**1.2 VZV research methods**

VZV contains the smallest genome among the eight human herpesviruses, consisting of a 125-kb double-stranded DNA genome that encodes 70 unique open reading frames (ORFs). The function of most of these ORFs, however, was largely unknown until recent years. This is in part due to the absence of both a genetic tool to efficiently generate mutant clones for loss-of-function studies and a true animal model for large scale screening of *in vivo* virulence factors (Cohen et al., 2007).

Obstacles in mutagenizing VZV include its large genome size, narrow host range, and marked differences in replication cycles when studied *in vitro* versus *in vivo* (Arvin, 1996; Cohen, 2001). A once prevalent technique to create recombinant VZV variants was the fourcosmid system, made by cloning overlapping segments of the VZV genome into four large cosmids (Cohen & Seidel, 1993; Mallory et al, 1997; Niizuma et al., 2003). Co-transfection of these cosmids, one of which containing a mutation in the desired ORF, created a recombinant VZV variant. However, this method alone faced many challenges. For example, research was thwarted because co-transfection of the large cosmids into permissive mammalian cells and multiple homologous recombination events within a single cell were necessary to generate the full-length viral genome (Zhang et al, 2008).

More recent developments have helped to circumvent these problems by cloning the entire VZV genome as a bacterial artificial chromosome (VZVBAC) (Nagaike et al., 2004). This approach provides easy and efficient manipulation of the viral genome and rapid isolation of recombinant viruses, making the systemic deletion of every ORF in the genome feasible. A firefly luciferase cassette is also inserted into the VZVBAC to produce a novel luciferase VZVLuc BAC. This allows us to not only generate VZV ORF deletion mutants, but also monitor its subsequent growth in cultured cells.
