Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a A schematic diagram of transformation of CRISPR-Cas13a and CRISPR-Cas9 with targeting bla IMP-1 into bla IMP-1 -expressing E. coli STBL3. b E. coli STBL3 expressing bla IMP-1 from a plasmid (plasmid-borne bla IMP-1 ) and chromosome (chromosome-borne bla IMP-1 ) were prepared, and transformed with CRISPR-Cas13a or CRISPR-Cas9, both with spacer targeting bla IMP-1 or no spacer (nontargeting). The resulting transformants were plated on an LB plate containing kanamycin (Km) to test sequence-specific bacterial killing by CRISPR-Cas13a and CRISPR-Cas9. Km was used to maintain the plasmids. c The number of bacteria on the plate obtained in the experiment of b was counted. The statistical significance was determined by two-sided Student’s t -test. Each bar represents the mean with standard deviation ( n = 3). d A schematic diagram of the experiment to test CRISPR-Cas13a-dependent cell growth inhibition. Anhydrotetracycline (aTc)-inducible bla IMP-1 - or rfp -expression plasmid (pKLC56) was co-transformed with pKLC21 plasmid expressing CRISPR-Cas13a with spacer targeting bla IMP-1 or rfp into E. coli STBL3. The resulting transformants were then cultured and the aTc induction in the presence of the antibiotics Km and Cm (Km and Cm for maintaining plasmids) was carried out at the indicated time points. Thereafter, the OD values were measured every hour. e Growth curves were plotted. Each line of the growth curves represents the mean with standard deviation. f The number of viable cells was counted to calculate the ratio of cell death caused by bla IMP-1 -targeting CRISPR-Cas13a. The bacterial culture prepared in d was diluted to 1/100, then aTc was added to induce bla IMP-1 . Km and Cm were also added to maintain the CRISPR-Cas13a and the aTc-inducible bla IMP-1 plasmids. Thereafter, the bacterial cultures were sampled at the indicated time points, and the number of surviving bacteria was counted on fresh LB plates. Each bar represents the mean with standard deviation of four biological replicates. p -values were determined by two-sided Student’s t -test. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Transformation Assay, CRISPR, Expressing, Plasmid Preparation, Sequencing, Standard Deviation, Inhibition, Cell Culture

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a Graphical concept model depicting the mode of action of CapsidCas13a, with bla IMP-1 -targeting CRISPR-Cas13a packaged into E. coli M13 phage capsid (EC-CapsidCas13a- bla IMP-1 ) for delivery into host bacterial cells during the normal course of viral infection. In the presence of the target gene ( E. coli carrying bla IMP-1 ), CRISPR-Cas13a will be activated and the subsequent non-sequence-specific RNase activity will result in host cell death. In the absence of the target gene ( E. coli without bla IMP-1 ), CRISPR-Cas13a will not be activated and there will be no subsequent cell death. b – d Spot assay with generated CapsidCas13a(s) on bacterial lawn of E. coli NEB5α F′ I q to test bactericidal activity. A clear lysis zone of the spotted area represents bacterial killing. NEB5α F′ I q carrying bla IMP-1 expression vector and empty vector (control) were infected with tenfold serial dilutions of EC-CapsidCas13a- bla IMP-1 in the presence or absence of L-arabinose for inducing bla IMP-1 expression ( b ); CapsidCas13a(s) programmed to target different carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , bla VIM-2 , bla NDM-1 , and bla KPC-2 ) and colistin resistance genes ( mcr-1 and mcr-2 ) were spotted on a series of NEB5α F′ I q strains expressing each of the resistance genes ( c ); comparison of bactericidal activity between CRISPR-Cas9 and CRISPR-Cas13a. Nontargeting and bla NDM-1 -targeting CapsidCas13a and CapsidCas9, in tenfold serial dilutions, were spotted on lawn of NEB5α F′ I q with or without expression of bla NDM-1 on plasmid or chromosome ( d ). All assays were replicated three times.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: CRISPR, Infection, Sequencing, Activity Assay, Spot Test, Generated, Lysis, Expressing, Plasmid Preparation, Bla VIM Assay

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: Programmed CapsidCas13a altered the composition of bacterial population. A mixed cell population was prepared by mixing E. coli NEB5α F′ I q (control) with equal numbers of NEB5α F′ I q expressing bla IMP-1 and mcr-2 , respectively. The cell mixtures were then independently treated with bla IMP-1 -targeting, mcr-2 -targeting, and nontargeting CapsidCas13a. Note that each AMR gene-targeting CapsidCas13a reduced the corresponding target cell population. The percentage represents the mean of three biological replicates. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Expressing

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: Examination of the therapeutic effect of EC-CapsidCas13a- bla IMP-1 using a Galleria mellonella infection model. Administration of EC-CapsidCas13a- bla IMP-1 (MOI 100) into G. mellonella larvae infected with R10-61 (carbapenem-resistant clinical isolates of E. coli carrying bla IMP-1 ) significantly improved host survival compared to controls, EC-CapsidCas13a-nontargeting ( p = 0.044), and phosphate-buffered saline (PBS; p = 0.0016). The p -value between two groups infected with R10-61, and treated with EC-CapsidCas13a-nontargeting and PBS was 0.30. The p -values are calculated by log-rank test. The results are presented as the aggregate values of three independent experiments performed using ten larvae per group. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Infection

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a A schematic illustration of generation of PICI-based EC-CapsidCas13a targeting bla IMP-1 . Mitomycin C induction promotes the packaging of PICImid carrying a CRISPR-Cas13a system and kanamycin (Km) resistance gene into the capsid of helper phage Φ80. b – d Bactericidal activity test of PICI-based EC-CapsidCas13a::KanR- bla IMP-1 (in tenfold serial dilutions) against E. coli MC1061 with or without the expression of target gene was carried out on LB agar plates ( b ); and the test results were judged by observing bacterial growth on LB bottom agar plates supplemented with Km ( c ), or observation of cell lysis on drug-free LB bottom agar plates ( d ); noting that the former assay had an enhanced sensitivity by about three orders of magnitude against the bacteria carrying target gene. e – j The PICI-based EC-CapsidCas13a(s) were applicable to detect various carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , and bla VIM-2 ) regardless of their location on either the plasmid or chromosome ( e , f ), whereas EC-CapsidCas9 could detect genes located on the chromosome but not on the plasmid ( g ); and the PICI-based EC-CapsidCas13a(s) also effectively detected toxin-encoded genes ( h ), differentiated different genes located on the same plasmid ( i ), and were also applicable to clinical isolates ( j , left panel) as being verified by PCR ( j , right panel). k SA-CapsidCas13a::TetR- mecA generated by packaging mecA -targeting CRISPR-Cas13a into capsid of S. aureus phage 80α exhibited mecA -specific bactericidal activity against MRSA, but not S. aureus strains deficient in mecA . All assays were replicated three times. Uncropped images of the gels are available in the Supplementary Information.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: CRISPR, Activity Assay, Expressing, Lysis, Bla VIM Assay, Plasmid Preparation, Generated

Journal: The Journal of Infectious Diseases

Article Title: The Global Regulon sarA Regulates β-Lactam Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus In Vitro and in Endovascular Infections

doi: 10.1093/infdis/jiw386

Figure Lengend Snippet: Oxacillin minimum inhibitory concentrations (MICs) determined by the Etest (A), relative expression of mecA in the absence (B), and the presence of half MICs of oxacillin (C) and penicillin-binding protein 2a (PBP2a) agglutination (D) of study methicillin-resistant Staphylococcus aureus parental and sarA mutant strain sets. B, Relative transcription levels of mecA represent the mean (+SD) of 3 biological replicates in vitro. The fold changes represent the normalized mecA expression (2−ΔCt) to the housekeeping gene gyrB. *P < .05, compared with their respective parental strains. Open bars, parental strain; shaded bars, sarA mutant. D, mecA-positive (ATCC 43300) and mecA-negative (ATCC 25923) controls. Interpretation of agglutination intensity: +++, high; ++, moderate; +, low; and −, none.

Article Snippet: Staphylococcus aureus Strains and Plasmids Used in This Study Determination of Oxacillin Minimum Inhibitory Concentrations (MICs) The oxacillin MICs of study MRSA strain sets were determined by using both the standard Etest method (BioMerieux, La Balme-les-Grottes, France), according to the manufacturer's recommended protocols, and the standard broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute [ 23 ].

Techniques: Expressing, Binding Assay, Agglutination, Mutagenesis, In Vitro

Journal: The Journal of Infectious Diseases

Article Title: The Global Regulon sarA Regulates β-Lactam Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus In Vitro and in Endovascular Infections

doi: 10.1093/infdis/jiw386

Figure Lengend Snippet: Oxacillin minimum inhibitory concentrations determined by the Etest (A) and biofilm formation (B) of the methicillin-resistant Staphylococcus aureus JE2 parental strain, its sarA mutant, mecA mutant, ΔsarA/psarA (sarA complemented in the sarA mutant strain), and ΔsarA/pmecA (mecA complemented in the sarA mutant strain). Results of biofilm formation are shown as the mean of the OD490nm (+SD) from 3 biological replicates, each of which was done in triplicate. *P < .05, for the strains, compared with the JE2 parental strain.

Article Snippet: Staphylococcus aureus Strains and Plasmids Used in This Study Determination of Oxacillin Minimum Inhibitory Concentrations (MICs) The oxacillin MICs of study MRSA strain sets were determined by using both the standard Etest method (BioMerieux, La Balme-les-Grottes, France), according to the manufacturer's recommended protocols, and the standard broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute [ 23 ].

Techniques: Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: Structure and analysis of E. coli O157 LPS molecules. ( A ) Schematic representation of E. coli O157 LPS molecule, showing O-antigen, outer core, inner core, and lipid A. Dotted lines indicate the level of LPS truncation resulting from each mutation (Kdo, 3-deoxy-D-manno-octulosonic acid; PPEtN, pyrophosphorylethanolamine; Hep, heptose; GlcNAc, N-acetylglucosamine; Glc, glucose; Gal, galactose). ( B ) LPS was isolated from wild-type and mutant cells of E. coli O157 43895 and analyzed by silver-stained Tricine–SDS-PAGE, as described in the Materials and Methods section. The expected location of O-antigen regions and the core is indicated on the left side of the gel.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Mutagenesis, Isolation, Staining, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: Biofilm formation of E. coli O157 43895 WT and mutant derivatives. ( A ) Pellicle formation at 37 °C. Pellicle rings of a 48 h old floating biofilm of E. coli O157 43895. Mutants were detected by crystal violet (CV) staining. ( B ) Biofilm measurement using a standard microtiter assay. E. coli O157 43895 WT and mutants were incubated at 37 °C in MSM with 4% glucose. Bound CV was solubilized with 95% ethanol and quantified by absorbance at 595 nm. Assays were performed six times for each strain. Mean values with standard deviation are shown. Statistical analysis used one-way ANOVA test and Dunnett post-test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Mutagenesis, Staining, Incubation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS-core truncation on the swimming motility and flagella biosynthesis of E. coli O157 43895. Swimming motility of wild-type and LPS mutants was assessed using soft-agar swarm plates. After 40 h incubation, the diameter of the motility halo was measured. Experiments were repeated three times. Presence of flagella was detected by immunoblot (lower panel). Mean values with standard deviation are shown. Statistical analysis used two-tailed unpaired t -test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Incubation, Western Blot, Standard Deviation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS-core truncation on the curli biosynthesis of E. coli O157 43895. E. coli O157 43895 and mutants were grown on Congo-red (CR) indicator plates at 37 °C for 24 h. Presence of curli on the cell surface was detected by CR binding.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS core-OS truncation on cell adherence and invasion. ( A ) The adherence of LPS-core and flagella mutants of E. coli O157 43895 to epithelial cells was examined. The monolayers of MAC-T cells on coverslips in 24-well plates were co-cultured with the bacteria at 37 °C for 3 h. The cells were washed five times with PBS and fixed with 4% paraformaldehyde for 15 min, washed again with PBS, stained with Giemsa stain, and viewed under the microscope (magnification ×1000). ( B ) E. coli O157 43895 and the mutants were tested for epithelial-cell invasion using a MAC-T gentamicin protection assay, as described in the Materials and Methods section. Bacteria were co-cultured with MAC-T monolayers at a multiplicity of infection (MOI) of 10. The experiment was repeated three times with three replicates per strain, and CFUs were determined by plate count and were transformed to log10 value. Mean values with standard deviation are shown. Statistical analysis used one-way ANOVA test and Dunnett post-test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Cell Culture, Bacteria, Staining, Giemsa Stain, Microscopy, Infection, Transformation Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: E. coli O157 strains and plasmids.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000, 1,500, or 150 copies of IAV (H1N1), and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later. Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, * p < 0.05. ( c ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV (H1N1), and intracellular vRNA levels were measured with RT-qPCR 2 days later. The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. ** p < 0.01. ( d ) PK-15 ( Stat2 k/o)/TMPRSS2 #15, PK-15 ( Stat2 k/o), PK-15/TMPRSS2 #23, or normal PK-15 cells treated with one ng/mL pig IFNβ or left untreated for 24 hr and then infected with 15,000 copies of IAV (H1N1). vRNA levels in the culture supernatant were measured with RT-qPCR 2 days after infection. The relative value was calculated according to the values in normal PK-15 cells without pig IFNβ treatment. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 80,000, 8,000, or 800 copies of SIV (H1N1). and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a Mann–Whitney U -test. ** p < 0.01.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or VeroE6/TMPRSS2 cells were infected with 75,000 copies of IAV(H1N1), and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. The relative value was calculated according to the values in normal PK-15 cells. Results are the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, *** p < 0.001.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV(H1N1) in the presence of different concentrations of nafamostat mesylate, and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of measurements (n - 6) from one assay and are representative of at least three independent experiments. ( b ) The EC 50 of nafamostat mesylate against IAV (H1N1) in PK-15/TMPRSS2 #23 cells was calculated using Prism.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation

Journal: Archives of virology

Article Title: Natural antisense transcripts of UL123 packaged in human cytomegalovirus virions

doi: 10.1007/s00705-013-1793-5

Figure Lengend Snippet: HCMV antisense RNAs are packaged into virions. (A) Schematic diagram of the structure of sense and antisense transcripts of the UL123 region in laboratory strain Towne. The upper diagram shows the UL123 sense transcript, starting from the PSS in productive infection, encoding the initial and most abundant viral protein, referred to as IE1 (also named IE72). The lower diagram shows a summary of UL123ast in previously reported GM-Ps latently infected with AD169 (originating from nt 171,867) and novel UL123ast observed in HELs productively infected with Towne (originating from nt 171,870). (B) Sense-antisense RNA levels of the viral genes UL21.5, UL123 and the cellular gene GAPDH in virions. The y-axis represents the log10-transformed copy number of each RNA. (C) The virion protein pp65 was used as an indicator of HCMV entry by IFA assay. Cells on cover slips were stained with monoclonal antibodies to pp65 (left) and the nuclear dye Hoechst (right). (D) Sense-antisense RNAs levels of UL21.5 and UL123, either in the presence or absence of ActD by real time RT-PCR assay. The average Ct values, which are inversely proportional to the amount of the target nucleic acid in the samples, were compared for each RNA between these two groups. Data were analyzed using Student’s unpaired t-test (one-tailed) and are presented as mean ± standard deviation (SD) (n = 2). Values of *p<0.05 were considered statistically significant

Article Snippet: A confluent monolayer of human embryonic lung fibroblast cells (HELs) was infected with HCMV strain Towne (ATCC VR-977) at an MOI of 2.

Techniques: Infection, Transformation Assay, Staining, Bioprocessing, Quantitative RT-PCR, One-tailed Test, Standard Deviation

Journal: Archives of virology

Article Title: Natural antisense transcripts of UL123 packaged in human cytomegalovirus virions

doi: 10.1007/s00705-013-1793-5

Figure Lengend Snippet: Determination of TSSs by 5′RACE analysis of UL123ast in Towne-infected HEL cells

Article Snippet: A confluent monolayer of human embryonic lung fibroblast cells (HELs) was infected with HCMV strain Towne (ATCC VR-977) at an MOI of 2.

Techniques: Sequencing, Clone Assay

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: Four cynomolgus macaques were challenged with MPXV IIb virus on day 0 with 1 × 10 7 pfu of MPXV by the rectal route. Two animals were euthanized on day 11 and two were followed for 83 days for clinical and immunological monitoring ( a ). Five clinical signs were monitored for 34 days ( n = 2): the presence of rectal exudate, loss of appetite, presence of lesions, lymphadenomegaly, and weight loss >10% were recorded for each animal. b Viral load assessment in rectal and seminal fluids ( n = 4 for rectal fluid and n = 2 for seminal plasma). MPXV DNA was quantified by qPCR and is expressed as viral DNA copies/mL. The lower limit of detection (LLOD) was 90,000 copies/mL, and the lower limit of quantification (LLOQ) was 100,000 copies/mL. c The rectal and seminal fluid samples containing viral DNA were tested for virus infectivity in Vero cells. Viral infectivity is quantified in pfu/mL. The LLOD was established at 10 PFU/mL, indicated by the dotted line. All samples below this LLOD were considered negative and are represented by a value of 1. d Orthopoxvirus-binding IgG concentrations are reported in arbitrary units (arb. unit./mL). Serum (left) and seminal plasma (right) from two NHPs ( n = 2) were evaluated for the total anti-MPXV protein IgG concentration before challenge and at various time points up to days 62 and 83 post-challenge for serum and seminal plasma, respectively. Each symbol represents one animal, and the different shapes represent different antigens. The dashes represent the mean. Ag: antigen. Source data are provided as a Source Data file.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Virus, Clinical Proteomics, Infection, Binding Assay, Concentration Assay

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: Representative Hematoxylin-Eosin staining of the rectal mucosa on day 11 post-challenge from a cynomolgus macaque infected with MPXV. The analysis was performed on 2 animals. a Ulcerated rectal mucosa at the margin of the anus showing ulcerative, necrotizing rectitis (*) characterized by b hyperplastic mucosa (arrowhead) and c fibrin and extravasated erythrocytes (hemorrhage) (arrow head). d rectal submucosa, with numerous inflammatory cells (arrow head), mostly e neutrophils (calprotectin staining), f T cells (CD3 staining), g follicular-like B-cell aggregates (CD20 staining), and h minimal diffuse macrophage infiltration (CD68 staining). i RNA scope using a specific MPXV probe in the submucosal ulcerated tissue and adjacent epithelium (purple staining localized mostly in the ulcerated area). j RNAscope with the negative control probe DapB. Scale-bars: a = 500 µm, b – j = 100 µm.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Staining, Infection, RNAscope, Negative Control

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: a Study design on NHP immunization and challenge periods in weeks, and a description of the four groups of cynomolgus macaques. One group was vaccinated before the challenge with two doses of MVA administered 4 weeks apart (MVA-PrEP, n = 4; gray). Another group was vaccinated with a single dose four days after the MPXV IIb challenge (MVA-PEP, n = 4; black). In addition, we challenged one group of convalescent NHPs previously exposed via the rectal or ID route alone or in combination 37–46 weeks before the challenge and described previously (Conv., n = 6; orange) and one control group (CTRL, n = 4; red). The four groups were challenged with 1 × 10 7 pfu of MPXV by the rectal route as indicated in blue. MVA-PrEP animals were monitored during the MVA immunization phase, with blood samplings for immunogenicity analysis. Following the MPXV challenge, blood, rectal fluid, seminal plasma, and skin swab samples were collected. The blue vertical dotted line represents the challenge. The image was created in BioRender. Herate, C. (2025) https://BioRender.com/lkzvios . b Rectal fluids collected during the challenge phase were evaluated for viral DNA content by qPCR. Viral load is expressed as viral DNA copies/mL. Each graph represents the viral kinetics of one group during 27 days. The LLOD was 36,000 copies/mL and the LLOQ 100,000 copies/mL. c Rectal fluid containing viral DNA was tested for virus infectivity in Vero cells and measured in pfu/mL. The LLOD was established at 10 PFU/mL, indicated by the dotted line. All samples below the LLOD were considered negative and are represented by a value of 1. When qPCR was negative, samples were considered negative and are represented by a value of 1. d Area under the curve (AUC) of viral DNA in rectal fluids during the 27 days of follow-up and statistical analysis. (CTRL, n = 4; red), (MVA-PEP, n = 4; black) (MVA-PrEP, n = 4; gray) (Conv., n = 6; orange). **: p = 0.0079. Each circle represents one animal. e Viral load measured by qPCR in skin swabs (left) and seminal plasma (right) sampled at day 11 and statistical analysis. The mean value is represented by the horizontal bar. (CTRL, n = 4 for skin and seminal fluid; red), (MVA-PEP, n = 3 for skin and n = 4 for seminal fluid; black) (MVA-PrEP, n = 3 for skin and n = 4 for seminal fluid; gray) (Conv., n = 5 for skin and n = 6 for seminal fluid; orange). *: p = 0.0333. Each circle represents one animal. Statistical analyses were performed using the Kruskal-Wallis test followed by a two-tailed non-parametric Mann-Whitney test (* p < 0.05, ** p < 0.01). Source data are provided as a Source Data file.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Control, Immunopeptidomics, Clinical Proteomics, Virus, Infection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: a The concentrations of five MPXV (upper panel) and homologous VACV (lower panel) protein-binding IgG were quantified in the serum of NHPs that were either immunized with MVA and/or challenged with MPXV. The data are expressed in arbitrary units (arb. unit./mL). Total IgG concentrations in NHP serum were monitored during the immunization phase for the MVA-PrEP group and after the challenge phase. For the remaining three groups, IgG follow-up began two weeks prior to the challenge. b Seroneutralization titers were measured in the serum collected at week -1 and week 4 post-challenge using Vero cells and live MPXV or VACV. The mean PRNT50 with standard deviation were calculated using infectious MPXV IIb (left panel): *: p = 0.0159, MPXV Ia (middle panel): *: p = 0.0159 or VACV-107 (right panel): *: p = 0.0190 and **: p = 0.0095. (CTRL, n = 4; red), (MVA-PEP, n = 4; black), (MVA-PrEP, n = 4; gray), (Conv., n = 6 at W-1 and n = 5 at W4; orange). c , d Cellular immunity conferred by MVA vaccination and/or MPXV challenge. (CTRL, n = 4; red), (MVA-PEP, n = 4; black) (MVA-PrEP, n = 4; gray) (Conv., n = 6 and n = 5 from W14; orange). Intracellular staining of PBMCs, collected during the MVA immunization phase and after MPXV challenge, after an overnight MPXV or MVA stimulation in vitro. Panels represent the mean percentage with standard deviation of CD4 + CD154 + cells expressing IFN-γ ( c ) and IL17A ( d ) upon MPXV 2b (left) or MVA (right) stimulation. For ( b – d ), each circle represents one animal. When represented, statistical analyses were performed using the Kruskal-Wallis test followed by a two-tailed non-parametric Mann-Whitney test (* p < 0.05, ** p < 0.01). Ag: antigen; Stim. Stimulation. Source data are provided as a Source Data file.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Protein Binding, Standard Deviation, Staining, In Vitro, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: CD4+ and CD8+ cells producing TNF-α, IL-2, and IFN-γ are represented for each animal (one pie chart per animal) at W12 post-immunization (W−1 post-challenge) and W15 post-immunization (W2 post-challenge). The polyfunctionality of CD4 + CD154 + (left) and CD8 + CD137 + (right) cells upon MPXV IIb stimulation is shown. Non-responder cells are not represented. Cells secreting only one of the three cytokines are in blue, two of them in green, and three in orange. Source data are provided as a Source Data file.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques:

Journal: Nature Communications

Article Title: Efficacy of modified-vaccinia Ankara vaccine as pre- and post-exposure prophylaxis against monkeypox sexual transmission in non-human primate model

doi: 10.1038/s41467-025-62681-2

Figure Lengend Snippet: Correlation matrix between virological parameters measured after the MPXV challenge and humoral and cellular parameters measured 1 week before the challenge. Spearman's r values from −0.7 to −1 and from 0.7 to 1 are shown. Fl.: fluid.

Article Snippet: Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039 ) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO 2 atmosphere.

Techniques: