THE DEVELOPMENT OF A SUBTYPE B AMPV REVERSE GENETICS SYSTEM

A reverse genetics system is the only available method to introduce specific mutations into the AMPV genome. The virus has a negative sense genome but even transfecting a positive sense copy into AMPV susceptible cells will not lead to recovery of virus. This is because the virus polymerase is only able to copy and transcribe the RNA genome when as a component of the ribonuclear protein complex (RNP complex) which also includes the nucleocapsid (N) protein, phosphoprotein(P) and M2 protein. If plasmids coding for N, P, M2, the polymerase and a full length genome copy are transfected into suitable cells together, under the control of appropriate promoters, virus will be generated.To date no system for the AMPV subtype B had been reported. In the system described, plasmids for N, P, M2, the polymerase and the full genome were constructed under the control of the T7 promoter. Producing intact clones containing the virus polymerase gene proved highly technically demanding due to the issue of them containing sequences toxic to bacteria, thus leading to sections of the gene being spontaneously deleted. Nonetheless, once all clones had been generated and transfected into Vero cells previously infected with a fowlpox recombinant virus expressing T polymerase, AMPV was recovered.