[Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein]

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein (P) and matrix protein (M) in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts (CEF) cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDV-GFP) expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive ailantoic fluid were used for further egg passages. All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1 x 10(4) EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers (10(9) EID50/mL). In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1 x 10(6) EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.