The Mechanism Of SOCS3 In NDV Escapes Host Innate Immunity Based On Transcriptome Sequencing

Newcastle disease?ND?caused by Newcastle disease virus?NDV?is one of the most important infectious diseases.Previous studies have shown that the non-structural protein V protein of NDV plays an important role in antagonizing host innate immunity,but there are few reports on how NDV regulates host protein to escape from host innate immunity.Therefore,by exploring the changes of differentially expressed genes induced by NDV infection in the host,the molecules that affect NDV replication were screened out and the mechanism was studied,which may provide a theoretical basis for revealing the pathogenic mechanism of NDV and the escape of the host innate immunity.As a lymphoid organ,the bursa of Fabricius?BF?plays a pivotal role in destroying invading pathogens.Virulent NDV strains can cause rapid atrophy of the BF;however,there is limited knowledge regarding the BF innate immune response to NDV infection.In this study,4-week-old chickens were randomly chosen and inoculated with 0.2 mL of plaque-forming units?PFU?of F48E9 via the intranasal route,and the negative control group was injected with the same volume of PBS.We randomly collected the bursa of fabricius at 48 and 72 hpi and the negative control group for transcriptome sequencing analysis.A total of 256 differential expression genes?DEGs?were identified at 48 hpi,and 216 DEGs were upregulated genes and40 DEGs were downregulated genes.A total of 1,419 DEGs were identified at 72 hpi,and 1132 DEGs were upregulated and 287 DEGs were downregulated.The significantly enriched KEGG pathways?p<0.05?at 48 hpi and 72 hpi were analysised.In total,28 KEGG pathways were identified during the process of NDV infection at these two time points.Among the 28 KEGG pathways,there were nine at 48 hpi and19 at 48 hpi.Nine of the 28 pathways are associated with metabolism and ten with the immune response,and most of these signaling pathways were related to immune response and metabolism pathway.NDV infection induced a strong innate immune response in the bursa of fabricius,but failed to clear the invading virus.In order to explore how NDV escaped the host immune response,we analyzed co-expressed DEGs at 48 and 72 hpi,and found that the suppressor of cytokine signaling 3?SOCS3?gene was consistently expressed at these two time points.The SOCS3 inhibits type I interferon-dependent antiviral signaling pathway by utilizing a feedback loop.However,the potential molecular mechanism by which NDV activates SOCS3 expression is unclear.In order to explore the role of SOCS3 in the process of NDV infection,we firstly found F48E9 infection could induce the expression of SOCS3 in the CEF cells,and the expression of SOCS3was correlated with the titer and virulence of the virus.Next,we found that non-structural proteins V protein of F48E9 played an important role in inducing the expression of SOCS3,and the c-terminal of V protein played a key role.The proliferation of NDV was significantly promoted or inhibited by overexpression or inhibiting the expression of SOCS3.Mechanism research showed that SOCS3 can affect the phosphorylation of STAT1 to influence the interferon stimulated genes?Interferon-stimulated genes,ISGs?expression of OASL and MX1 to influence the proliferation of NDV,and the MEK/ERK signaling pathway also plays a significant role in inducing the expression of SOCS3 during NDV infection.Studies have shown that miRNAs play a significant role in viral infection.In this study,bioinformatics was used to analyze and screen out predictive miRNAs that target and regulate the expression of SOCS3?gga-miR-455-5p?.Double luciferase reporter gene assay,qRT-PCR and Western Blot was used to detect the regulate between gga-miR-455-5p and SOCS3.Moreover,NDV infection could inhibit the expression of miR-455-5p in CEF cells.Transfected with miR-455-5p mimics or inhibitors can significantly inhibit or promote the proliferation of NDV.Mechanism studies showed that gga-miR-455-5p not only regulates the expression of SOCS3,but also degradats the expression of NDV NP gene and enhancing the expression of IFN?,OASL and MX1.In conclusion,by analyzing transcriptional data of chicken bursa of fabricius at48 and 72 h infected by F₄₈E₉,this study demonstrated that NDV infection in CEF cells can induce SOCS3 expression through MEK/ERK signaling pathway and inhibition of gga-miR-455-5p expression,thereby helping the virus escape from the host innate immune response through inhibition of JAK-STAT signaling pathway.