| TMUV was isolated for the first time in China in 2010,resulting in a sharp drop in duck egg production in East and North China,which seriously affected the development of duck industry in China.The pathogen was identified as Tembusu virus,which is the only flavivirus found to be pathogenic to ducks.Mosquito-borne transmission is the classic mode of transmission of the virus.The results of previous studies showed that TMUV has a significant preference for the peripheral blood mononuclear phagocytic system after infecting chickens and ducks through mosquito-borne routes,and this process is a key step of TMUV replication,pathogenesis and transmission in ducks.Therefore,it is necessary to comprehensively and deeply analyze the response and mechanism of duck peripheral blood leucocytes in the early stage of TMUV mosquito-borne infection.This study investigated the response of duck peripheral blood leucocytes to TMUV infection,and found that there were differences between in vivo and in vitro infection with TMUV.It shows that the infection may be regulated and influenced by internal factors during in vivo infection,which highlights the necessity and importance of in vivo research.In view of the lack of understanding of the duck immune system in the current field and the limited immune cell marker genes,this study used the single-cell transcriptome sequencing technology(sc RNA-seq)based on the 10×Genomics platform carried out the detection and analysis of the panoramic response of duck peripheral blood leucocytes in the early stage of TMUV mosquito vector infection.In order to obtain the transcriptional profile of duck peripheral blood leukocytes at the single-cell level,peripheral blood leukocytes were isolated from five 45-week-old SPF male ducks,and single-cell transcriptome data were obtained by sequencing.Using the t-SNE method for dimensionality reduction,the cells were initially divided into 12 clusters.Further cellular annotation found that duck immune cells could not be accurately identified by using human immune cell marker genes and characteristic transcriptional profiles.Therefore,in this study,the main types of duck peripheral blood leukocytes were identified by using the limited duck immune cell marker protein-specific monoclonal antibody combined with morphological observation.On this basis,further cell heterogeneity analysis was carried out for each main types of cells.Four clusters can be obtained after reaggregation of granulocytes.Compared with human granulocyte gene profiles,duck and human granulocyte marker genes are less conserved.Pseudotime analysis found that with the differentiation of cells,high levels of inflammatory response signaling and cell death occurred.However,the expression of the genes involved in cell proliferation was reduced.The monocytes were reclustered to obtain five clusters,which were subjected to GO enrichment analysis and pseudotime analysis,respectively.One branch point and three cell states with two cell fates were observed.In the process of cell differentiation,the biological processes of immune responses,macrophage differentiation,and cell migration were activated.The biological processes of metabolic processes and energy production were repressed.Five clusters were obtained by reclustering of lymphocytes,which were subjected to GO enrichment analysis and plotted cell pseudotime trajectories,two branch points and five cell states with three types of cell fates.On this basis,this study subdivided duck peripheral blood leukocytes into 14 clusters,and obtained the molecular marker genes of each main clusters.In this study,sc RNA-seq technology was further used to study the effect of TMUV on host peripheral blood leucocytes in the first stage of mosquito vector transmission in vivo.In vitro and in vivo experiments combined with single-cell transcriptomic data analysis revealed that the dynamic transition of granulocytes and monocytes from steady state to antiviral state may be critical for the body to successfully control TMUV infection.In this study,the homeostasis of duck peripheral blood leukocytes in vivo and the heterogeneity after infection with TMUV was analyzed by sc RNA-seq technique,and to explore the panorama and mechanism of the early response of peripheral blood leukocytes to TMUV infection.It provides basic data for the comprehensive and in-depth study of the composition of duck immune system and immune response mechanism. |
