Construction And Preliminary Application Of A Two-Plasmid Rescue System For Newcastle Disease Virus

Reverse genetics of RNA viruses is a method that involves constructing a full-length c DNA clone of the viral genome,followed by artificial manipulation and modification of the RNA virus at the DNA level,and ultimately obtaining infectious progeny virus through in vitro transcription.Newcastle disease virus(NDV),a single-stranded negative-sense RNA virus,requires transfection of helper plasmids expressing the NP,P,and L proteins,as well as c DNA clone plasmids,during the virus rescue process.The expression of these components within the cell leads to the formation of RNP complexes,triggering transcription of the viral genome RNA and synthesis of the various structural proteins,culminating in the assembly of complete virus.NDV attenuated vaccine has significant advantages as a viral vaccine vector,such as high safety,genetic stability,no integration into host genes,and convenient administration.To achieve artificial manipulation of the virus,the establishment of a reverse genetics operation technique and virus rescue system is indispensable.The appearance of the RNA polymerase II reverse genetics operation system greatly simplifies the reverse genetics operation of negative-sense RNA viruses and enhances the efficiency of virus rescue.However,the traditional four-plasmid system has low rescue efficiency and requires the genome plasmid and three helper plasmids to enter a single cell for replication and expression in order to complete virus assembly.To reduce the number of transfected plasmids and improve the efficiency of Newcastle disease virus(NDV)rescue,in this study we constructed a full-length c DNA vector of the NDV La Sota strain genome and a multi-promoter expression vector that can simultaneously express NP,P,and L proteins.A double-plasmid high-efficiency NDV rescue system based on the CMV promoter was established.According to the genome sequence of the NDV La Sota strain,the full genome was divided into five overlapping gene fragments for segmental cloning.The adjacent fragment overlap parts were spliced together in vitro using restriction enzyme cutting sites to obtain the complete NDV genome full-length c DNA.Hammerhead ribozyme(Ham Rz)sequence and hepatitis delta virus ribozyme(Hdv Rz)sequence were introduced at the 5’ and 3’ ends,respectively.The construct was cloned into the eukaryotic expression vector p CI containing the human cytomegalovirus(CMV)promoter.In addition,the NP,P,and L genes were separately amplified and fused with promoters and poly A signals to construct the triple-promoter auxiliary plasmid p CI-NPL,which could co-express NP,P,and L proteins.Western blot and indirect immunofluorescence were used to confirm the independent and correct expression of NP,P,and L proteins.The NDV La Sota strain full-length c DNA cloning plasmid p CI-La Sota and auxiliary plasmid PCI-NPL were co-transfected into BHK-21 cells in a 2:1 ratio using the calcium phosphate transfection method.After 5 days,the transfected cell supernatant was collected and inoculated into the cloaca of 9-11-day-old SPF chicken embryos.After incubation for 5 days,the cloacal fluid was collected for the hemagglutination test.Positive cloacal fluid was confirmed by RT-PCR and indirect immunofluorescence using NDV-positive serum,indicating the successful rescue of the recombinant virus r La Sota.Therefore,the novel double-plasmid rescue system for NDV La Sota strain was successfully constructed.To validate the effectiveness of the dual-plasmid rescue system as an NDV vector development platform,an enhanced green fluorescent protein(EGFP)expression cassette was cloned and inserted between the P and M genes of the NDV genome using the dual-plasmid rescue system.Recombinant virus r La Sota-EGFP expressing EGFP was successfully rescued,and obvious spontaneous green fluorescence was observed under a fluorescence microscope,with Western blot confirming correct expression of EGFP.Based on the EGFP reporter gene,the rescue efficiency was evaluated.And the results showed that the rescue efficiency of the dual-plasmid system was higher than that of the traditional four-plasmid rescue system.The results of hemagglutination titer,EID50,MDT,and ICPI,detection showed that the biological characteristics of the recombinant viruses r La Sota and r La Sota-EGFP were similar to those of the parental wild-type strain.Compared with the traditional four-plasmid system,the dual-plasmid rescue system can provide higher efficiency in the actual production process,laying the foundation for the rescue of NDV and other paramyxoviruses.