The application of reverse genetics to the study of pathogenicity in avian pneumovirus

Avian pneumovirus (APV) is a virus causing respiratory disease in birds, particularly in turkeys, resulting in significant losses to poultry farmers as well as suffering to the birds. The virus has eight genes that can cause infected cells to produce nine proteins each of which are then modified in various ways. Some of the proteins are able to make copies of the viral genome (a ribonucleic acid [RNA] molecule that comprises the eight genes with extra pieces at the ends and between them). Other proteins combine with the genome and membranes from the cell to form virus particles that may then infect new cells. The proteins affect the disease caused by the virus both by direct damage to cells and by the reaction of the bird's immune system to them. The immune system helps to clear infections by viruses but also is often responsible for signs of disease as the infected cells are destroyed. The importance of different proteins in the production of disease and also in the production of an effective immune response by weakened viruses used as vaccines is not understood. This is important because the vaccine viruses can become disease producing again. Genetic engineering enables deoxyribonucleic acid (DNA) copies of the virus genome to be copied into virus RNA genomes and eventually into infectious virus. It is possible to make changes in the DNA copies of the virus genome by changing, deleting or inserting nucleotides that are linked together to make up the DNA. These modified DNA copies can then be used to generate viruses and the properties of these viruses studied in cell cultures and turkeys. It provides a means of designing vaccines in a rational manner. The project aims to investigate the importance of various changes observed between different viruses either as a result of mutations or of different modifications of the proteins in different cell types on the tissues in the turkeys where the virus replicates and the disease caused. The importance of changes in vaccine strains for protection will also be studied with the ultimate aim of identifying mutations that will allow virus to be produced that can stimulate a protective immune response but is incapable of causing disease. We already know that deletion of one gene alters the effect of the virus on cells in culture, and in a related virus deletion of a similar gene affects whether the virus grows best in the nose and throat or lungs of various animals. We will study this protein, which has no known function, in more detail. Ultimately, as well as expanding knowledge about the functions of the virus genes in the interaction of the virus with its host information from this study should be useful in designing more effective vaccines benefiting farmers (and turkeys!).

Avian pneumovirus (APV) causes respiratory disease of economic importance to the poultry industry worldwide. Vaccines have been produced but are prone to reversion to virulence. The genetic basis of attenuation of APV is not understood. Our unpublished data shows that genome mutations, alterations in post-translational modifications or a combination of these may be involved. Virus is often attenuated by culture in mammalian cells which can be associated with differences in glycosylation of at least one of the viral surface glycoproteins. It is hypothesized that the site of replication of the virus may be restricted in attenuated viruses and that this enables the birds to develop a protective immune response before disease can develop. The two laboratories have identified a number of mutations associated with attenuation in different genetic backgrounds of APV and these are located in a restricted number of virus genes. Some alterations have been identified in the virus intergenic regions which may have an effect on virus gene expression. Using a reverse genetics system for APV developed collaboratively by the two applicant laboratories (Naylor et al., 2005, J Gen Virol, 85, 3219-3227) we will identify the mutations responsible for the attenuated phenotype in APV. Using the reverse genetics system specific, site-directed, mutations will be introduced into the virus genome either by PCR based methods or QuikChange mutagenesis (Stratagene) on subclones followed by cloning into a full-length clone we have modified to contain useful restriction sites. We will introduce singly and together a range of specific mutations into the APV genome from two different isolates and assess the effects on pathogenicity in birds. Using a dicistronic minigenome system (Randhawa et al., 1997, J Virol 71, 9849-9854) we will assess the impact of the intergenic mutations on expression of a downstream gene by measuring the relative expression levels of two reporter genes. Northern blot analysis and ribonuclease protection studies will enable measurements to be made on expression of actual genes in mutant viruses. The reverse genetics system also allows the insertion or deletion of genes. The green fluorescent protein ORF bounded by appropriate viral regulatory sequences will be introduced into full-length viral clones using assembly PCR. This virus will be used to follow the spread of virus in infected birds with wild type and mutant genomes. This will clarify the nature of any tropism restriction that exists with the viruses. This work will be carried out using virus propagated in different cell lines to assess the importance of host-derived factors in the initial stages of infection in vivo. In addition, we investigate the function of the SH protein in cell-cell spread of virus. In contrast to other viruses deletion of the SH gene alone in APV causes a phenotypic change with enhanced cell-cell fusion being produced in cell culture. Studies in cell culture on the importance of two apparent domains in the APV SH protein on its function will be carried out using viruses modified as above with alterations to conserved residues or systematic replacement of amino acids, the syncytial phenotype being used as a functional screen. The way in which this phenotype is established, either by loss of a fusion inhibiting function or of a function required for development of a non-syncytial phenotype, will be studied by induction of SH expression at different times after infection by SH deleted virus. Suitable cell lines will be established expressing functional or non-functional SH protein expressed in an inducible manner from the same locus in the chromosomal DNA using the tet Flp-In system (Invitrogen). This will provide information on the regulation of fusion activity and the function of the SH protein. The sub-cellular location of SH protein will be identified by immunofluorescence using virus expressing a tagged SH protein.