Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: Schematic of HAdV-3p E3-9K mutant virus genotypes. Mutant viruses were generated by recombination of bacmid pKSB2Ad3wt with pE3-9K shuttle plasmids carrying the desired mutations of ORF E3-9K, digestion of bacmid with Mlu I to release the mutated HAdV-3 genome, transfection into A549 cells, and propagation of resultant mutant virus as described in the Materials and Methods section. Recombinants contain Sal I and Bam HI restriction enzyme sites and a flippase recognition target (FRT) scar site. The HAdV-3p-WT control virus was generated by linearization of pKSB2Ad3wt by digestion with Mlu I without prior recombination or mutation. HAdV-3p-E-3-9K-rec, the recombination control virus, was generated by recombination of pKSB2Ad3wt with unaltered pE3-9K shuttle plasmid. An E3-9K knock out mutant, HAdV-3p-E3-9K-KO, was generated by recombination of pKSB2Ad3wt with a pE3-9K shuttle plasmid that contained TAG stop codons in place of all the in-frame ATG codons present in ORF E3-9K. HAdV-3p-E3-9K-NULL was generated by recombination of pKSB2Ad3wt with a pE3-9K shuttle plasmid carrying the HAdV-7h-encoded null version of this ORF (GenBank accession # Z48954 and AF321310).

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Generated, Transfection, Plasmid Preparation, Knock-Out

Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: HAdV-3p E3-9K mutant virus-induced cytopathic effect in A549 cells. A549 cells were infected with HAdV-3 control viruses (HAdV-3p-WT, HAdV-3p-E3-9K-rec) or HAdV-3p-E3-9K mutant viruses (HAdV-3p-E3-9K-KO, HAdV-3p-E3-9K-NULL) at a MOI of 1 PFU/cell and analyzed by light microscopy 4 days pi. Classical adenovirus CPE was characterized by “webbing” or “lacy” appearance of cells followed by rounding and detaching of cells from the culture plate surface.

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Infection, Light Microscopy

Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: Growth phenotype of HAdV-3p E3-9K mutant viruses in A549 cell monolayers. A549 cells in 6-well plates were infected with approximately 20 PFU per well of HAdV-3p E3-9K mutant viruses ( A ) or HAdV-C rec 700 and pm734.1 as a positive control (B) and overlayed with a semi-solid agarose/medium overlay. At 13 days pi (A) or 6 days pi ( B ), cells were fixed and stained with crystal violet to visualize plaques.

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Infection, Positive Control, Staining

Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: Dissemination of HAdV-3p E3-9K mutant viruses in A549 cell monolayers. A549 cells were infected at a MOI of 1, 0.1, or 0.01 PFU/cell with HAdV-3p E3-9K mutant viruses ( A ) or HAdV-C viruses rec 700 and pm734.1 ( B ), replenished with liquid medium, fixed at 5 days pi, and stained with crystal violet to visualize virus-induced cell monolayer destruction.

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Infection, Staining

Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: HAdV-3p E3-9K mutant virus egress from A549 cells. A549 cells were infected with HAdV-3p E3-9K mutant viruses ( A - B ) or HAdV-C rec 700 and pm734.1 ( C - D ) at a MOI of 10 PFU/cell, replenished with liquid medium, and total (A,C) or extracellular (B,D) infectious virus yields were determined by plaque assay at the indicated times pi. A-B is representative of two independent experiments.

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Infection, Plaque Assay

Journal: BMC Research Notes

Article Title: No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infection

doi: 10.1186/1756-0500-5-429

Figure Lengend Snippet: Viability of HAdV-3p E3-9K mutant virus-infected A549 cells. A . A549 cells were infected with HAdV-3p E3-9K mutant viruses at a MOI of 10 PFU/cell. Medium and trypsinized cells were pooled at indicated times pi, stained with an equal volume of 0.4% trypan blue solution, and approximately 200 cells were counted using a light microscope and hemocytometer. The percentage of viable cells was determined by dividing the number of unstained cells by the total number of cells counted. Error bars indicate standard error of the mean for three independent experiments. Mock-infected cells were used as a negative control. B. HAdV-C rec 700 and pm734.1 controls. Representative trypan blue exclusion assay results consistent with those reported by Tollefson et al. for the same viruses.

Article Snippet: A549 cells (ATCC #CCL-185) were grown in 8% (v/v) newborn calf serum-supplemented Eagle Minimum Essential Medium (EMEM) (A549 Growth Medium).

Techniques: Mutagenesis, Infection, Staining, Light Microscopy, Negative Control, Trypan Blue Exclusion Assay