Generation of Avian Pneumovirus Modified Clones for the Development of Attenuated Vaccines

Abstract (one page maximum, single spaced), include: List the original objectives, as defined in the approved proposal, and any revisions made at the beginning or during the course of project: The main goal described in our original proposal has been the development of a molecular infectious clone of the avian metapneumovirus subtype B (aMPV-B) and the modification of this clone to create mutated viruses for the development of attenuated vaccines. The Achievements and Appendix/Part I sections of this report describes the accomplishments in creating such a molecular clone. These sections also contain the results of a longitudinal study that we made in Israel, demonstrating the infiltration of field strains of aMPV into vaccinated flocks and emphasizing the need for the development of better vaccines. We also describe our unexpected findings regarding the ability of aMPV to establish persistent infection in cell cultures. Although this direction of research was not described in the original proposal we feel that it is highly important for the understanding of aMPV pathogenesis. For example, this direction has provided us with evidence showing that aMPV replication can augment influenza replication. Moreover, we observed that viruses that were produced from chronically-infected cells show reduced ciliostasis. Accordingly, we carried vaccination trials using such viruses. In the original grant proposal we also offered that the American lab will clone and express immunomodulators in the context of an aMPV -based replicon that the Israeli lab has generated. However, as we reported in our annual reports, further analysis of this replicon by the Israeli lab has revealed that the level of expression achieved by this vehicle is relatively poor; thus, the American lab has focused on sequencing the genomes of different aMPV-C isolates that differ in their virulence (including vaccine strains). Achievements and Appendix/Part II sections of this report include the summary of this effort. Background to the topic: The aMPVs belong to the paramyxoviridae family and cause mild to severe respiratory tract diseases mainly in turkeys and also in chickens. Four aMPV subgroups, A, B, C and D, have been characterized; in Israel aMPV-A and B are the common subtypes while in the USA type C is the prevalent one. Although vaccine strains do exist for aMPVs, they do not always provide full protection against virulent strains and the vaccines themselves may induce disease to some extent. Improved vaccines against aMPV are needed, to achieve better protection of the poultry industry against this pathogen. Major conclusions, solutions, achievements: We isolated aMPV-B from a diseased flock and accomplished the sequencing and cloning of its full-genome. In addition, we cloned the four genes encoding the viral replicase. These should serve as the platform for generation of modified aMPV-Bs from molecular clones. We also identified aMPVs that are attenuated in respect to their ciliostatic activity and accordingly showed the potential of such viruses as vaccine strains. For aMPV-C, the different mutations scattered along the genome of different isolates with varied virulence have been determined. Implications, both scientific and agricultural: The newly identified pattern of mutations in attenuated strains will allow better understanding of the pathogenicity of aMPV and the generation of aMPV molecular clones, together with isolation of strains with attenuated ciliostatic activity should generate improved vaccine strains Abstract (one page maximum, single spaced), include: List the original objectives, as defined in the approved proposal, and any revisions made at the beginning or during the course of project: The main goal described in our original proposal has been the development of a molecular infectious clone of the avian metapneumovirus subtype B (aMPV-B) and the modification of this clone to create mutated viruses for the development of attenuated vaccines. The Achievements and Appendix/Part I sections of this report describes the accomplishments in creating such a molecular clone. These sections also contain the results of a longitudinal study that we made in Israel, demonstrating the infiltration of field strains of aMPV into vaccinated flocks and emphasizing the need for the development of better vaccines. We also describe our unexpected findings regarding the ability of aMPV to establish persistent infection in cell cultures. Although this direction of research was not described in the original proposal we feel that it is highly important for the understanding of aMPV pathogenesis. For example, this direction has provided us with evidence showing that aMPV replication can augment influenza replication. Moreover, we observed that viruses that were produced from chronically-infected cells show reduced ciliostasis. Accordingly, we carried vaccination trials using such viruses. In the original grant proposal we also offered that the American lab will clone and express immunomodulators in the context of an aMPV -based replicon that the Israeli lab has generated. However, as we reported in our annual reports, further analysis of this replicon by the Israeli lab has revealed that the level of expression achieved by this vehicle is relatively poor; thus, the American lab has focused on sequencing the genomes of different aMPV-C isolates that differ in their virulence (including vaccine strains). Achievements and Appendix/Part II sections of this report include the summary of this effort. Background to the topic: The aMPVs belong to the paramyxoviridae family and cause mild to severe respiratory tract diseases mainly in turkeys and also in chickens. Four aMPV subgroups, A, B, C and D, have been characterized; in Israel aMPV-A and B are the common subtypes while in the USA type C is the prevalent one. Although vaccine strains do exist for aMPVs, they do not always provide full protection against virulent strains and the vaccines themselves may induce disease to some extent. Improved vaccines against aMPV are needed, to achieve better protection of the poultry industry against this pathogen. Major conclusions, solutions, achievements: We isolated aMPV-B from a diseased flock and accomplished the sequencing and cloning of its full-genome. In addition, we cloned the four genes encoding the viral replicase. These should serve as the platform for generation of modified aMPV-Bs from molecular clones. We also identified aMPVs that are attenuated in respect to their ciliostatic activity and accordingly showed the potential of such viruses as vaccine strains. For aMPV-C, the different mutations scattered along the genome of different isolates with varied virulence have been determined. Implications, both scientific and agricultural: The newly identified pattern of mutations in attenuated strains will allow better understanding of the pathogenicity of aMPV and the generation of aMPV molecular clones, together with isolation of strains with attenuated ciliostatic activity should generate improved vaccine strains.