Construction and analysis of alphaherpesviruses expressing green fluorescent protein

Over the past 10 years, the use of fluorescently tagged herpesviruses has evolved from relative obscurity into a common component in the arsenal of many molecular herpesvirology laboratories. In this chapter we provide methods for construction and analysis of recombinant alphaherpesviruses using conventional co-transfection and homologous recombination procedures. In recent years many herpesviruses have been cloned into bacterial artificial chromosomes (BACs), which has facilitated their manipulation by sophisticated bacterial molecular genetic techniques [Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H., and Koszinowski, U. H. (1997) Proc Natl Acad Sci USA 94,14759-63; Smith, G. A., and Enquist, L. W. (2000) Proc Natl Acad Sci USA 97,4873-8; Tischer, B. K., von Einem, J., Kaufer, B., and Osterrieder, N. (2006) Biotechniques 40,191-7]. These technological breakthroughs have allowed for the genetic analysis of virus gene products, including those that are essential for virus replication, with unprecedented ease. The main caveat to this approach is that one requires their virus strain of interest cloned into a BAC. If the virus strain under consideration has not been introduced into a BAC, it is far from trivial to do so. While comparatively antiquated, the procedures provided in this article can be used with any strain. Here we focus on pseudorabies virus (PRV), a swine pathogen, which is the alphaherpesvirus most amenable to genetic manipulation using this transfection-based approach.