Study On In Vitro Infected Model Of Tembusu Virus

Tembusu virus(TMUV)belongs to flaviviridae,flavivirus genus.Tembusu virus disease is a new acute infectious disease with high incidence and strong infectivity.It mainly causes dramatic decrease in feeding intake of ducks,sudden decrease in egg production,or even discontinuation of production,which brings serious economic losses to poultry industry in China.Due to the rapid changes in the disease of Tembusu,the anti-epidemic products against the virus need to be updated in time.Therefore,the mechanism of proliferation,transmission and pathogenesis of the strain of Du/CH/LSD/110128 should be further understood,but it is not applicable at present.To study the in vitro experimental model of this strain,it is necessary to establish a model of in vitro infection of the Tembusu.RT-PCR was used to detect that Du/CH/LSD/110128 strain of Tembusu virus could proliferate in avian cells DF-1,LMH,CEF,DEF,monkey cells Vero,human non-transformed cell lines 293 T,human transformed cell lines A549 and HCT116,but did not cause cytopathic effect(CPE).Du/CH/LSD/110128 strain virus can cause bleeding injury of duck embryo body after passage on duck embryo.However,the attenuated virus of chicken embryo can only proliferate in Vero cells,and does not cause duck embryo hemorrhage.CF1 and CF100 can be stably used as an in vitro model for studying the mechanism of increasing the value of this strain;DEF cytotoxicity of this strain and CF1 have no difference in virus proliferation in duck-derived cell DEF,but there is a difference in CPE,so it can be used as an in vitro study model of the pathogenic mechanism of strains on duck-derived cells;DEF cytotoxicity can also proliferate in human cells and trigger cytopathic effects in DEF cells.Based on the above results,this experiment selected DEF cytotoxicity to infect human non-transformed cell line 293 T as an in vitro infection model,using CRISPR/Cas9-based genome-wide gene function deletion screening technology to find the key to the pathogenic effect of human cells infected with Tembusu virus in host restriction factor.Bioinformatics analysis High-throughput sequencing results in 276 significantly down-regulated genes.The down-regulated genes may be genes that promote cell death after 293T-GeCKO cells infected with Tembusu.These down-regulated genes may be produced by virus-infected cells present in cells.The limiting factor of the lesion.In this study,KEGG cell signaling pathway analysis and GO functional annotation analysis were performed on these genes.It was found that biological processes such as Insulin secretion,Glucagon signaling pathway,intracellular calcium activated chloride channel activity and calcium iontransport may be important mechanisms for human cells to resist TMUV lesion.In this study,protein-protein interaction network analysis was carried out for 276 significantly down-regulated genes.It was found that POLR2 C may play a central regulatory role in the above process.In this study,we explored the transformation of Tembusu virus strains stored in our laboratory with different species of hosts and the interaction of non-transformed cells.We preliminarily determined the in vitro infection model of Tembusu virus for different research purposes,which laid a foundation for the follow-up study of the pathogenic mechanism of Tembusu virus.