Newcastle disease(ND) is a serious avian disease distributed throughout the world that can cause substantial economic losses and remains a major threat to the poultry industry in every country. The aetiological agent,Newcastle disease virus NDV is a member of the genus Avulavirus in the family Paramyxoviridae (11). The genome of NDV is a non-segmented,single -stranded,negative-sense RNA of 15 186,15 192 or 15 198 (2) nucleotides in length,which contains six genes encoding the nucleocapsid protein (NP),phosphoprotein (P),matrix protein (M),fusion protein (F),haemagglutinin-neuraminidase (HN) and large polymerase protein (L). The NP protein binds to the genomic RNA to form a remarkably stable nucleocapsid core structure to which P and L proteins are attached. This ribonucleoprotein (RNP) complex,rather than naked viral RNA,is the template for replication and transcription by the viral polymerase (P and L protein). The NDV genes are linked in tandem in the order 3′-NP-P-M-F-HN-L-5′and are separated by junction sequences that consist of three elements,known as gene-end (GE),intergenic (IG) and gene-start (GS) sequences. For most members of the subfamily Paramyxovirinae,including NDV,efficient replication is dependent on the genome length being a multiple of six. This requirement is known as the 'rule of six' (14).
Reverse genetics manipulation technique is based on intracellular transcription of viral full-length RNAs and simultaneous expression of viral proteins required to form the typical viral RNP complex. The first recovery systems,based on a lentogenic vaccine strain (LaSota) of NDV,were reported simultaneously by two independent groups in 1999 (13, 15). The strategy to recover NDV was to transfect cells with four plasmids encoding the full-length antigenomic viral RNA,the NP protein,and RNA polymerase proteins (P and L) respectively. This transfection resulted in the production of RNPs,leading to the generation of infectious NDV.
Because the rescued virus was wholly derived from cloned cDNA,gene manipulation such as deletion and insertion mutations could be done to investigate virus-host interactions,the molecular basis of NDV pathogenesis,and the strategy for eliminating NDV. Also,using NDV to express foreign gene of other pathogen has been a hot area in vaccine development. Since Krishnamurphy (8) reported that NDV could be used as an expression vector,several genes of other viral protective antigens had been inserted into the NDV genome (6, 12). In this study,we established the reverse genetics system for an NDV ZJI strain of goose origin and inserted the GFP reporter gene between the P and M genes to evaluate the possibi-lity of the expression of a foreign gene in the velogenic NDV and to use the GFP-tagged NDV for investiga-ting the pathogenesis of virulent NDV.
Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene
- Received Date: 14 August 2006
- Accepted Date: 17 October 2006
Abstract: Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF)cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.