Avian metapneumovirus studies using reverse genetics

Falchieri, Marco Avian metapneumovirus studies using reverse genetics. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Avian metapneumovirus (AMPV) is an enveloped negative sense single stranded RNA virus which is a major endemic respiratory pathogen of global domestic poultry. Since reverse genetic (RG) techniques have been applied to this pathogen several reports have investigated the effects of single and multiple genomic mutations and gene deletions or insertions on viral biology. The aim of this study was to gain a better understanding of the viral capacity to accept and in some cases express homologous and heterologous extra sequences. Initially an AMPV subtype A was modified to introduce a homologous 200 bp sequence within the G gene and this recombinant was suggested to be used as a positive control for validating all stages of a previously established RT-nested PCR. Different Green Fluorescent Protein (GFP) AMPV recombinants were then prepared each one containing the reporter gene in a different intergenic position and then were assessed for expression, stability and viability. In particular, quantification of the expression was calculated using a quantitative ELISA. All recombinants showed high stability, while good viability was observed in all the positions, except when GFP was inserted between nucleocapsid (N) and phosphoprotein (P). The highest expression was detected in the virus with the insertion between N and P as expected, according to the transcriptional model for non-segmented negative stranded viruses. However GFP was produced at high levels even when inserted at the trailer end intergenic positions. Poor expression was seen for all the other positions. The vectoring abilities of subtype A strains were further investigated to accept and express foreign genes, specifically GFP gene and both spike (S1) and nucleocapsid (N) genes of infectious bronchitis virus (IBV). After viruses had been recovered by RG, all recombinants were proven to express the inserted genes efficiently and were all found to be highly stable during passage in vitro. Subsequently IBV recombinants were tested as candidate vaccines by eye-drop inoculation of one-day-old chickens. When chicks were challenged with IBV, partial protection results were observed, as assessed by greater motility of tracheal cilia from animals receiving the recombinants. Finally the development of a new RG system was attempted in order to extend this type of studies to the B subtype. This subtype is distributed worldwide and growing field evidence suggests it to be more able to infect commercial chickens compared to subtype A. For this reason it would be convenient to have an RG available also for B viruses. The construction of a DNA copy of the viral genome was attempted using site-directed mutagenesis and ligation techniques, resulting in more than 85% of the genome cloned. Unfortunately full genome cloning proved to be not possible, as severe problems were encountered, including construct instability and cloning bacteria intolerance to viral sequences.

Item Type:	Thesis (Doctor of Philosophy)
Additional Information:	Date: 2012-09 (completed)
Uncontrolled Keywords:	AMPV, Reverse genetics
Divisions:	Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User:	Symplectic Admin
Date Deposited:	08 Aug 2013 08:49
Last Modified:	16 Dec 2022 04:38
DOI:	10.17638/00009753
Supervisors:	Naylor, CJ Jones, RC
URI:	https://livrepository.liverpool.ac.uk/id/eprint/9753