Journal: Biotechnology and Bioengineering

Article Title: Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

doi: 10.1002/bit.27200

Figure Lengend Snippet: Characterization of expressed type‐C endogenous retrovirus (ERV) sequences in parental CHO‐K1 cells. (a) Illumina sequencing reads of the total cellular mRNA (UT mRNA, dark colors), or of the viral genomic RNA extracted from viral particles (UT VP, light colors) obtained from CHO‐K1 cell cultures, were mapped on group 1, 2, and 3 type‐C ERV sequences. Reads were mapped to a consensus of the possibly expressed group 1 sequences and on two distinct loci for group 2 (ETC386F) and group 3 (ETC506F) sequences. The ERV sequence sizes are represented by the x ‐axis in base pairs. The lines under the schematic representation of group 2 and 3 ERVs loci indicate probable loss of function mutations occurring in these ERV sequences, blue for frameshift mutations, red for stop codon mutations and gray for deletions, with the deletion size indicated in base pairs. (b) Quantification of the average number of reads per kb of proviral sequence that mapped to the three different ERVs presented in panel A, from a total of 25 Gb and 208 Mb of mappable genomic sequences for cellular mRNA and VP RNA, respectively. (c) Confocal pictures of interphase CHO‐K1 cells subjected to DNA‐FISH (left pictures) or RNA‐FISH (right pictures), using a probe specifically targeting group 1 type‐C ERV (top pictures) or a nonspecific negative control probe (bottom pictures). Pictures were pseudocolored for visualization purposes, chromosomal DNA being represented in red and the DNA fluorescence in situ hybridization (FISH) signals of integrated retroviral sequences shown as green dots. For the RNA‐FISH, DNA staining is shown in blue, whereas ERV type‐C group 1 RNA signals are shown in red. The bright purple dot represents the nascent group 1 RNA signal at the transcription locus. Complete FISH analysis is presented in Figure S1 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To do so, the viral genomic RNA was extracted from the VPs released in the cell culture supernatants, followed by Illumina sequencing analysis and mapping of the reads to their corresponding ERV DNA sequences.

Techniques: Illumina Sequencing, Sequencing, Genomic Sequencing, Negative Control, Fluorescence, In Situ Hybridization, Retroviral, Staining

Journal: Cells

Article Title: The Envelope Residues E152/156/158 of Zika Virus Influence the Early Stages of Virus Infection in Human Cells

doi: 10.3390/cells8111444

Figure Lengend Snippet: Table showing particle-to-PFU ratios. Viral RNA extracted from viral stock P2 were subjected to quantification by RT-qPCR using E primers. Obtained Ct values were plotted in a standard curve (serial dilutions of plasmid copies) in order to get the number of viral RNA molecules per mL. These results were compared to viral stock quantifications by standard plaque-forming assay, which then gave the particle-to-PFU ratios, also named vRNA-to-PFU ratios. Values represent means and standard errors of two to four independent experiments.

Article Snippet: Total RNA including genomic viral RNA was extracted from cells (Qiagen, Hilden, Germany) and reverse transcription was performed using 500 ng of total RNA, random hexamer primers (intracellular viral RNA) or E reverse primer (virus particles) and moloney mouse leukemia virus reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 °C for 50 min. Quantitative PCR was performed on a CFX96 qPCR System (Bio-Rad, Hercules, CA, USA).

Techniques: Plasmid Preparation

Journal: Cells

Article Title: The Envelope Residues E152/156/158 of Zika Virus Influence the Early Stages of Virus Infection in Human Cells

doi: 10.3390/cells8111444

Figure Lengend Snippet: Viral fusion in A549-Dual™ cells. Pre-chilled cells were incubated at 4 °C with ZIKV at MOI of 1. After 1-h incubation, cells were shifted to 37 °C. Chloroquine was then added to the culture medium. Viral RNA was measured by RT-qPCR 30 h at 37 °C. Error bars represent standard errors of two independent experiments. *: p value < 0.1

Article Snippet: Total RNA including genomic viral RNA was extracted from cells (Qiagen, Hilden, Germany) and reverse transcription was performed using 500 ng of total RNA, random hexamer primers (intracellular viral RNA) or E reverse primer (virus particles) and moloney mouse leukemia virus reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 °C for 50 min. Quantitative PCR was performed on a CFX96 qPCR System (Bio-Rad, Hercules, CA, USA).

Techniques: Incubation, Quantitative RT-PCR