strain genotype construction details reference kt2440 wild type pseudomonas putida kt2440 n  (ATCC)

Journal: mBio

Article Title: The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

doi: 10.1128/mBio.01289-19

Figure Lengend Snippet: Neutrophils harbor and transfer MCMV ex viv o and in vivo . (A) Experimental design. (B) PECs were evaluated for the presence of GFP-expressing virus. Live GFP-expressing cells were gated for CD11b + cells followed by Ly6-G + gating. (C) Total DNA was extracted from 1 × 10 6 purified neutrophils or Flowthrough cells. qPCR was performed to evaluate the number of MCMV genomes present. (D) Infectious center assay for determining the number of infected neutrophils. A total of 1 × 10 6 purified neutrophils were incubated with an uninfected fibroblast monolayer for 5 days, and the number of plaques was evaluated. (E) MCMV genomes were quantified from whole blood isolated from mice that were adoptively transferred 1 × 10 6 neutrophils from infected mice. Values are average titers ± standard deviations (SD) (error bars). Statistical significance was determined by Student’s t test and indicated as follows: ns, not significant; ***, P ≤ 0.001.

Article Snippet: Illumina sequencing reads (150 × 150 paired-end sequencing reads) were mapped to the parental MCMV C5X reference genome (wild-type [WT] Smith strain).

Techniques: In Vivo, Expressing, Virus, Purification, Infection, Incubation, Isolation

Journal: mBio

Article Title: The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

doi: 10.1128/mBio.01289-19

Figure Lengend Snippet: CXCR2 stimulation increases MCMV dissemination. (A) MCMV was administered i.p. during peak neutrophil response induced with IL-17A or vehicle control (not treated). (B and C) Viral titers of whole-organ homogenates were determined in the spleen (B) and lungs (C) of mice treated with IL-17A 5 days postinfection. n = 3 with 4 or 5 mice per group. (D) The salivary glands (SGs) of mice were harvested 5 days postinfection (dpi). Total DNA was harvested and subjected to qPCR for total MCMV burden in the SG. (E) Depletion of neutrophils reduces IL-17A-enhanced MCMV dissemination to the spleen. Plaque assays of spleens of mice 5 dpi. that received phosphate-buffered saline (PBS; i.e., vehicle), IL-17A alone, neutrophil-depleting antibody (anti-Ly6G [αLy6G]) coupled with IL-17A treatment (IL-17A & αLy6G), or neutrophil-depleting antibody alone prior to infection with MCMV. Data are from two pooled experiments. Data are normalized to the mean PFU/gram of tissue of the PBS control for each experiment. Values are average titers ± SD. Statistical significance was determined by Student’s t test or one-way ANOVA with Tukey’s multiple comparison of means and indicated as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: Illumina sequencing reads (150 × 150 paired-end sequencing reads) were mapped to the parental MCMV C5X reference genome (wild-type [WT] Smith strain).

Techniques: Control, Saline, Infection, Comparison

Journal: mBio

Article Title: The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

doi: 10.1128/mBio.01289-19

Figure Lengend Snippet: Generation of recombinant MCMV expressing vCXCL-1 Tol . (A) Bacterial artificial chromosome (BAC) schematic of vCXCL-1 Tol insertion into the mck2 locus. (B) HindIII RFLP analysis of the BACs. The triplet banding patterns are boxed in red. (C) Western blot analysis of the viral supernatant of MCK2-2A-vCXCL-1 Tol MCMV. (D) Single-step growth curve (MOI of 5.0). DPI, day postinfection. (E) Multistep growth curves (MOI of 0.05). Values are average titers ± SD (error bars).

Article Snippet: Illumina sequencing reads (150 × 150 paired-end sequencing reads) were mapped to the parental MCMV C5X reference genome (wild-type [WT] Smith strain).

Techniques: Recombinant, Expressing, Western Blot

Journal: mBio

Article Title: The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

doi: 10.1128/mBio.01289-19

Figure Lengend Snippet: vCXCL-1 Tol increases virulence of MCMV in NSG mice which correlates with increased viral loads in the spleen. (A) Kaplan-Meier survival curve comparing NSG mice infected with either RMvCXCL-1 Tol or Smith MCMV. (B and C) At the termination of the experiment (12 days postinfection), MCMV titers in the spleen (B) or SG (C) were determined. Bars represent the average titers ± SD (error bars). Each symbol shows the viral titer for an individual mouse. Data are from three separate experiments with four to six mice per group for each virus. Statistical significance was ascertained using the Student’s t test and indicated as follows: ns, not significant; ****, P ≤ 0.0001.

Article Snippet: Illumina sequencing reads (150 × 150 paired-end sequencing reads) were mapped to the parental MCMV C5X reference genome (wild-type [WT] Smith strain).

Techniques: Infection, Virus