Identification of non-essential loci within the Meleagrid herpesvirus 1 genome.

Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.