Expression of transgenes from newcastle disease virus with a segmented genome

Paramyxoviruses belong to the Paramyxoviridae family of the order Mononegavirales. They have a nonsegmented negative-stranded RNA genome and can cause a number of diseases in humans and animals. We generated a recombinant Newcastle disease virus (NDV) possessing a two-segmented genome. Each genomic segment is flanked by authentic NDV 3' and 5' noncoding termini allowing for efficient replication and transcription. A reporter gene encoding green fluorescent protein (GFP) was inserted into one segment, and a red fluorescent protein dsRed gene was inserted into the other segment in order to easily detect the replication and transcription of segments in infected cells. The rescued viruses grew well and were stable in embryonated chicken eggs over multiple passages. We were able to detect the expression of both reporter genes in the same cell infected with the virus possessing a segmented genome, and viral particles can contain either one or two types of RNA segments. We also rescued a two-segmented virus expressing GFP and the severe acute respiratory syndrome-associated coronavirus spike S protein, which is about 200 kDa. The chimeric virus extends the coding capacity of NDV by 30%, suggesting that the two-segmented NDV can be used for development of vaccines or gene therapy vectors carrying long and multiple transgenes.