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: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: PNA probe hybridization assay for specific detection of uropathogens. A) We have designed PNA probes specific to the i) E. coli and ii) P. mirabilis species, the iii) Enterobacterales order, and the iv) eubacterial kingdom, in order to be able to detect all predominant uropathogens. We ensure that the designed probes can detect signal from target bacteria over blank urine background (N) by measuring probe fluorescence in the presence of E. coli (EC), P. mirabilis (PM), K. pneumoniae (KP), and P. aeruginosa (PA) ( p ‐values are calculated using unpaired one‐tailed t ‐tests; * p < 0.05, ** p < 0.01, *** p < 0.001, no asterisks between bars indicates no significant difference). B) Our assay works across a wide range of i) lysis temperatures and ii) hybridization temperatures (green: selected temperatures). C) Bulk‐based pheno‐molecular AST of reference E. coli ATCC 25922 and multi‐drug resistant E. coli BAA 2471 using hybridization detection of 16S rRNA is feasible, but requires >90 min of culture/antibiotic exposure to differentiate the effect of gentamicin on the susceptible and the resistant strains of E. coli . Data presented as mean +/− SD, n ≥ 3 or n ≥ 2 (bulk pheno‐molecular AST).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Hybridization, Bacteria, Fluorescence, One-tailed Test, Lysis

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Single‐cell detection of bacterial 16S rRNA from urine using microfluidic droplets. A) i) Urine samples of distinctly different color and turbidity can be discretized using flow‐focusing to generate monodisperse droplets (scale bars ≈50 µm) of ii) 4 ± 1 pL volume. B) Droplet fluorescence peak traces i) without E. coli , droplets emit baseline fluorescence signal, and have a positive droplet rate of 0.0079% (also known as the average limit of blank). ii) In the presence of 10 7 CFU mL −1 E. coli , droplets emit a higher fluorescence signal, and have a positive droplet rate of 6.67%. C) Droplet‐based quantification of E. coli in urine across four orders of magnitude within the clinically relevant dynamic range for UTIs (10 4 to 2 × 10 7 CFU mL −1 ), R 2 = 0.992 D) i) Reduction in droplet volume from 30 to 4 to 1 pL results in lower background fluorescence signals (scale bars in white ≈100 µm). ii) Compared to larger 30 pL droplets, 4 pL droplets facilitate faster generation of differentiable fluorescence signal over the reduced local background, as quickly as within 15 min. Data in (C,D(i)) presented as mean +/− SD, n ≥ 2 except for 2 × 10 7 CFU mL −1 input bacterial concentration in (C).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Fluorescence, Concentration Assay

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Accelerating antimicrobial susceptibility assessment via quantitative measurement of 16S rRNA from single cells. A) LIF detection of droplets containing E. coli cells suspended in MH broth i) without 30 min culture results in the expected 8% positive droplet frequency (7.02% observed), ii) following 30 min culture results in higher positive droplet intensities (indicative of higher 16S rRNA production) and 7.70% frequency, and iii) and after 30 min culture along with bactericidal gentamicin results in lower positive droplet intensities (indicative of relatively lower 16S rRNA production) and 3.12% frequency. B) Resistant E. coli can be differentiated from reference E. coli spiked into urine by comparing the positive droplet percentage from cells subject to antibiotic and no‐antibiotic conditions (“Normalized Positive Droplet Population”) for culture/drug exposure durations as low as 10 min. C) Resistant and susceptible strains of E. coli can be differentiated using our platform for three different antibiotics spanning distinct classes—gentamicin (aminoglycoside), ciprofloxacin (fluoroquinolone), and ampicillin (beta lactam). Error bars represent 1 standard deviation. The p ‐values are calculated from unpaired one‐tailed t ‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Standard Deviation, One-tailed Test

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: DropDx clinical comparison study of 50 deidentified patient samples from Johns Hopkins Hospital. A) Each sample was simultaneously tested using clinical standard ID/AST tests as well as with 2 DropDx devices for measurements without and with ciprofloxacin. For ID, we used a combination of EC, EB, and UNI probes. B) Our 7‐outcome DropDx workflow is used to determine if there is a Gram‐negative bacterial infection present, whether the infecting pathogen is E. coli , whether the infecting pathogen is in the Enterobacterales order, or whether the infecting pathogen is a different (Gram‐negative) bacteria and to assess the susceptibility of the infecting pathogen to ciprofloxacin. λ is the proportion of droplets that contain a single cell to all droplets. C) Unbiased thresholding for each diagnostic metric was conducted in pilot studies using ROC curve analysis, and the final data groups and resulting ROC curves are plotted for i) differentiating culture‐positive from culture‐negative samples (AUC: 0.964), for ii) differentiating E. coli from non‐ E. coli samples (AUC: 1.000), for iii) differentiating Enterobacterales from non‐ Enterobacterales samples (AUC: 0.956), and for D) differentiating ciprofloxacin resistant from susceptible samples (AUC: 0.951). Importantly, DropDx's single‐cell pheno‐molecular AST results in a categorical agreement of 95.3% with no major errors. Error bars represent mean and standard error. The p ‐values are calculated from unpaired one‐tailed t‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Comparison, Infection, Bacteria, Diagnostic Assay, One-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: 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: