Development and use of avian pneumovirus reverse genetics systems

• Main Author: Edworthy, Nicole Lynn
• Published: University of Warwick 2008
• Subjects: 571.99211; QH426 Genetics
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.524224

Avian pneumovirus (APV) has remained an important pathogen of domestic fowl since its isolation in the 1970s. A reverse genetics system for APV was developed that affords direct manipulation and analysis of the molecular biology, pathogenicity, and tropism of APV. Using a synthetic minigenome system, the M2-1 protein was found to enhance transcription but not be essential for replication and the APV M2-2 protein was shown to inhibit transcription of a reporter gene. The viral cis-acting sequences were mutated to determine their role in transcription. Initially, a series of mutations originating from vaccine candidates were introduced into the gene end sequence of the LUC gene. The levels of LUC reporter protein expression in the mutants was 40-70% of normal, thus demonstrating a mechanism for reduction of virus immunogenicity as the result of a single point mutation. Heterologous rescue of the APV minigenome was carried out using plasmids expressing the RSV, PVM and hMPV proteins and showed that homologous protein: protein interactions were necessary for minigenome transcription. An APV cloned virus rescue system (Naylor et al., 2004) was used to create APV viruses which contained the gene encoding enhanced green florescent protein (eGFP) either within intact APV, or in mutants lacking the SH and G genes or lacking the SH gene alone. It was demonstrated that the SH and G genes are not essential for APV replication in vitro and in vivo and that the APV genome is capable of accepting insertions of foreign material. Expression of eGFP from the recombinant viruses was investigated in vivo in turkeys at 3 and 5 days post infection. eGFP was found in the sinus tissue of the birds infected with the virus containing the full complement of virus genes in addition to that encoding eGFP.