The Establishment Of A Platform For Defective Interfering Particles Inhibiting The Replication Of Newcastle Disease Virus

The minigenome(MG)of Newcastle disease virus(NDV)retains the key sites recognized by NDV polymerase,and the rest of virus genome is replaced by reported gene.The transcription of NDV MG depends on viral NP,P,and L proteins.The genetic element of MG is similar to that of the defective viral genomes(DVGs).Due to the low fidelity of viral polymerase in replication process of the genome,DVGs are resulting in partial deletion of the genome,but retaining the key elements of viral polymerase reproductive and transcriptional recognition.The virus particles containing DVGs are also called defective interfering particles(DI or DIPs).In recent years,DIPs have been applied in clinic as vaccine adjuvant,which provide a reference for us to develop more efficient ND vaccine for further control the spread of ND epidemic.At present,the study on DIPs in NDV is still blank,and whether DIPs existed in NDV is unknown.In this study,MG was used instead of DVGs to establish a platform for DIPs to inhibit NDV replication.By infecting BHK-21 cells expressing MG with NDV,it was found that MG could be packaged into DIPs and then inhibit the replication of NDV.The establishment of this platform lays a solid foundation for the development of NDV DIPs vaccine,and has importantly theoretical and practical significance for the prevention of ND.The main contents and results of this study included the following several aspects.1.Construction and optimization of NDV MG system.Firstly,the genetic composition of NDV MG was determined,including Leader sequence,NP 3'UTR,reporter gene(EGFP),L 5'UTR and Trailer sequence.Then plasmids expressing MG,NP,P and L proteins under T7promoter and CMV promoter were constructed respectively,and combined with each other,and then co-transfected into BHK-21 cells.The efficiency of combined MG systems was analyzed by fluorescent microscopy and flow cytometry.Through the analysis of rescue efficiency,it was found that the efficiency of MG system composed of MG under T7 promoter and helper plasmids under CMV promoter was significantly higher than that of other combinations.Finally,the reporter gene EGFP of MG was replaced by Cherry,and Cherry was still highly expressed,indicating that the MG system was generally applicable no matter which fluorescent gene was replaced by the reporter gene.2.Optimization of conditions of MG expression driven by NDV.In order to drive the expression of MG efficiently by NDV,we explored the following conditions in BHK-21 cells:(1)The ability of different NDV strains to drive MG expression;(2)The exploration of the interval time between transfection and infection;(3)The exploration of the amount of virus infection;(4)The exploration of expression time of NDV infection driving MG.The expression of EGFP in BHK-21 cells was detected by fluorescenct microscopy and Western Blot.It was found that NDV C22 strain had the strongest ability to drive MG expression when BHK-21 cells were transfected with MG for 4 h and infected with the 2 MOI virus.And with the increase of NDV-driven MG expression time,the expression of the reporter gene also gradually increased,and the expression of EGFP was the highest after 60 h.3.The effect of DIPs on NDV replication.DIPs were packaged using C22 strain with chimeric EGFP(r C22-EGFP)and MG plasmid expressing Cherry(MG-Cherry).Follow the conditions we have explored before,BHK-21 cells were infected with 2 MOI r C22-EGFP 4 h after transfection with MG-Cherry.After 60 h,the supernatant of cells was collected and infected into chicken embryos.Then,BHK-21 cells were infected with the obtained allantoic fluid(named MG-C22).The expression of Cherry and EGFP was detected by fluorescenct microscopy and Western Blot,and it was found that MG-Cherry could be efficiently packed into DIPs.Then BHK-21 cells were infected with the same amount of MG-C22 and r C22-EGFP,and the virus content was detected by Western Blot,q RT-PCR,and TCID₅₀.The results showed that DIPs inhibited the replication of NDV.Futhermore,the ICPI of MG-C22 and r C22-EGFP showed that DIPs reduced the virulence of NDV.In conclusion,this study optimized the NDV MG system,determined the conditions for NDV to efficiently drive the expression of MG,and successfully packaged MG into DIPs.Further study found that DIPs could inhibit the replication of NDV and reduce the viral virulence.