**3.2 The VZVBAC with a luciferase marker (VZVLuc)**

To generate a VZV strain expressing luciferase, a firefly luciferase expression cassette was inserted into the intergenic region between ORF65 and ORF66 of the VZVBAC genome. This clone was transfected into MeWo cells to produce the VZVLuc strain. 24 hours later, cell culture media was replaced with media containing 150 µg/ml D-luciferin. After incubation at 37°C for 10 minutes, bioluminescent signals were observed and quantified using an *In Vivo* Imaging System (IVIS).

Upon analysis (Fig. 4), the growth of VZVLuc closely resembled that of its parental VZVBAC (data not shown). This confirms that the addition of a luciferase reporter to the viral BAC did not change its growth properties. Unlike its parental strain however, only cells infected with VZVLuc expressed high levels of luciferase activity (Fig. 4A) and emitted a strong bioluminescence after the addition of D-luciferin (Fig. 4B).

To explore the possibility of using bioluminescent signals as an indicator of viral growth, bioluminescent assays were compared to the conventional infectious center assay. Plates were inoculated with wild-type (WT) VZV and VZVLuc. Their viral titers were quantified daily via both methods for seven days and the data collected was used to construct viral growth curves (Fig. 4C). As the figure illustrates, the intensity of the bioluminescent signals strongly correlated with the viral titers generated by an infectious center assay. Thus, this data supports BLI as an alternative method for growth curve assays and quantifying viral titers.

Fig. 4. Analysis of the VZVLuc strain. (A) Luciferase assay. MeWo cells were infected with VZVBAC/VZVLuc for two days; luciferase activity was measured. The cells infected with VZVLuc showed a high level of luciferase activity, while the parental VZVBAC strain showed no activity. (B) Bioluminescence measure. Two wells of MeWo cells were infected with VZVBAC (upper left), and two were infected with VZVLuc (upper right). Two days postinfection, D-luciferin was added to the cultured wells. Bioluminescence was measured and could only be detected in VZVLuc-infected cells. The intensities are indicated by an intensity scale bar at the top; higher intensities are represented by warmer colors, and lower intensities are represented by cooler colors. The infection was verified by the 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.

### **3.3 Bioluminescence imaging for studying VZV in SCID-hu mice**

Another useful application of bioluminescence imaging is the live-image analysis of VZV replication in severe combined immunodeficient mice with human tissue xenografts (SCID-hu

clone was transfected into MeWo cells to produce the VZVLuc strain. 24 hours later, cell culture media was replaced with media containing 150 µg/ml D-luciferin. After incubation at 37°C for 10 minutes, bioluminescent signals were observed and quantified using an *In* 

Upon analysis (Fig. 4), the growth of VZVLuc closely resembled that of its parental VZVBAC (data not shown). This confirms that the addition of a luciferase reporter to the viral BAC did not change its growth properties. Unlike its parental strain however, only cells infected with VZVLuc expressed high levels of luciferase activity (Fig. 4A) and emitted a strong

To explore the possibility of using bioluminescent signals as an indicator of viral growth, bioluminescent assays were compared to the conventional infectious center assay. Plates were inoculated with wild-type (WT) VZV and VZVLuc. Their viral titers were quantified daily via both methods for seven days and the data collected was used to construct viral growth curves (Fig. 4C). As the figure illustrates, the intensity of the bioluminescent signals strongly correlated with the viral titers generated by an infectious center assay. Thus, this data supports

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BLI as an alternative method for growth curve assays and quantifying viral titers.

Fig. 4. Analysis of the VZVLuc strain. (A) Luciferase assay. MeWo cells were infected with VZVBAC/VZVLuc for two days; luciferase activity was measured. The cells infected with VZVLuc showed a high level of luciferase activity, while the parental VZVBAC strain showed no activity. (B) Bioluminescence measure. Two wells of MeWo cells were infected with VZVBAC (upper left), and two were infected with VZVLuc (upper right). Two days postinfection, D-luciferin was added to the cultured wells. Bioluminescence was measured and could only be detected in VZVLuc-infected cells. The intensities are indicated by an intensity

scale bar at the top; higher intensities are represented by warmer colors, and lower intensities are represented by cooler colors. The infection was verified by the 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

Another useful application of bioluminescence imaging is the live-image analysis of VZV replication in severe combined immunodeficient mice with human tissue xenografts (SCID-hu

VZVBAC VZVluc VZVBAC VZVluc

bioluminescence assay (green curve and right scale) were compared.

**3.3 Bioluminescence imaging for studying VZV in SCID-hu mice** 

*Vivo* Imaging System (IVIS).

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bioluminescence after the addition of D-luciferin (Fig. 4B).

mice). Because VZV only infects human cells, *in vivo* studies of VZV pathogenesis have been limited to the use of immunodeficient mice with human tissue implants. However, although SCID-hu mice are established as appropriate models for studying VZV pathogenesis (Besser et al, 2003; Ku et al., 2005; Zerboni et al., 2005), collecting quantitative data has been a major challenge. Since measuring viral growth required the mice to first be euthanized, it was impossible to monitor the progression of the viral infection in the same mouse. In addition, viral titers tend to vary from animal to animal because of the differently sized implants (Moffat & Arvin, 1999), thus hindering not only the frequency of data collection, but also the accuracy as well. These factors greatly impeded efforts to study large numbers of VZV variants and made it difficult to discern minor phenotypic differences leading to pathogenesis.

The development of BLI has been extremely helpful to circumvent these obstacles. Luciferase provides a visible marker for detecting VZV in human tissues within living animals. By using VZVLuc, the SCID thymus-liver mouse model, and *In Vivo* Imaging System (IVIS, Xenogen), the spread of the VZV infection can be frequently monitored in the same mouse over an extended period of time; thereby, allowing the generation of credible growth curves to gain accurate insights into VZV's growth kinetics *in vivo*.

We applied this method to explore VZV replication and measure its spread *in vivo*. Human fetal thymus and liver tissue were implanted under the left kidney capsule of the SCID mouse. Over the course of the next few months, the implanted tissue developed into a thymus-like organ consisting mainly of T cells. VZV-infected cells were then inoculated into the SCID-hu mice with thymus-liver implants. VZV replication was measured *in vivo* after the injection of the luciferin substrate, using an IVIS. Each mouse was imaged daily, starting four hours after inoculation (i.e day zero), for eight days (Fig 5A).

Our data depicts the daily increase in bioluminescence emitted from the infected implants (Fig. 5B). The quantified signals were plotted to generate an *in vivo* growth curve (Fig. 5C). As shown, VZV grew rapidly in human T cells, doubling approximately every 12 hours and peaking at seven days postinfection. The exponential growth curve is then followed by a steady state where the viral infection reaches the saturation limit of the implant.

We also tested the VZVLuc viruses for their spread and detection in human fetal skin xenografts *in vivo*. Similar to the process outlined above, human skin tissues were introduced into SCID mice. Four weeks after implantation, VZVLuc virus was inoculated into the skin tissues and viral growth was monitored every two to three days for 15 days using an IVIS. High luciferase activity was detected in the implants (data not shown), verifying VZVLuc's ability to grow in skin tissue *in vivo*.

In short, by engineering VZV to express luciferase enzymes, bioluminescence imaging can be used to monitor the progression of viral growth and quantify viral replication in organ cultures and SCID-hu mice. Compared to the traditional infectious center assay, BLI not only saves time and labor, but also significantly increases the reproducibility of results (Doyle et al., 2004). Moreover, the presence of luciferase activity indicates viral replication in cells and not free-viral particles (Zhang et al., 2010), making BLI the most suitable method for studying this particular cell-associated virus. Consequently, the development of BLI has greatly facilitated our ability to investigate aspects of VZV infection in the SCID-hu mouse model and has significantly advanced our understanding of VZV pathogenesis and viruscell interactions (Zerboni et al., 2010; Arvin et al, 2010; Zhang et al; 2010; Moffat & Arvin; 1999; Arvin, 2006).

Fig. 5. Monitoring VZVLuc virus replication in SCID-hu mice. (A) SCID-hu model. 1. Human fetal thymus/liver tissues were implanted under SCID mouse kidney capsule. 2. Two to three months later, the implant was inoculated with VZVLuc. 3. Viral replication in human T cells was detected by IVIS. (B) Replication and progression of VZVLuc in human thymus/liver implants in SCID mice. Three SCID-hu mice with thymus/liver implants were inoculated with VZVLuc. Using IVIS, each mouse was scanned daily (from day 0 to day 8). Measurements were taken 10 minutes after i.p. injection with luciferin substrate. Only images from one mouse are shown. Warmer colors indicate higher viral load; colder colors indicate lower viral load. (C) VZV growth curves *in vivo*. Bioluminescence from three SCIDhu mice in the above experiment (B) was measured and VZV growth curves in human thymus/liver implants were generated.
