly discovered resistance in wild tomatoes is associated with one or two mutations on the MP gene of ToBRFV.

First report of Coniella granati causing leaf spot of pomegranate (Punica granatum L.) in Hungary.

Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllo, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) - 8 - (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 - (3.6) - 3.9 × 10.2 - (13,1) - 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.

Screening of Solanum (sections Lycopersicon and Juglandifolia) germplasm for reactions to the tomato brown rugose fruit virus (ToBRFV).

The reaction of 636 Solanum (sections Lycopersicon and Juglandifolia) accessions were evaluated under greenhouse conditions after mechanical inoculation with a Jordanian isolate of the new tobamovirus tomato brown rugose fruit virus (ToBRFV). Local and systemic infections were assayed by symptoms evaluation and virus detection via biotests and RT-PCR. All cultivated tomatoes (Solanum lycopersicum) and the great majority of wild tomato accessions proved susceptible to ToBRFV. They showed a wide range of symptoms (mosaic, leaf deformations, mottling, shoestring, and stunting). Twenty-six accessions representing S. lycopersicum var. cerasiforme, S. pimpinellifolium, S. habrochaites, and S. chilense were tolerant. High levels of resistance have been demonstrated in three accessions of S. ochrantum, a close relative to wild tomatoes (member of the sect. Juglandifolia) not only to ToBRFV but also to the tobamoviruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). After mechanical inoculation, the three tobamoviruses could be detected only in inoculated leaves in the accessions LA2160, LA2162, and LA 2166, which remained symptomless. However, two other S. ochrantum accessions PI 473,498 and PI 230,519 reacted unusually. They were demonstrated highly resistant to TMV and ToMV, but proved transiently susceptible to ToBRFV showing mild systemic mosaic followed by total recovery from symptoms and the virus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s41348-021-00535-x.

First report of sweet potato chlorotic stunt virus infecting sweet potatoes in Hungary.

Sweet potato chlorotic stunt virus (SPCSV), a crinivirus in the family Closteroviridae, is a quarantine pest in Europe and one of the most economically important viruses of sweet potato (Ipomoea batatas (L.) Lam) crops globally. It forms synergies with other viruses in sweet potato, leading to yield loss of 30-100% (Qin et al., 2014). In summer 2020, 62 symptomatic and 38 symptomless sweet potato vines were randomly collected in farmers' fields in the south (Ásotthalom, Szeged) and central (Galgahévíz) parts of Hungary and transplanted in an insect-proof greenhouse. Six of the plants expressed SPCSV-like symptoms, including stunting, vein clearing and leaf purpling (Suppl1). To check for common viruses of sweet potato (Suppl2), total RNA and DNA were extracted from leaves of each of the 100 plants using Trizolate reagent (UD-GenoMed, Debrecen, Hungary) and Zenogene kit (Zenon Bio, Szeged, Hungary), respectively. Primer pair Ch2N (Suppl2) was designed using Primer3 (v. 0.4.0) to amplify a 194 bp fragment of SPCSV RNA1. Presence of the RNA viruses was checked by qPCR using qPCRBIO SyGreen 1-step qPCR kit (PCR Biosystems, London, UK), while DNA viruses were checked by PCR using DreamTaq DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), followed by 1% agarose gel electrophoresis. Four samples (labelled A5.1, A6.1, A6V9-1, A6V9-2) out of the 100 tested positive for SPCSV. Two of them (A6V9-1 and A6V9-2) were co-infected with SPCSV, a badnavirus sweet potato pakakuy virus (SPPV) and a potyvirus sweet potato virus 2 (SPV2), while the other two (A5.1 and A6.1) lacked SPV2. Plants infected with SPCSV, SPV2 and SPPV displayed more severe symptoms. To confirm the results, cDNA synthesized from the four SPCSV positive samples using RevertAid first strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania) underwent PCR (94oC 4 min, 94oC 1 min, 53oC 30 s, 72oC 70 s and 72oC 10 min for a total of 30 cycles) using primers CL43U and CL43L for the viral heat shock protein 70 gene (Maliogka et al., 2020). An expected band size of 486 bp was obtained in all cases. The amplicon from sample A6.1 was sequenced and found to be identical to SPCSV Guatemalan isolate GT:B3:08 (acc. JF699628). RNA1 and RNA2 complete sequences from sample A6.1 were obtained via PCR amplifications of cDNA using primers (Suppl2) designed (from acc. KC888966 for RNA1 and acc. KC888963 for RNA2) to amplify overlapping fragments of West African strain of SPCSV. QIAquick gel extraction kit (QIAGEN, Hilden, Germany) was used to purify the PCR fragments, which were then cloned into pGEM-T Easy Vector (Promega, Madison, USA) and sequenced using Sanger sequencing technique (Biomi, Gödöllo, Hungary). BLASTn search revealed that RNA1 of our isolate Hun_01 (acc. MW892835) had 99.63% sequence identity to SPCSV isolate su-17-10 (acc. MK802073), while RNA2 of Hun_01 (acc. MW892836) was 99.68% similar to SPCSV isolate min-17-1 (acc. MK802078) and isolate 24-1 (acc. MK802080). Phylogenetic analysis using MegAlign (v. 7.1.0, 44.1) showed a close relationship between our isolate and those isolated in China, suggesting that they may have a common origin (Suppl1). Severe stunting and leaf yellowing symptoms developed in I. setosa indicator plants grafted with SPCSV infected sweet potato scions. qPCR test for the virus confirmed its presence in the I. setosa leaves. To the best of our knowledge, this is the first report on the occurrence of SPCSV in Hungary and the third in Europe (Valverde et al. 2004; EPPO 2021).

Role of Lacosamide in Preventing Pentylenetetrazole Kindling-Induced Alterations in the Expression of the Gamma-2 Subunit of the GABAA Receptor in Rats.

BACKGROUND: Epilepsy remains challenging to treat still no etiologic treatment has been identified, however, some antiepileptic drugs (AEDs) are able to modify the pathogenesis of the disease. Lacosamide (LCM) has been shown to possess complex anticonvulsant and neuroprotective actions, being an enhancer of the slow inactivation of voltage-gated sodium channels, and it has the potential to prevent epileptogenesis. Recent evidence has shown that LCM indirectly improves the function of GABAA receptors. Receptors at most GABAergic synapses involve the gamma-2 subunit, which contributes to both phasic and tonic inhibition, and its presence assures benzodiazepine sensitivity. Moreover, mutant gamma-2 subunits were associated with generalized epilepsy syndromes. In animal models, the expression of the gamma-2 subunit of the gamma-aminobutyric acid A receptor (GABAAg2) was shown to be increased in pentylenetetrazole (PTZ)-induced chemical kindling in Wistar rats. OBJECTIVE: This study hypothesized that LCM might affect the kindling process by influencing the expression of GABAA receptors in the hippocampus. METHODS: The gene and protein expression levels of the GABAAg2 were studied using RT-qPCR and immunofluorescent staining. RESULTS: It was found that LCM treatment (10 mg/kg i.p. daily for 57 days) reduced the maximal intensity of the PTZ-induced seizures but did not prevent kindling. On the other hand, LCM treatment reverted the increase of mRNA expression of GABAAg2 in the hippocampus and prevented the decrease of GABAAg2 protein in the hippocampal CA1 region. CONCLUSION: LCM could exhibit modulatory effects on the GABAergic system of the hippocampus that may be independent of the anticonvulsant action.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary.

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.

Molecular characterization of a beet ringspot nepovirus isolated from Begonia ricinifolia in Hungary.

A nepovirus was isolated from Begonia ricinifolia showing chlorotic ringspot and line pattern symptoms. The purified virus had spherical particles of ca. 30 nm and contained a single coat protein subunit of ca. 56 kDa. The complete nucleotide sequence of the bipartite viral genome was determined. RNA 1 is 7394 nucleotides long, flanked by 5' and 3' untranslated regions (UTR), and followed by a 3' poly-A tail. It contains a single 6810 nt long open reading frame (ORF), which is translated into a 255 kDa polyprotein composed of 2269 amino acids. The 4684 nt long RNA 2 has a 4053 nt long ORF which encodes a single polyprotein of 1350 amino acids with a molecular weight of 149 kDa. Sequence comparisons revealed that the virus isolated from B. ricinifolia has the highest sequence similarity to beet ringspot virus and should be considered as a strain of BRSV. This is the first report on the occurrence of BRSV in B. ricinifolia and the presence of this virus outside Scotland.

Nucleotide sequence analysis of peanut stunt virus Rp strain suggests the role of homologous recombination in cucumovirus evolution.

The complete nucleotide (nt) sequence of peanut stunt virus Robinia strain (PSV-Rp) was determined and compared to other PSV strains and to representatives of the genus Cucumovirus. Nt sequence comparison showed 74.1-84.6% identity with the known PSV strains. Phylogenetic analysis revealed the different origin of the two genes encoded by RNA3. While the 3a gene clustered with PSV-W, the coat protein gene clustered with PSV-Mi. Recombination breakpoint analysis revealed two recombination points on RNA3. Based on these results, the establishment of a fourth PSV subgroup is proposed. This work revealed that homologous recombination occurred during the evolution of PSV.