| Duck Tembusu virus disease is a new acute infectious disease caused by duck Tembusu virus(DTMUV),which causes a sharp drop in egg production in laying ducks and breeding ducks,with ovariitis and neurological symptoms as the main clinical features.DTMUV is the first flavivirus that can cause serious infection of duck flocks,which not only seriously affects the duck industry,but also poses a potential threat to public health and safety.The blood-brain barrier(BBB)is distributed between the blood and brain tissue.It is a complex structure composed of brain microvascular endothelial cells,basement membrane,pericytes and astrocyte foot processes.It plays a very important role in the stability and normal physiological activities of the central nervous system,which is a natural barrier to prevent blood toxins and macromolecular substances from entering the brain tissue.At present,most of the research on DTMUV focuses on the ovary of the laying duck,and the research data on the central nervous system is very scarce.Viruses invade the central nervous system,and the BBB is the key.This study intends to explore the mechanism of DTMUV-induced central nervous system damage in ducks from the perspective of virus invasion.This paper explored the way that DTMUV crosses the BBB and enters the central nervous system in the early stage of infection,and also analyzes the reasons for the destruction of the BBB in the middle stage of infection.Experiment Ⅰ Development of the blood-brain barrier in ducksDuck is the representative of waterfowl in China,and its brain structure is different from that of mammals: the main body of the brain is the striatum,and it has a developed midbrain.The morphological structure and development of the duck blood-brain barrier have not yet been reported.Therefore,this paper firstly studied the development of duck BBB,and observed the structure of the blood-brain barrier and the distribution of capillaries in the embryonic and post-embryonic stages(E14 ～ P60)of ducks by transmission electron microscopy and immunohistochemistry.The results showed that the formation of the duck blood-brain barrier is a gradual process.The structure of the duck blood-brain barrier changed significantly during the embryonic period,matured rapidly at E21 ～ P1,and still had some changes in the ultrastructure level after hatching.The BBB in the embryonic duck midbrain develops faster than the cerebrum and cerebellum.Experiment Ⅱ DTMUV infection induced non-suppurative encephalitis in ducksThe brain is the main target organ of flaviviruses and one of the organs where the virus persists for the longest time.Duck age is also an important factor affecting the susceptibility of ducks to the virus.In this paper,histological and ultramicroscopic pathological studies were performed on the nervous system of ducklings and adult ducks after challenge.DTMUV causes nonsuppurative encephalitis,especially in ducklings.The results of electron microscopy and molecular biology experiments showed that DTMUV had entered the brain in the early stage of infection.The virus content in the brain reached a peak at 9 dpi,and as the diseased duck recovered,the virus content in the brain gradually decreased.The results of pathological examination showed that DTMUV infection caused extensive pathological damage in duck brain.Observed at the histological level: no obvious pathological changes were observed in the early stage of infection;pathological phenomena such as microglial activation,glial nodules,neuronophagia,infiltrated lymphocytes and hemorrhage and other pathological phenomena could be clearly observed in the middle and late stages of infection.At the ultrastructural level,severe vacuolation in the brain could be observed:neuronal swelling and rupture,degeneration of the myelin sheath of nerve fibers,astrocyte foot process swelling,microglia phagocytosis of damaged cells and tissues,abnormal synaptic structure and edema around the capillaries.Virus particles could be observed in the brain of ducklings in the early stage of infection,and the neurological symptoms were obvious in the middle and late stages of infection,accompanied with the destruction of the BBB.Almost no virus particles were observed in the adult duck brain,and the neurological symptoms were not obvious.Although duck brain capillaries already have the corresponding barrier function after hatch from eggs,the ultrastructural level was still in the stage of improvement.Combined with the results of Experiment Ⅰ,it shows that the difference in the level of BBB development is one of the factors that severe encephalitis in ducklings.Experiment Ⅲ DTMUV infection caused the destruction of the blood-brain barrier in ducksIn the second experiment,it was found that in the middle and late stage of DTMUV infection,the permeability of the duck blood-brain barrier was significantly enhanced,and the structure of the blood-brain barrier was significantly damaged.Aiming at this phenomenon,this paper analyzed the reasons for the destruction of BBB.Morphologically,DTMUV virions and their replicating vesicles were directly observed in duck brain microvascular endothelial cells,suggesting that the direct effect of DTMUV on duck brain microvascular endothelial cells may affect the function of BBB.Through RNA-Seq analysis,it was found that in the middle stage of infection,the inflammation-related pathways such as cytokines,cell adhesion molecules and TNF in the brain were significantly enriched,indicating that DTMUV infection caused a large number of inflammatory substances in the brain.Based on the results of RNA-Seq,the detection of tight junctions,cell adhesion molecules and inflammatory factors at the molecular level throughout the disease process found that in the stage of obvious neurological symptoms,the expression of tight junction-related molecules in the brain was generally down-regulated,and the expression of cell adhesion molecules and inflammatory factors were generally down-significantly regulated.According to the above results,it is speculated that the "inflammatory factor storm" caused by DTMUV entering the brain is the main reason for the destruction of the BBB.Experiment Ⅳ The pathway of DTMUV invading the central nervous system across the BBBIn the early stage of DTMUV infection,the BBB was intact,but virus particles could already be detected in the brain,indicating that DTMUV had entered the brain before the BBB was destroyed.How does DTMUV invade the CNS across the BBB?In this experiment,the infection kinetics of DTMUV in brain microvascular endothelial cells and its effect on BBB permeability were studied by using Transwell system to construct in vitro BBB model.The results showed that DTMUV could replicate efficiently in brain microvascular endothelial cells,but the replication efficiency was low.In the in vitro BBB barrier model,BBB permeability was not significantly altered after challenge.However,in the early stage of infection,virus particles were observed in the lower chamber of the cell culture,and the virus titer in the lower chamber was consistent with that in the upper chamber with time,indicating that the virus particles may be released from the basal side of brain microvascular endothelial cells into the lower room.The above model suggests that DTMUV virion in the blood could be released into the central nervous system from the basal side of the brain microvascular endothelial cells.At the same time,DTMUV infection could up-regulate cell adhesion molecules in brain and brain microvascular endothelial cells,suggesting that DTMUV infection of endothelial cells could promote lymphocyte migration to this site.Studies on DTMUV infection of spleen and blood-derived lymphocytes show that DTMUV can effectively infect lymphocytes.DTMUV takes advantage of the migratory properties of lymphocytes to inflammatory sites,"riding" lymphocytes,adhering to the walls of brain capillaries and then entering the central nervous system.In conclusion,DTMUV may cross the BBB and invade the central nervous system through the transcellular and "Trojan horse" manner. |
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