The Effects Of Recombinant NDV By V-Protein Of Virulent And Lentogenic Strain On Virus Replication And Cell Apoptosis

Newcastle disease(ND)is one of the most serious avian infectious diseases in the world and has a great impact on the poultry industry in many countries.The pathogen that causes the disease is Newcastle disease virus(NDV)thatis the virus has envelope,non-segmented,single-stranded,negative RNA chain.Studies have found that Interferon(IFN)has a certain antagonistic effect on NDV,and the V protein of NDV can antagonize the innate immune response mediated by the host IFN signaling pathway,but the specific mechanism is still unclear.To investigate the effects of V proteins of virulent and lentogenic strain on virus replication,IFN antagonist and cell apoptosis during infection,the LA-V and HB-Veukaryotic expression plasmids that expressing V proteins of virulent and lentogenic strains were constructed in this study.The infection efficiency of recombinant viruses r L-LA and r L-HB inserted with exogenous V protein was verified and evaluated.Then the innate immune levels of the recombinant virus were measured,including the expression levels of the critical proteins p-STAT1 and MDA5 in the IFN pathway,the production of IFN-?,the transcription levels of the downstream antiviral proteins 2'5'-OAS,MX1 and PKR,as well as the transcription levels of various innate immune molecules in the IFN pathway.Finally,the levels of cell apoptosis,membrane fusion and nuclear fragmentation,and caspase 3 cleavage after virus infection were detected.The test results are as follows:1.The detection and verification of virus infection efficiency.At the basis of measuring the transcription level of NP protein in the growth titer of the recombinant virus and the infection efficiency at different time,the expression level of V protein in the constructed eukaryotic expression plasmids of LA-V and HB-V and the ability to infect cells were detected and verified in this experiment.The results showed that the growth titers of the recombinant LA-V and HB-V virus were higher than the parent virus r L,and the insertion of exogenous V protein could play a role in virus infection and promoting the proliferation and replication of the virus.This experiment also found that the infection efficiency of LA-V was higher than that of HB-V,but theyboth had the ability of promote replication,and the V protein of the lentogenic strain bounding cells was higher than the V protein of the virulent strain.2.The determination of innate immune levels of virus.The results of the determination of innate immune level showed thatthe P-STAT1 and MDA5 expression of r L-LA was lower than r L and r L-HB,r L-LA could more suppress IFN-?.Their transcription levels of IFN are also lower than r L-HB.The results showed that the ability of r L-LA antagonist was stronger than r L-HB.r L-LA could replicate in the cell for a long time.These results suggested that V protein might play an important role in the immune escape mechanism of the virus.The insertion of V protein could inhibit the transcription level of immune molecules to a certain extent,reduce the antiviral effect,and make cells unable to play the role of antiviral.3.Detection of apoptosis after virus infection membrane fusion,apoptosis of DF1 and A549 cells after virus infection,karorrhexis and the cleavage of apoptotic protein(caspase 3)were detected in this experiment.The test results showed that,with trypsin,parental toxicity r L failed to cause cell fusion,and the recombinant virus observed cell fusion at 24 h.Even without trypsin,and the recombinant virus still induced cell fusion at 48 h.By Hoechst staining,the number of cells in r L-HB infection group was the least and the nuclear fragmentation was the most obvious.The level of caspase 3 cleavage increased after virus infection,and the highest level of caspase 3 cleavage protein was found 24 h after r L-HB infection.To sum up,V protein could promote virus replication and proliferation.V protein could affect the early production of IFN.At the same time,V protein might assist F protein to promote cell fusion during virus infection.The results of this experiment can provide reference for the pathogenic mechanism of NDV virulent and lentogenic strains,and it has great significance for exploring the pathogenic mechanism of virus.