Abstract
This chapter deals with (1) the preparation of herpes simplex virus (HSV) virion DNA of a quality and purity suitable to be used for the generation of infectious virus, and (2) its use in the preparation of infectious virus. An important development in the understanding of virus genetics and gene products has been the ability to carry out reverse genetics. This is dependent on the ability to manipulate the genome in vitro and reconstitute infectious virus. Our understanding of DNA viruses and positive stranded RNA viruses (where DNA and RNA/cDNA, respectively, are generally infectious) is considerably greater than for negative stranded RNA viruses, where until recently, it had been impossible to generate virus.from either RNA or cDNA. Within the herpesviridae, knowledge of the function of HSV gene products is one of the more advanced owing to the relatively straightforward techniques required to generate virus from HSV-DNA, and to introduce desired mutations by cloning small parts of the genome, manipulating them, and then reintroducing the mutations by a process of cotransfection and in vivo recombination with intact virus DNA. Other α-herpesviruses, such as EHV-1 and PRV, are equally amenable to such manipulation, and knowledge of their gene products is also well advanced. In contrast, this technology is only now, and with much less success, being applied to other members of the family, such as EBV, HCMV, and VZV, and knowledge of their genetics is much less advanced. The use of cosmids to reconstitute intact virus will aid in the advance of knowledge for these viruses. For examples of uses of recombinant DNA technology, the reader is referred to other chapters (especially those on cloning and mutagenesis). I will concentrate on the techniques currently in use in my laboratory, but will also mention other techniques in use elsewhere that may be more appropriate in certain cell types.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
MacPherson, I. and Stoker, M G (1962) Polyoma transformation of hamster cell clones an investigation of genetic factors affecting cell competence Virology 190, 221–232.
Graham, F L and van der, E B (1973) A new technique for the assay of the infectivity of human adenovirus 5 DNA Virology 52, 456–467.
Stow, N D and Wilkie, N M (1976) An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA J Gen Virol 33, 447–458.
Preston, V G (1981) Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome. J Virol 28, 150–161.
Rixon, F J and Mclaughlin, J (1990) Insertion of DNA sequences at a unique restriction enzyme site engineered for vector purposes into the genome of herpes simplex virus type 1 J Gen Virol 71, 2931–2939.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
MacLean, A.R. (1998). Preparation of HSV-DNA and Production of Infectious Virus. In: Brown, S.M., MacLean, A.R. (eds) Herpes Simplex Virus Protocols. Methods in Molecular Medicine, vol 10. Humana Press. https://doi.org/10.1385/0-89603-347-3:19
Download citation
DOI: https://doi.org/10.1385/0-89603-347-3:19
Publisher Name: Humana Press
Print ISBN: 978-0-89603-347-4
Online ISBN: 978-1-59259-594-5
eBook Packages: Springer Protocols