Avian Metapneumovirus Reverse Genetics Developments and their Effect on the Performance of Recombinant Live Vaccines

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 vectoring abilities of subtype A strains have been also investigated. The aim of this study was to develop a new RG system in order to extend this type of studies to the B subtype, gain a better understanding of the viral capacity to accept and express heterologous extra sequences. Initially was performed a nucleotide and amino acid comparison between subtypes A and B, to establish whether subtype A RG components could be partially or fully substituted. Eventually a cDNA copy of subtype B genome was generated and the virus rescued using subtype A and B RG components. The vectoring abilities of subtype B was firstly investigated to accept and express both spike (S1) and nucleocapsid (N) genes of infectious bronchitis virus (IBV). After viruses have been recovered by RG, all recombinants were found to be highly stable during passage in vitro and subsequently tested as candidate vaccines by eye-drop inoculation of one-day-old chickens. When chicks were challenged with IBV, negligible protection and replication results were observed. To improve the ability of the recombinant to induce protective immunity, the sequence of subtype B cDNA was modified in order to increase the replication in the respiratory tract. The new recombinants were rescued and challenge in vivo, showing an increase of the protection. Other strategies were adopted to improve the protection, as increasing the expression of the foreign genes and multiple genes insertions, but in most of the cases the virus rescue proved to be not possible.