Pathological Observation And Transcriptome Analysis Of Thymus Injury In PRRSV-infected Piglets

Porcine reproductive and respiratory syndrome virus（PRRSV）infection leads to reproductive disorders in sows,respiratory damage and immunosuppression in piglets.It seriously endangers the health of pigs and the economic development of pig industry.Thymus is an important central immune organ of pigs,which is very important in resisting pathogen invasion and maintaining immune function.PRRSV infection cause thymus damage,which is an important reason for decreased immunity and secondary infection in pigs.However,the specific mechanism of thymus injury has not been clarified.In this study,a piglet model infected with PRRSV was constructed.Histopathological observation and immunohistochemical staining were used to explore the specific lesions of thymus caused by PRRSV infection.Based on Illumina platform,the transcriptome sequencing of piglet thymus in control group and infection group was carried out.The aim was to explore the relationship between differentially expressed genes and signal pathways and thymus injury.The results were as follows:1.PRRSV piglet infection modelIn this study,Eleven 40 day old ETONG piglets in the experimental group were intramuscularly injected with PRRSV WUH3 virus suspension,and the challenge dose was 3×10⁵ TCID₅₀.Three piglets were injected with normal saline according to body weight as the control group.The body temperature of PRRSV-infected piglets increased from the 4th day.On the 8th day,the maximum body temperature of piglets was 42-43℃.The clinical symptoms of piglets were anorexia,reluctance to walk,cough and dyspnea.Virus nucleic acid was detected in lungs,thymus,lymph nodes,tonsils and spleen of infected piglets,and PRRSV antibody was detected in serum.Gross autopsy and histopathological observation showed that the lung was interstitial pneumonia.Combined with above results,the PRRSV-infected piglet model was successfully established.2.Autopsy and histopathological changes of immune organsThymus of PRRSV-infected piglets became smaller.Hilar lymph nodes,mandibular lymph nodes,inguinal lymph nodes and spleen became larger.Bleeding spots were scattered on the surface of thymus and tonsil of infected piglets.The surface of lymph nodes and spleen was dark red due to bleeding.Histopathological observation showed that thymic lymphocyte necrosis and lymphopenia in infected piglets.Lymph node hemorrhage,blurred structure of cortex and medulla,and massive reduction of lymphocytes.There were a large number of necrotic lymphocytes in the center of lymph nodes in tonsil.Red pulp congestion of spleen.Combined with the above results,PRRSV infection caused serious damage to the immune organs of piglets.3.Histopathological changes of thymusThe main lesions of thymic cortex were cortical atrophy,cell apoptosis and necrosis,vascular proliferation and macrophage proliferation.Thymic corpuscle hyperplasia could be seen in thymic medulla.In addition,the interstitium of thymic lobules widened.Immunohistochemistry and immunofluorescence staining showed that PRRSV was located in the cytoplasm of macrophages.Combined with the histopathological observation of thymus,it is speculated that PRRSV invades thymic macrophages of piglets,resulting in thymocyte apoptosis and necrosis.The immune defense function of animal body makes blood vessels proliferate.Monocytes in the blood migrate to the thymus and develop into macrophages,resulting in the increase of macrophage.The virus further replicates and then releases,infecting macrophages.4.Transcriptional analysis of thymus in PRRSV-infected pigletsIn GO enrichment analysis,angiogenesis,regulation of angiogenesis,immune effrctor process and positive regulation of cell migration were significantly enriched,which was related to thymic cortical angiogenesis and macrophage proliferation.In addition,Toll-like receptor signaling pathway,IL-17 signaling pathway and TNF signaling pathway were significantly enriched.It is suggested that these signaling pathways play a role in the pathogenesis and immune escape of PRRSV.The expression of TLR8 and IRF5 in Toll-like receptor signaling pathway was significantly up-regulated.Monocyte-macrophage chemokines CCL2,CCL3L1,CCL5 and their receptors CCR1,CCR2 and CCR5 were significantly up-regulated.It is suggested that these cytokines may be related to the pathological processes of thymic cortical cell apoptosis and necrosis,vascular proliferation and macrophage proliferation.In conclusion,PRRSV infection caused damage to immune organs of piglets,especially thymus tissue.The biological processes such as angiogenesis and positive regulation of cell migration enriched by transcriptomics are related to the proliferation of blood vessels and macrophages in the thymus.The expression level of monocyte-macrophage chemokines in thymus of infected piglets was significantly up-regulated.It is suggested that these cytokines may participate in the pathological changes of thymus,which provides a certain basis for understanding the molecular mechanism of thymus injury caused by PRRSV infection.