Effect Of Interferon Stimulated Gene 15 On The Replication Of Porcine Deltacoronavirus

Porcine deltacoronavirus(PDCoV)is a newly discovered coronavirus that causes acute diarrhea,dehydration and even death in pigs.At present,PDCoV has spread to major pig breeding countries in the world.Due to the lack of specific therapeutic drugs and preventive vaccines,it has caused serious losses to the global pig breeding industry.Ininnate immunity is the body’s first line of defense against microbial infection,and the host activates cells to produce antiviral factors against viral invasion by upregulating interferon-stimulating Genes(ISGs)in response to innate immunity.ISG15 is one of the earliest and most highly expressed ISGs,which can induce the production of type I interferon and other antiviral factors in host cells,and has a negative regulation effect on the replication and assembly of many viruses.However,the role of ISG15 in the process of PDCoV infection and its effect on viral replication is not yet clear.This study explores the influence of ISG15 on PDCoV replication,and the main research contents and results are as follows:1.In this study,ISG15 stable overexpression and silencing cell lines of LLC-PK1 cells were constructed respectively.The results showed that PCDNA3.1-m Cherry-ISG15 overexpression vector was successfully constructed by designing the ISG15 overexpression primer,and then the recombinant cell line LLC-PK1-ISG15-OE with stable overexpression of ISG15 was screened by using genetic mycin(G418).The expression of ISG15 was 12 times higher than that of normal cells(P<0.001).At the same time,three sh RNA interference sequences were designed.The recombinant vector pcDNA3.1-Double U6-XK1-BE2,pcDNA3.1-Double U6-XK1-BE3 and pcDNA3.1-Double targeting porcine ISG15 gene were successfully constructed using pcDNA3.1-Double U6-XK1-BE3 and pcDNA3.1-Double U6-XK2-BE3;The recombinant vector was transfected into LLC-PK1 cells,respectively,and the recombinant LLC-PK1 cell line with stable silencing ISG15 was screened out.The recombinant cell line LLC-PK1-ISG15-KD-3 with the best silencing effect was screened by RT-PCR,RT-q PCR and WB,and its ISG15 expression level was only 0.33 times that of the normal cell(P<0.001).2.The influence of ISG15 on PDCoV replication was explored.The results showed that within 30 h after LLC-PK1 cell inoculation,the expression of ISG15 increased gradually with the time of virus infection,and increased to 2164 times at 30 h,but the expression of ISG15 decreased sharply after 30 h.The expression of ISG15 increased gradually with the infection dose,but decreased sharply when the infection dose increased to 2 MOI.The replication of PDCoV was significantly inhibited in LLC-PK1 cells stably overexpressing ISG15(P<0.01 or <0.001),and at 6 h,12 h,18 h and 24 h after inoculation,the expression level of PDCoV m RNA in overexpressed cell lines was 0.698 of that in normal cell lines(Students’ not test,P<0.001),0.628(P<0.001),0.643(P<0.001)and 0.580times(P<0.001);Replication was significantly promoted in silenced ISG15 recombinant cell lines(P<0.01 or P<0.001),at 6,12,18 and 24 h after inoculation,the expression level of PDCoV m RNA in silent cell lines was 2.913 that of normal cell lines(P<0.001),2.848(P<0.001),2.919(P<0.001)and 2.897 times(P<0.001);The results of IFA and WB also confirmed the same trend.In conclusion,ISG15 can inhibit the replication of PDCoV,which will provide experimental basis and new ideas for elucidating the interaction between PDCoV and ISG15 and antiviral research.