The generation of cell lines permitting isolation of genetically intact human cytomegalovirus(HCMV)

Human cytomegalovirus (HCMV) is a clinically important human pathogen that can cause severe disease in immunologically deficient individuals. Studies into the virus are hindered by the rapid and reproducible mutations that occur in vitro, resulting in viruses which behave differently from those found in vivo. Mutations rapidly occur in the RL13 and UL128L gene regions, leading to increased growth kinetics and altered cellular tropism respectively. In order to work with genetically intact HCMV, genomes have been BAC cloned, and repaired to genetically match the original clinical sample. This does not, however, prevent the emergence of new mutants. To overcome this, RL13 and UL128L are conditionally suppressed during virus growth using a tetracycline repressor based system. Although effective, this requires the genome to be available as a BAC clone, preventing their use with primary clinical isolates. This thesis aimed to generate cell lines to enable work with genetically stable clinical isolates without the need for BAC cloning, to produce an indicator cell line for the rapid detection of replicating virus, and to convert existing laboratory-adapted viruses to more closely represent wild-type isolates. As an alternative to tet repression, cell lines expressing shRNA targeting RL13/UL128L were used to suppress protein expression during infection, however these attempts were unsuccessful. Indicator cell lines were produced that expressed GFP following infection, however the level of GFP induction was too weak for these lines to be used reliably. However, a UL128 expressing cell line was successfully produced, which complemented the loss of this gene from our existing bank of lab-adapted viruses. As a result, it became possible to infect a range of cell types with existing viruses, therefore enabling studies into viral dissemination, pathogenesis and disease prevention, without the need to re-generate existing constructs in new virus backgrounds