Reverse Genetics Systems Of NDV: Basic And Applied Research Straine

The establishment of reverse genetic for non-segment negative single strand RNA viruses enabled Newcastle disease virus (NDV) to be the extremely attractive recombinant live viral vaccine candidates. In this study, NDV LaSota vaccine strain that has been widely used for control and prevention of Newcastle disease in chicken was rescued from genome cDNA clone by reverse genetic. The genome of LaSota, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. The plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large polymerase protein into cells infected with vTF7-3, a recombinant vaccinia virus expressing T7 RNA polymerase. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen free (SPF) chicken eggs. The rescued NDV, rLaSota, was indistinguishable from the parental wild-type virus. The rLaSota kept similar characteristics with parent LaSota vaccine strain, including high growth titer (10^10.09 EID₅₀/ml allantoic fluid) and low pathogenicity (MDT=96, ICPI=0.35-0.37 and IVPI=0), and efficient eliciting high titer HI antibody (2^7-9) response in new born chicken, and protecting immunized chickens from lethal challenge with highly virulent strain F48E9. The results indicated that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.A recombinant NDV expressing the enhanced green fluorescent protein (EGFP) was generated by applying the same reverse genetics techniques. The EGFP 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 LaSota. 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 EGFP in live infected cells confirmed further the recovery of a recombinant LaSota (rLaSota-EGFP) expressing this reporter gene. After 20 successive passages in...