**8. Conclusions**

76 Bacterial Artificial Chromosomes

corresponding deletion mutants (Zhang et al., 2010). Three of these four ORFs (ORF10, ORF14, ORF47) have previously been identified as tissue-tropic factors (Cohen & Seidel, 1994; Heineman & Cohen, 1995; Moffat et al., 1998). Our findings verified these previous studies and additionally identified ORF7 as a novel skin-specific virulence factor. To confirm our original finding here, we also produced a premature stop-codon mutant (ORF7S) by mutating the 5th codon from TGT to the TGA stop codon. Like ORF7D, ORF7S displayed wild-type growth in MeWo cells, but had a growth defect in SOC, indicating that

As mentioned previously, after a primary VZV infection, the virus will remain dormant in the sensory ganglia of its host. When reactivated, VZV will erupt from the sensory neurons and infect surrounding skin tissue, causing characteristic rashes and severe pain due to nerve damage. Therefore, identifying the VZV factors responsible for not only skin-tropism,

We proposed that the VZV genome also encodes factors required for efficient invasion and egress from specific tissues during natural infection, such as neurotropic factors. Using our newly created VZV deletion mutants, we screened each of the 18 dispensable VZV ORFs to determine which are implicated with VZV neuronal infection. First, the mutant DNAs were confirmed to be replication competent in a primary permissive cell line, the human retinal epithelial ARPE19 cells (Schmidt-Chanasit et al., 2008). Then, the deletion mutants were screened in human neuroblastoma, SH-SY5Y, following a similar transfection approach as described earlier for MeWo cells, to establish their essentiality for replication in human neurons. Of the mutants tested, only the ORF7 deletion mutant was unable to form viral plaques (data not yet published). To confirm this, we also infected differentiated neuroblastoma and human embryonic stem cell-derived neurons with wild type and 7D cell-free particles. As expected, the WT infection exhibited robust proliferation, while the 7D infection yielded no visible plaques. Our finding here establishes ORF7 as the only known VZV factor required

While the current VZV vaccine is sufficient to prevent chickenpox, many issues still surround this live attenuated vaccine. Firstly, bulk vaccine production using the vOka strain is difficult and costly due to vOka virus's relatively low yield (Schmid & Jumaan, 2010; Gomi et al, 2002). The vaccine is also not entirely effective at eliminating chickenpox outbreaks. Despite seroconversion after vaccination, varicella infections still occur in some children and adults exposed to wild-type VZV (Schmid & Jumaan, 2010; Bernstein et al., 1993, White et al., 1991). Most significantly however, the currently marketed vaccine strain, v-Oka, while highly attenuated in the skin, still retains its neurovirulence (Hambleton et al., 2008). This means that the virus will continue to establish latency in the sensory nerve ganglia of the immunized host and can potentially reactivate later to trigger herpes zoster and post-herpetic neuralgia. Furthermore, while drug treatments available to date can alleviate some symptoms of VZV-elicited diseases and shorten the disease duration, they

ORF7 may function as a VZV skin-tropic factor.

but also neurotropism is of great importance.

**7.1 Screening for VZV neurotropic factors** 

for viral spread in human neurons.

**7.2 Future applications** 

**7. Ongoing research** 

The use of a bacterial artificial chromosome system has proven to be an invaluable tool in human herpesvirus studies, without which, our genome-wide VZV mutagenesis could not have been possible. Not only can BACs clone the large viral genomic DNA, their slow replication rate and relative ease and accuracy of producing and reproducing stable viral mutants make BACs the ideal method for functional analysis of the VZV genome. Furthermore, the addition of a luciferase marker has greatly improved the efficiency, accuracy and reproducibility of our results. Combined, this luciferase BAC approach has truly facilitated genetic studies of VZV and provided vital insights into the replication and pathogenesis of the virus.

In this study, a global functional analysis of the entire VZV genome was carried out, focusing on the identification of ORFs essential for viral replication in cultured MeWo cells and human fetal skin organs. In all, our study has distinguished novel functional annotations for 36 VZV genes and shed light on the essentiality of each of the 70 unique VZV ORFs. More importantly, our findings have identified ORF7 as both a skin-tropic and neurotropic factor, implicating the ORF7 deletion virus as an ideal vaccine candidate to prevent both VZV-elicited diseases, chickenpox and shingles.

As our research progresses, future VZVLuc BAC studies will continue to provide more exciting discoveries and help identify new antiviral targets. Soon, effective vaccines and improved therapy for the prevention and treatment of a wide array of infections will be tangible.
