Investigating Genetic Components of Meleagrid Herpesvirus 1 for Viral Vector Applications for the Poultry Industry

Hall, Robyn Nora (2013). Investigating Genetic Components of Meleagrid Herpesvirus 1 for Viral Vector Applications for the Poultry Industry PhD Thesis, School of Veterinary Science, The University of Queensland.

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Author Hall, Robyn Nora
Thesis Title Investigating Genetic Components of Meleagrid Herpesvirus 1 for Viral Vector Applications for the Poultry Industry
Formatted title
Investigating Genetic Components of Meleagrid Herpesvirus 1 for Viral Vector Applications for the Poultry Industry 
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Timothy Mahony
Joanne Meers
Elizabeth Fowler
Total pages 392
Language eng
Subjects 1002 Environmental Biotechnology
070712 Veterinary Virology
Formatted abstract
Restrictions on prophylactic and therapeutic interventions in the poultry industry, particularly the use of antibiotics, means there is a heavy reliance on vaccines for disease prevention, however vaccines can be costly to purchase and administer. One alternative to administering multiple vaccines is the use of multivalent recombinant vaccines to protect against multiple pathogens from a single vaccination. Recombinant delivery vectors can also be used to deliver other transgenes such as cytokine genes that may improve bird health non-specifically. Meleagrid herpesvirus 1 (MeHV-1) is an ideal vaccine vector for the poultry industry because it is apathogenic, non-immunosuppressive, is not spread horizontally from vaccinated chickens, can be administered by in ovo vaccination using automated devices and has a large DNA genome with the potential capacity to carry large amounts of foreign DNA.

To fully realise the potential of MeHV-1 as a delivery vector requires a thorough understanding of the genetic background of the virus and the development of efficient methods for the generation of recombinants. There is a paucity of genetic information on MeHV-1 specifically, and much of the current knowledge has been extrapolated from closely related viruses such as Gallid herpesvirus 2 or Human herpesvirus 1. This project proposed to address this lack of knowledge by determining the essential and non-essential genes of MeHV-1, thereby facilitating the construction of recombinant vaccines by identifying suitable transgene insertion sites within the MeHV-1 genome.

Five infectious bacterial artificial chromosome (BAC) clones of MeHV-1 had previously been constructed within Dr Mahony’s laboratory. The construction of herpesvirus BACs enables single viral genomes to be stably maintained within bacteria, thereby making them amenable to well-established powerful bacterial mutagenesis methods. Characterisation of the five parent MeHV-1 BACs by restriction endonuclease mapping, full genome sequencing and in vitro replication studies identified genetic and phenotypic differences, despite the clones having been generated during a single experiment. These studies provide further evidence that herpesviruses, particularly the mardiviruses, exist as mixed genetic populations. The cloning of herpesviruses as BACs selects for single genomes out of the mixed genetic background, and clones may vary considerably both genetically and phenotypically from the wild-type virus pool.

To determine the gene requirements of MeHV-1, one of these BACs was used as a target for transposition, first with the Tn5 transposition system and subsequently with a MuA system. These studies demonstrated that MeHV-1 is partially resistant to Tn5 transposition, and that BACs vary in their susceptibility to different transposition systems. Using the Tn5 and MuA transposition systems, 76 MeHV-1 gene disruption clones were generated. These insertions disrupted 30 individual genes. The requirement for two genes, both present in dual-copy within the MeHV-1 genome, was not able to be determined. Fourteen genes were classified as non-essential for in vitro replication, identifying these locations as putative transgene insertion sites. Gene-disrupted mutants were found to vary considerably in their replication capacities, potentially allowing targeted use of certain mutants for different applications, such as for early embryo delivery of transgenes. Additionally eight intergenic insertion sites were determined to be non-essential, including two within BAC vector sequences. Fourteen MeHV-1 genes were confirmed to be essential for in vitro replication.

Due to the identification of major deletions within the five existing MeHV-1 BAC clones, a new MeHV-1 BAC was constructed using a novel synthetic assembly method. Thirteen overlapping fragments of the MeHV-1 genome were generated by PCR and were assembled with a yeast-bacterial shuttle vector using transformation-associated recombination machinery of yeast. Using this assembly method, an infectious MeHV-1 construct was identified, although this construct appeared to have minor deletions within the repeat genomic regions. This construct is the largest infectious synthetic virus generated to date. These methods are widely applicable to the generation of large DNA constructs, including recombinant vaccines and herpesvirus BACs, without the need for serial passage under selection.

These studies have greatly contributed to the current level of knowledge of the biology and genetics of MeHV-1 and the cloning of mardiviruses as BACs. The potential transgene insertion sites identified may be further developed for generation of recombinant MeHV-1-based vaccines and delivery constructs. The synthetic assembly methods developed offer alternative techniques for BAC construction and mutagenesis and more broadly, are applicable to the generation of any large DNA construct. It is hoped the findings from these studies can be implemented in the development of future recombinant multivalent vaccines to improve poultry health within both the Australian and international poultry industries. 
Keyword Meleagrid herpesvirus 1
Bacterial Artificial Chromosomes
Turkey herpesvirus

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Created: Mon, 28 Apr 2014, 18:05:40 EST by Robyn Hall on behalf of Scholarly Communication and Digitisation Service