Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene / 中国病毒学

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.

Deletion of Nucleotides of NS Gene from 263 to 277 Decreases the Viral Anti-IFN Ability of H5N1 / 微生物学通报

Since 2000,most of H5N1 subtype influenza A virus had a unique mutation of NS gene with 15base pair deletion from 263 to 277. In order to investigate the bio-characteristics of this mutation,two different NS recombinants,RWSN-248 and RWSN-m248,were generated via plasmid rescue from A/WSN/33(H1N1) and A/SD/04(H5N1). RWSN-248 had a higher viral titer than RWSN-m248 in MDCK and COS-1 cells that have an IFN response,but they had the similar growth ability in Vero cells that lack an IFN response. Both of two recombinants grew well in embryonated chicken eggs and had the similar viral titer and MDT. The results above revealed that the deletion from 263 to 277 sites of NS gene did not influence viral virulence to but decreased viral anti-IFN ability of H5N1.