Pathogenicity Of Novel Duck Orthoreovirus And Its Mechanism Of Induction Of Ferroptosis In Macrophages

Duck spleen necrosis was a new infectious disease caused by Duck orthoreovirus(DRV)infection,which first occurred in China in 2006.The spleen damage caused immunosuppression,which increases the susceptibility of ducks to other pathogens and increases the mortality rate of the disease.In previous reports,the mortality rate of duck spleen necrosis was usually 5-15%.In 2017,an infectious disease characterized by severe spleen necrosis occurred in a large-scale duck farm in Shandong Province,and the mortality rate reached 50%,which seriously endangered the safety of duck breeding.It was identified that Novel duck orthoreovirus(N-DRV)was the pathogen that caused the new epidemic of duck spleen necrosis.As an important immune organ,the spleen has a pivotal role in immune response,pathogen clearance,and injured cells clearance.The invasion of the spleen by pathogenic infection can promote the replication,spread and development of the disease.N-DRV infection can cause severe spleen necrosis,but the pathogenesis by which it induces spleen damage remains unclear.To date,relevant studies on the mechanism of N-DRV-induced spleen injury have received scant attention in the research literature.In order to systematically study the mechanism of N-DRV-induced splenic necrosis,this research clarified the invasiveness of N-DRV on immune organs such as the spleen,and determined that N-DRV mainly damages spleen macrophages and lymphocytes.The differentially expressed genes related to cell damage were screened by RNA-Seq,and the main mode of cell damage was determined based on this.Combined with molecular biology techniques such as fluorescent probe detection and RNAi,the important role of ferroptosis in spleen injury induced by N-DRV infection was verified,and identified the regulatory mechanism of N-DRV-induced macrophage ferroptosis.This study will contribute to a deeper understanding of the mechanism by which N-DRV and other orthoreoviruses induce cell damage.The research has been organised in the following three aspects:1.Etiology and pathogenicity of N-DRVIn this research,three N-DRV strains(SDYC,SDLS and XT18)were isolated from clinical cases of splenic necrosis,and the whole genome sequence were obtained by Next-generation sequencing(NGS).Combined with the other representative strains of waterfowl origin in the NCBI database,a systematic analysis of homology alignment,genetic evolution,nucleic and amino acid variation was carried out.The results showed that the nucleotide homology of each gene fragment between N-DRV isolate SDYC strain and DRV isolates(TH11 strain,091 strain,HN5d strain)was 86.1?99.2%,and the amino acid homology was 95.1?100%.The three N-DRV strains located in different lineages on the encoding genes of?B,?C,?A and?NS,suggesting that there may be differences in the sources of these gene fragments.Our visualization of the genome showed that the SDYC strain had obvious mutations in the?B,?A,?A,?NS protein coding regions and other waterfowl-derived isolates,and was significantly different from the classical DRV strain in the?A coding gene.The proteins encoded by ARV contain conserved motifs with different biological functions.On?and?group and?A,?B,?NS,p10,p17 proteins,the functional motifs of N-DRV and classical DRV isolates were highly conserved,but on the amino acid motifs of?C protein,the N-DRV isolate and the classic DRV-HN5d strain had large variation at amino acid site of 119?177 and 151?325,which were responsible for antigenicity and receptor binding,respectively.As for the protein structure analysis,we found that SDYC strain had multiple amino acid variations in?-helix and?-sheet structures in?B and?A proteins.1-day-old ducklings were infected with N-DRV by intravenous injection,and the pathogenic mechanism of N-DRV was explored through pathology,histopathology,immunocytochemistry and observation of viral load in organs.Pathological studies suggested that N-DRV has strong invasive power to immune organs such as spleen,thymus,and bursa.The spleen of ducklings showed swelling and scattered necrotic spots at 2 dpi.At 5?7dpi,the spleen was severely necrotic,with necrotic foci occupying the entire surface of the spleen.The pathological process of the spleen was as follows:local tissue necrosis and disintegration,and lymphocytes decreased at 5 dpi.At 7 dpi,the spleen tissue was necrotic and disintegrated,and lymphocytes were greatly reduced due to necrosis.As for 9 to 11 days after infection,local necrotic tissue formed granulomas,splenic sinuses expanded,and macrophages infiltrated.When the disease progressed to 15 dpi,the necrotic areas of the spleen were replaced by fibrous tissue.In the immunocytochemistry test,the positive cells in the spleen were mainly macrophages and lymphocytes.The TUNEL detection found that the peak of cell damage appeared at 7-9 days after infection,and lots of macrophages were apoptosis during this period.In addition,pathogenic studies on breeding ducks indicated that N-DRV infection can cause follicular atrophy,theca and fallopian tube hemorrhage,significantly reduce the fertilization rate and hatching rate of breeding eggs.The above results established that NDRV was the pathogen of the newly prevalent duck spleen necrosis,N-DRV was highly invasive to the spleen and other immune organs,mainly damages spleen macrophages and lymphocytes,and N-DRV can be transmitted vertically through eggs,which endangers the safety of provenance.2.Research on the mechanism of N-DRV-induced spleen cell damageIn order to explore the dynamic changes of gene expression in spleen lymphocytes after N-DRV infection,this study isolated the spleen lymphocytes of ducklings at 1,2,3,5 and 7days after N-DRV infection,and carried out RNA-Seq analysis.After the data quality control was qualified,we set up two indicators of the expression fold changes and the significance of expression changes(Fold change>1 and p<0.05)to screen differentially expressed genes(DEGs),and carried out GO functional classification and KEGG signal pathway enrichment analysis.The data showed that at different time points after N-DRV infection,a total of263?658 differential genes were screened,including 117?492 up-regulated genes and 80-349down-regulated genes.These DEGs were mainly related to the immune system,cell growth and necrosis,signal transduction and other biological functions.In terms of cell damage,a total of 156 DEGs related to cell death were screened,mainly involving apoptosis,necrosis and ferroptosis,while the signaling pathways involved in the regulation of cell death were mainly HIF-1,PI3K-Akt,and TNF signaling pathways.Although apoptosis,necrosis and ferroptosis were all involved in N-DRV-induced cell damage,the significant difference comparison showed that ferroptosis played a pivotal role in N-DRV-induced cell damage.RNA-Seq analysis provided new insights into the dynamic changes of cell damage-related genes after N-DRV infection,suggesting an important role of ferroptosis in spleen necrosis.On the other hand,these data will contribute to a deeper understanding of cell damage and host response after orthoreovirus infection.3.Mechanism of N-DRV-induced ferroptosis in macrophagesPrevious RNA-Seq analysis had established that ferroptosis was significant in N-DRV infection-induced spleen damage.In order to determine the ferroptosis of spleen cells induced by N-DRV,and to further clarify the mechanism of ferroptosis,we used chicken macrophages HD11 cells for in vitro verification.We found that N-DRV infection could induce HD11 cells to produce ROS,and the amount of ROS was positively correlated with the N-DRV infection dose and time.Mitochondrion of infected cells showed characteristic morphological changes of ferroptosis with fractured cristae and reduced number of cristae.Combined with the increased intracellular Fe²⁺concentration and the differential proteins and m RNA expression of ferroptosis marker gene,it was confirmed that N-DRV infection can induce ferroptosis in HD11 cells.Further analysis indicated that ferroptosis-related DEGs were mainly located in iron metabolism regulatory pathways(TFRC,TF,ACO1,STEPA3,SLC40A1,etc.).Using transferrin receptor(TFRC)as the target gene,we found that RNAi knockdown of TFRC expression significantly antagonized the increase of intracellular Fe²⁺and ROS production induced by N-DRV infection,indicating that N-DRV mainly induces ferroptosis in HD11 cells mainly through the iron metabolism regulatory pathway.This research provided new insights that N-DRV can induce ferroptosis in macrophages through the regulation of iron metabolism.The signal transduction and the final execution mechanism of ferroptosis needs to be further verified.In conclusion,this research clarified the damage mechanism of N-DRV on the spleen,discovered and verified the pivotal role of ferroptosis in N-DRV-induced spleen damage,and revealed the regulatory pathway of N-DRV-induced ferroptosis in macrophages.This research make up for gaps in ferroptosis research in poultry field,and will contribute to a deeper understanding of the regulation mechanism of spleen injury and ferroptosis.