Whole Genome Sequence Analysis Of Newcastle Disease Virus SH15 Strain And Its Analysis Of Oncolytic Effect To Colon Cancer Cells

Newcastle disease virus(NDV),as an acute infectious disease that threatens the development of poultry industry,has so far spread to more than 100 countries around the world,causing huge economic losses.At the same time,NDV as a study of oncolytic virus has a history of more than 50 years,with safety(harmless to humans),specificity(to kill a variety of cancer cells and less impact on normal cells),operability(to build recombinant virus with reverse genetic technology),has become a hot spot in the study of oncolytic therapy.In this paper,NDV SH15 strainwas used as material due to its good oncolytic effect on Lo Vo(colon cancer cell)and A 549(lung cancer cell)in a time-dependent manner.Tumor capacity has strain specificity.The results of biological experiments showed that the MDT of SH15 strain was 67 h and that of ICPI was 1.6,means it belong toMesogenic strain.The results of genome-wide analysis showed that the genome length of SH15 strain was 15 186 bp and accession number was KY247177,which belongs to class ? gene of class ?,suggesting that H203 on HN protein may be a potential key point for its oncolytic activity.Lo Vo and Lo Vo/Adr(colon cancer cells resistant to doxorubicin)were used to collect cell samples or culture supernatants at different time points after infection of SH15 strain.Virus replication was detected by microscopic photograph,CCK-8 and IFA test.Western Blotchecks the expression of PARP,Caspase-3,NP,Caspase-8,and Caspase-9.Apoptosis situation was examined by FCM,Hochest 33258 staining,TUNEL and Western Blot.Annexin V-FITC/PI staining was used to detect the viral replication.The expression of TNF-?,IFN-? and downstream MX1,IFIT3 and OAS were detected by real-time fluorescence quantitative PCR,and the corresponding protein was detected by ELISA.Hope to find the oncolytic mechanis m of SH15.The results are as follows:1.SH15 strain can effectively kill Lo Vo and Lo Vo/Adr,and the latter's killing effect is more obvious.Lo Vo/Adr had a sporadic adherent cell,and Lo Vo had half of the cells surviving.At 30 h after infection,Lo Vo/Adr was all in the floating state,while the Lo Vo field had a quarter cell adherents.The results of CCK-8 showed that Lo Vo activity decreased to 63.1% and Lo Vo/Adr activity decreased to 42.7% at 36 h after infection.The multi-step survival curve showed that the SH15 strain was nearly ten times as high as the replication rate in the two cells(36 h after infection).It was concluded that the difference of SH15 strain induced by different colon cancer cells was related to the rate of virus replication.2.The expression of Lo Vo and Lo Vo/Adr was mainly mediated by apoptotic pathway,and Lo Vo was dominated by exogenous apoptotic pathway(Caspase-8),while endogenous apoptosis pathway and exogenousapoptosis pathway(Caspase-8,Caspase-9)are both involvedin Lo Vo/Adr.Western blot was used to detect autophagy-related proteins(Beclin-1,LC3,and P62).It was found that there was no significant up-regulation of the two strains after infection with SH15 strain.Annexin V-FITC/PI staining confirmed that necrosis was not the main cause of both cell deaths.Apoptosis related proteins,such as PARP,Caspase-3,Caspase-8,Caspase-9 and other proteins,was inactivatedor activated after infected SH15.3.The difference of TNF-? expression between Lo Vo and Lo Vo/Adr virus was the main reason for the inconsistency of two kinds of cell death.The large number of TNF-? protein in Lo Vo/Adr resulted in rapid cell death.The mRNA level of TNF-? was significantly higher than that of Lo Vo at the 6th day after infection with Lo Vo/Adr(more than 12 times in the subsequent 12,18 h),and began to fall below Lo Vo after 24 h.The level of TNF-? protein,Lo Vo/Adr is always higher than Lo Vo.The results of TNF-?(including mRNA and protein level)of Lo Vo/Adrwere significantly higher than Lo Vo.4.The SH15 strain was able to induce the antiviral response of Lo Vo and Lo Vo/Adr,but the antiviral activity of Lo Vo was more obvious,while Lo Vo/Adr was weaker.The difference of replicationrate oftwo cells was determined with the difference ofanti-virus response.5.The IFN-? recognition on Lo Vo/Adr may be defective,resulting in extracellular accumulation of IFN-?affect the Lo Vo/Adr antiviral ability.