Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium.

Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

Summary To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium. K e y w o r d s

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twentyone transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58

The PARP inhibitor rucaparib blocks SARS-CoV-2 virus binding to cells and the immune reaction in models of COVID-19

\ua9 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.Background and Purpose: To date, there are limited options for severe Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 virus. As ADP-ribosylation events are involved in regulating the life cycle of coronaviruses and the inflammatory reactions of the host; we have, here, assessed the repurposing of registered PARP inhibitors for the treatment of COVID-19. Experimental Approach: The effects of PARP inhibitors on virus uptake were assessed in cell-based experiments using multiple variants of SARS-CoV-2. The binding of rucaparib to spike protein was tested by molecular modelling and microcalorimetry. The anti-inflammatory properties of rucaparib were demonstrated in cell-based models upon challenging with recombinant spike protein or SARS-CoV-2 RNA vaccine. Key Results: We detected high levels of oxidative stress and strong PARylation in all cell types in the lungs of COVID-19 patients, both of which negatively correlated with lymphocytopaenia. Interestingly, rucaparib, unlike other tested PARP inhibitors, reduced the SARS-CoV-2 infection rate through binding to the conserved 493–498 amino acid region located in the spike-ACE2 interface in the spike protein and prevented viruses from binding to ACE2. In addition, the spike protein and viral RNA-induced overexpression of cytokines was down-regulated by the inhibition of PARP1 by rucaparib at pharmacologically relevant concentrations. Conclusion and Implications: These results point towards repurposing rucaparib for treating inflammatory responses in COVID-19

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing

25openYesBackground The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species’ biology, behavior, and dispersal patterns were poorly investigated to date. Methodology/Principal findings To understand the species’ population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Conclusions/Significance Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of ‘Single Nucleotide Polymorphism’ markers. Considering its public health importance, it is crucial to further investigate the species’ population genetic dynamic, including a larger sampling and additional genomic markers.Kurucz, Kornélia; Zeghbib, Safia; Arnoldi, Daniele; Marini, Giovanni; Manica, Mattia; Michelutti, Alice; Montarsi, Fabrizio; Deblauwe, Isra; Van Bortel, Wim; Smitz, Nathalie; Pfitzner, Wolf Peter; Czajka, Christina; Jöst, Artur; Kalan, Katja; Šušnjar, Jana; Ivović, Vladimir; Kuczmog, Anett; Lanszki, Zsófia; Tóth, Gábor Endre; Somogyi, Balázs A; Herczeg, Róbert; Urbán, Péter; Bueno-Marí, Rubén; Soltész, Zoltán; Kemenesi, GáborKurucz, K.; Zeghbib, S.; Arnoldi, D.; Marini, G.; Manica, M.; Michelutti, A.; Montarsi, F.; Deblauwe, I.; Van Bortel, W.; Smitz, N.; Pfitzner, W.P.; Czajka, C.; Jöst, A.; Kalan, K.; Šušnjar, J.; Ivović, V.; Kuczmog, A.; Lanszki, Z.; Tóth, G.E.; Somogyi, B.A.; Herczeg, R.; Urbán, P.; Bueno-Marí, R.; Soltész, Z.; Kemenesi, G

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe

Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

Summary To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium. K e y w o r d s

Methylene Blue Is a Nonspecific Protein–Protein Interaction Inhibitor with Potential for Repurposing as an Antiviral for COVID-19

We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and for other medical applications as a small-molecule inhibitor of the protein–protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concern such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1–5 μM). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63–ACE2, hepatitis C virus E–CD81) as well as others (e.g., IL-2–IL-2Rα) with similar potency. This nonspecificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells, both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 μM) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19