Journal: Poultry Science

Article Title: Construction and modification of a low-copy plasmid-based infectious clone for GI-19 genotype IBV via Red/ET recombineering: A simplified and efficient reverse genetics system for co ronavirus

doi: 10.1016/j.psj.2026.106881

Figure Lengend Snippet: Validation of recombinant plasmids by restriction enzyme analysis and functional selection screening. (A) Restriction enzyme digestion of intermediate recombinant plasmids was performed using XhoI, XbaI , and KpnI . The observed banding patterns were consistent with the predictions generated by SnapGene software. Lanes 1–3 show the predicted digestion patterns of p15A-CmR-D90, p15A-CmR-D90-Δ5a/AmpR-ccdB, and p15A-CmR-D90-Δ5a/EGFP. Lanes 4–5, 6–7, and 8–9 correspond to the experimental digestion results of these respective plasmids. M: TaKaRa 1 kb DNA Ladder. All digested products were resolved by electrophoresis on 1% agarose gels and visualized with ethidium bromide staining. (B) Functional verification of the selection marker gene. The intermediate recombinant plasmid p15A-CmR-D90-Δ5a/AmpR-ccdB was transformed into two E. coli strains: GBred (CcdB-sensitive) and GBred- gyrA462 (CcdB-resistant). On LB agar plates containing ampicillin and chloramphenicol, only GBred- gyrA462 survived, confirming the toxicity of CcdB and functionality of the selection cassette. In the second recombination step, replacement of the AmpR-ccdB cassette by the target Δ5a/EGFP fragment yielded the final recombinant plasmid p15A-CmR-D90-Δ5a/EGFP. Upon counter-selection with CcdB, only host E. coli GBred harboring the correctly recombined plasmid survived on chloramphenicol-containing LB plates, while non-recombinant bacteria were eliminated.

Article Snippet: M: TaKaRa 1 kb DNA Ladder.

Techniques: Biomarker Discovery, Recombinant, Functional Assay, Selection, Generated, Software, Electrophoresis, Staining, Marker, Plasmid Preparation, Transformation Assay, Bacteria

Journal: Virus Research

Article Title: Mycoviruses diversity in the black kōji mold, Aspergillus luchuensis (section Nigri ) isolated from liquor-production environments in Japan

doi: 10.1016/j.virusres.2026.199724

Figure Lengend Snippet: Identification of mycoviruses in A. luchuensis strains. (A) Origin of Aspergillus strains for dsRNA screening and the results of analysis. The number in parentheses indicates the number of dsRNA positive and tested Aspergillus spp. strains (Nanaji and Fujimori, unpublished data, see also the text). The map is provided by the Geospatial Information Authority of Japan ( https://www.gsi.go.jp ). The dsRNA-sequencing strains are shown on the right with an additional Okinawa strain (JCM22320, ). (B) Colony morphologies of selected A. luchuensis strains carrying virus-like dsRNA elements. (C) dsRNA profiles of the selected A. luchuensis strains. Each dsRNA fraction was analyzed by 1.0% agarose gel electrophoresis. Fungal strains subjected to dsRNA-seq analysis are indicated in red. Expected dsRNA bands for an alternavirus (AlAV1) and a partitivirus (AlPV1) genome, as well as AlPV1 satellite-like elements, were shown on the right (see ). The asterisks indicate the expected dsRNA bands for two other partitiviruses, AlPV2 and AlPV3. A 1 kb dsDNA ladder size marker (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific, Waltham, MA, USA) was used as a size standard.

Article Snippet: A 1 kb dsDNA ladder size marker (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific, Waltham, MA, USA) was used as a size standard.

Techniques: Sequencing, Virus, Agarose Gel Electrophoresis, Marker

Journal: The Journal of Biological Chemistry

Article Title: DNA-dependent RNA polymerase incorporates β-D-N4-hydroxycytidine (NHC) linking Molnupiravir to host transcription-dependent mutagenesis

doi: 10.1016/j.jbc.2026.111409

Figure Lengend Snippet: Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and NHC-triphosphate. ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).

Article Snippet: Agarose gel electrophoresis was performed by using a 1 Kb Plus DNA Ladder (Invitrogen, Cat. 10787018) and 6x DNA Loading Dye (ThermoFisher, Cat. R0611) and revealed using ethidium bromide with ChemiDoc Touch Imaging System (Bio-Rad).

Techniques: In Vitro, Sequencing, Plasmid Preparation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Negative Control, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: DNA-dependent RNA polymerase incorporates β-D-N4-hydroxycytidine (NHC) linking Molnupiravir to host transcription-dependent mutagenesis

doi: 10.1016/j.jbc.2026.111409

Figure Lengend Snippet: cDNA synthesis from NHC-MP-containing RNA template by HIV-1 reverse transcriptase (RT) . ( A ) Scheme to summarize the experimental flow to investigate the NHC-mediated mutagenesis in this study. ( B ) cDNA levels in the HIV-1 RT reactions with RNA products synthesized by T7 RNA polymerase at two nucleotide pool conditions, (1) four rNTPs (ATP, GTP, UTP, and CTP) and (2) 3 rNTPs (ATP, GTP, and UTP) with NHC-TP. IVT products in triplicates were reverse transcribed by HIV-1 RT, and the RT products were analyzed by qPCR in duplicates. Mean with SD are represented with bars. ( C ) Agarose gel electrophoresis of one representative amplified DNA product. L: DNA ladder.

Article Snippet: Agarose gel electrophoresis was performed by using a 1 Kb Plus DNA Ladder (Invitrogen, Cat. 10787018) and 6x DNA Loading Dye (ThermoFisher, Cat. R0611) and revealed using ethidium bromide with ChemiDoc Touch Imaging System (Bio-Rad).

Techniques: cDNA Synthesis, Reverse Transcription, Mutagenesis, Synthesized, Agarose Gel Electrophoresis, Amplification