Journal: bioRxiv

Article Title: Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication

doi: 10.1101/2023.01.26.525530

Figure Lengend Snippet: (A) Schematic of the trans -complementation experiment which involved co-transfecting BHK-21 cells with mCherry replicons containing replication-defective 2C or 3B mutations together with a WT ptGFP, ptGFP-3B 3 T>K or ptGFP-3D GNN replicon. Fluorescent protein expression was monitored hourly for 24 hours. The data show (B) ptGFP positive cells per well or (C) mCherry positive cells per well at 8 hours post-transfection (n = 2 ± SD).

Article Snippet: BHK-21 cells obtained from the ATCC (LGC Standard) were maintained in Dulbecco’s modified Eagle’s medium with glutamine (Sigma-Aldrich) supplemented with 10 % FCS, 50 U / mL penicillin and 50 μg / mL streptomycin.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication

doi: 10.1101/2023.01.26.525530

Figure Lengend Snippet: (A) Schematic of the trans-complementation experiment which involved co-transfecting BHK-21 cells with mCherry replicons containing S-fragment deletions together with a WT ptGFP, ptGFP-3B 3 T>K or ptGFP-3D GNN replicon. Fluorescent protein expression was monitored hourly for 24 hours. The data show (B) ptGFP positive cells per well or (C) mCherry positive cells per well at 8 hours post-transfection (n = 2 ± SD).

Article Snippet: BHK-21 cells obtained from the ATCC (LGC Standard) were maintained in Dulbecco’s modified Eagle’s medium with glutamine (Sigma-Aldrich) supplemented with 10 % FCS, 50 U / mL penicillin and 50 μg / mL streptomycin.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication

doi: 10.1101/2023.01.26.525530

Figure Lengend Snippet: BHK-21 cells were co-transfected with BrU-labelled ptGFP replicon RNA together with 3D HA -labelled replicon RNA. At 4 hours post-transfection cells were fixed and 3D HA -BrU RNA complexes detected by proximity ligation assay (PLA) using anti-HA and anti-BrU primary antibodies together with PLA-labelled secondary antibodies. The in-situ PLA signal is detected as foci in the cell cytoplasm (pseudo-coloured magenta). Cell nuclei were stained with DAPI (pseudo-coloured blue). Images were captured on a Zeiss LSM-880 confocal microscope (bar 10 μm).

Article Snippet: BHK-21 cells obtained from the ATCC (LGC Standard) were maintained in Dulbecco’s modified Eagle’s medium with glutamine (Sigma-Aldrich) supplemented with 10 % FCS, 50 U / mL penicillin and 50 μg / mL streptomycin.

Techniques: Transfection, Proximity Ligation Assay, In Situ, Staining, Microscopy

Journal: Applied Microbiology and Biotechnology

Article Title: Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs

doi: 10.1007/s00253-023-12359-w

Figure Lengend Snippet: Confirmation of the genetic stability of the rescued recombinant viruses and their characterization. A Multiple sequence alignment of the VP2 region of WT, WTΔ3A, Y2098F, and Y2098FΔ3A after 12 passages in BHK-21 cells. The presence of phenylalanine (F) at 98th position in place of tyrosine (Y) confirms the presence of mutation in Y2098F and Y2098FΔ3A. B Multiple sequence alignment of the 3A non-structural protein region of WT, WTΔ3A, Y2098F, and Y2098FΔ3A after 12 passages in BHK-21 cells. The presence of deletion at 87–144 amino acids in WTΔ3A and Y2098FΔ3A confirms the deletion. C The titers of the rescued viruses—WT, WTΔ3A, and Y2098FΔ3A in the tissue culture supernatants, determined by TCID 50 analysis and mean titer expressed as Log 10 TCID 50 /ml

Article Snippet: Baby hamster kidney 21 (BHK-21) (clone 13; ATCC, USA, #CCL-10) cell line was maintained in Glasgow Modified Essential Medium (GMEM) (HiMedia, India, #AT1010) supplemented with 10% fetal bovine serum (HiMedia, India, #RM10681) and 60 µg/ml penicillin (Sigma, USA, #P3032), 100 µg/ml streptomycin (Sigma, USA, #S9137), and 100 µg/ml kanamycin (Sigma, USA, #K1377) at 37 °C in 5% CO 2 .

Techniques: Recombinant, Sequencing, Mutagenesis

Journal: Applied Microbiology and Biotechnology

Article Title: Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs

doi: 10.1007/s00253-023-12359-w

Figure Lengend Snippet: Antigenic characterization of the recombinant viruses. A Bright field and immunofluorescence microscopy images (400 × magnification) of BHK-21 cells infected with either WT or rescued negative marker viruses (WTΔ3A and Y2098FΔ3A) showing detectable fluorescence upon probing with 3B-specific mAb, suggesting virus replication. The fluorescence was absent in the mock-infected control cells. Scale bar: 20 µm. B Western blot analysis of lysate from the WT or recombinant virus-infected BHK-21 cells, probed with rabbit polyclonal serum against deleted 3A region, 3B-specific mAb (10H9D8), rabbit sera raised against 146S of FMDV serotype O, and 3D-specific mAb (6B8D11). Lane 1, mock infected; lane 2, WT; lane 3, WTΔ3A; lane 4, Y2098F; lane 5, Y2098FΔ3A

Article Snippet: Baby hamster kidney 21 (BHK-21) (clone 13; ATCC, USA, #CCL-10) cell line was maintained in Glasgow Modified Essential Medium (GMEM) (HiMedia, India, #AT1010) supplemented with 10% fetal bovine serum (HiMedia, India, #RM10681) and 60 µg/ml penicillin (Sigma, USA, #P3032), 100 µg/ml streptomycin (Sigma, USA, #S9137), and 100 µg/ml kanamycin (Sigma, USA, #K1377) at 37 °C in 5% CO 2 .

Techniques: Recombinant, Immunofluorescence, Microscopy, Infection, Marker, Fluorescence, Western Blot

Journal: Applied Microbiology and Biotechnology

Article Title: Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs

doi: 10.1007/s00253-023-12359-w

Figure Lengend Snippet: Growth kinetics of the recombinant viruses. A Plaque morphology of wild-type (WT), WTΔ3A, and Y2098FΔ3A recombinant viruses in BHK-21 cell monolayers. B Multistep growth kinetic study performed by infecting BHK-21 cells at 0.01 MOI with wild-type (WT), WTΔ3A, and Y2098FΔ3A viruses and titers in tissue culture supernatants determined by TCID 50 analysis. The average titers with standard deviations expressed as Log 10 TCID 50 /ml at indicated time points

Article Snippet: Baby hamster kidney 21 (BHK-21) (clone 13; ATCC, USA, #CCL-10) cell line was maintained in Glasgow Modified Essential Medium (GMEM) (HiMedia, India, #AT1010) supplemented with 10% fetal bovine serum (HiMedia, India, #RM10681) and 60 µg/ml penicillin (Sigma, USA, #P3032), 100 µg/ml streptomycin (Sigma, USA, #S9137), and 100 µg/ml kanamycin (Sigma, USA, #K1377) at 37 °C in 5% CO 2 .

Techniques: Recombinant