title: Molecular studies towards improved avian metapneumovirus vaccines
Creators: name: Clubbe, Jayne
Publisher: name: University of Liverpool Repository
Year: 2015

description: The work undertaken in this thesis centres around molecular studies on avian metapneumovirus (AMPV) subtypes A and B. The findings and outcomes may be of use in the future development of improved vaccines against the disease. Since the emergence of AMPV a range of live attenuated vaccines have been developed and these are used in the control of the disease throughout the world. However disease outbreaks still occur and improved vaccines are needed for the control, or better still the eradication of AMPV. Commercially, vaccines have been marketed with the belief that they are more efficacious in the species from which the progenitor virus was derived. To investigate this at a molecular level, the first part of the study compared full length (FL) genome sequences of chicken and turkey-derived AMPVs to identify potential species specific regions of sequence. A FL RT- PCR and sequencing system was developed for AMPV subtype B, and used along with an already established system for subtype A to determine the FL sequences from a range of field strains and commercially available vaccines. The nucleotide (nt) sequences and predicted amino acid (aa) sequences were aligned and compared. In parallel, turkey-derived viruses were multiply passaged in chicken tracheal organ culture (TOC) to investigate whether viruses would evolve towards potential species specific motifs. A conclusive comparison study for AMPV subtype A could not be completed due to problems sourcing subtype A chicken-derived viruses. For AMPV subtype B numerous sequence differences between viruses were identified, however none were specific for the host species. The second part of this study investigated one of the two major subtype A commercially available live vaccines, both derived from a highly virulent UK field strain, #8544. Previously, improvements of those vaccines, by use of reverse genetics technology (RG), was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based RG system. In the present study a FL DNA copy of one of the Vero grown, #8544 derived, live vaccines was modified, using the RG system, to create an exact FL DNA copy of #8544. This vaccine sequence was then re-introduced systematically, leading to the identification of a single coding substitution in the M2:2 reading frame responsible for replication and virus recovery in Vero cells. Ablation of M2:2 also resulted in replication and virus recovery in Vero cells. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The M2:2 modification of field virus #8544 will enable research leading to improved vaccines. RG systems are now commonly being employed to aid the development of improved vaccines. However, as yet a system does not exist for AMPV subtype B. In the final part of this study the FL genome sequence of AMPV subtype B was successfully cloned. This is a major step in the development of a functional AMPV subtype B RG system.