Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System

Generation and analysis of the VZV_Luc strain. (A) Luciferase assay. MeWo cells were infected with VZV_Luc or VZV_BAC for 2 days, and luciferase activity was measured. The cells infected with VZV_Luc showed a high level of luciferase activity, while the parental VZV_BAC strain had no activity. (B) Bioluminescence measurement. Two wells of a six-well dish of MeWo cells were infected with VZV_BAC (upper left), and two were infected with VZV_Luc (upper right). Two days postinfection, d-luciferin substrate was added to the cultured wells, and bioluminescence was measured using IVIS imaging. Bioluminescence can be detected only in VZV_Luc-infected cells. The intensities were indicated as pseudocolors, as shown by an intensity scale bar at the top; higher intensity is represented by a warmer color, and lower intensity is represented with a cooler color. The infection of these wells was verified by showing GFP-positive plaques (bottom panel). (C) Correlation of luminescence and plaque numbers. Growth curves generated by an infectious center assay (black curve and left scale) and a bioluminescence assay (green curve and right scale) were compared. (D) The growth curve comparison of VZV_Luc with and without the BAC vector. MeWo cells were infected with VZV with the BAC vector (green) and without the BAC vector (blue). Their grow curves were generated by bioluminescence assay. (E) Monitoring VZV replication in vivo. SCID mice with human thymus-liver implants were inoculated with 4 × 10³ PFU VZV-infected cells. At 7 days postinfection, a luciferase substrate, d-luciferin, was injected, and bioluminescence was observed 10 min later using IVIS imaging and overlaid on the mouse images.

Measuring VZV_Luc replication in SCID-hu mice. (A) Replication and progression of VZV_Luc in human thymus-liver implants in SCID mice. Three SCID-hu mice implanted with thymus-liver implants were inoculated with VZV_Luc. Using IVIS imaging, each mouse was scanned daily (from day 0 to day 8). Measurements were taken 10 min after intraperitoneal injection with d-luciferin substrate. Only images from mouse C are shown. Warmer colors indicate higher viral load; cooler colors indicate lower viral load. (B) VZV growth curves in vivo. Bioluminescence in three SCID-hu mice from the above experiment was measured, and VZV growth curves in human thymus-liver implants were generated.

Analysis of VZV ORF0 to ORF4. (A) Schematic diagram of the genomic organization of VZV ORF0 to ORF5 (1). Each ORF, from 0 to 4, was replaced by a Kan^r expression cassette to generate the deletion mutant as indicated for ORF4 (2). To create a rescue virus for the ORF4 deletion, ORF4 was cloned into pGEM-zeo-loxP, and the loxP-zeo-ORF4 cassette was amplified by PCR and used to replace the Kan^r cassette by homologous recombination (3). The zeocin-resistant gene (Zeo^r) can be removed from the viral genome by introducing Cre recombinase (4). (B) Digestion analysis of VZV wild-type and recombinant viral BAC clones. VZV DNA from the wild type (WT), the ORF0 to ORF4 deletion clones (0D to 4D), and the ORF0D and ORF4D rescue clones (0R and 4R) was digested with HindIII and loaded onto a 0.5% agarose gel. In order to clearly show both smaller fragments and large fragments, two photos were taken. The lower panel was taken at an earlier time, and the upper panel was taken at a later time. Sizing markers (MR) are indicated at the left. (C) Growth curve analysis of VZV deletion mutants in vitro. One hundred PFU of each deletion mutant and VZV_Luc (WT) were used to infect MeWo cells in six-well dishes in triplicate. Luminescence was measured using IVIS every day for 7 days after d-luciferin was applied to the cultured media. Total photon flux in each well (photons/s/cm²/steradian) was measured, and the values from triplicates were averaged. Growth curves were generated when the averaged photon counts for each day were plotted. (D) Growth curve analysis of VZV deletion mutants in vivo. SCID-hu mice with thymus-liver implants were inoculated with 4 × 10³ PFU VZV_Luc or VZV deletion mutant strains as indicated. VZV replication was measured by IVIS daily for 1 week as photon flux in the regions of interest above the thymus-liver implants. Each line represents an average of data from 2 or 3 mice. An uninfected SCID-hu thymus-liver mouse was injected with d-luciferin, and the background luminescence was measured (pink line). (E) Growth curve analysis of VZV ORF0 and ORF4 deletion rescue viruses in vitro. The conditions in this assay were same as those described for panel B. (F) Growth curve comparison of measurements by luminescence assay and infectious center assay. MeWo cells were grown in six-well dishes in triplicate. The cells were infected with 100 PFU of wild-type (WT), ORF0 deletion (ORF0D), and ORF0D rescue (ORF0R) viruses, respectively. Growth curve analyses were carried out by luminescence assays (LA) and infectious center assays (IA) as described above. Total photon counts in each well were measured using IVIS, averaged, and plotted on the left. Total plaques per well were counted, averaged, and plotted on the right.