Abstract
BackgroundMeleagrid 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.MethodsTn5 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.ResultsAttempts 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi10.1186/s12985-015-0362-9) contains supplementary material, which is available to authorized users.
Highlights
Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes are ideal vectors for the development of recombinant vaccines for the poultry industry
The requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time
The severe attenuation observed for MeHV-1 in this study suggests the UL21 gene is unsuitable for use in recombinant vaccine applications, it may be of use for generating replication-limited gene delivery constructs for poultry research applications
Summary
Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. 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. MeHV-1, bivalent vaccines containing MeHV-1/GaHV-3 or attenuated GaHV-2 strains, MD outbreaks continue to occur. The isolation of GaHV-2 field strains of increased virulence has been correlated with the loss of protective capacity of these vaccines, which reinforces the need for development of improved MD vaccines [4, 5]. It is likely that novel vaccines targeting GaHV-2 will be constructed using recombinant DNA technologies, for which MeHV-1 is ideally suited as a vector candidate
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