| Newcastle disease is a serious avian infectious disease caused by Newcastle disease virus(NDV).NDV belongs to the family Paramyxoviridae,genus Avulavirus,also known as avian paramyxovirus serotype 1(APMV-1).It causes respiratory,digestive and neurological disorders in birds.According to the clinicopathological features of the infected chickens,the virulent viruses are classified into two pathotypes:neurotropic and viscerotropic.It is widely believed that NDV invades central nervous system(CNS)through the blood-brain barrier(BBB)resulting in severe neurological symptoms.How does NDV pass through BBB is unknown.This study used the established chicken brain microvascular endothelial cells(BMECs)cultured BBB model in vitro to explore the mechanism of virus invasion into CNS.It would provide valuable clues to the prevention and control of NDV.The main results are as follows:1.NDV infection in chicken central nervous system.The high,medium and low-dose of the neurotropic virulent strain F48E9 were more severe clinically signs and dead than those of the intracranial infection group in SPF chickens.The virulent strain can be effectively replicated in the brain,cerebellum and spinal cord in both infection routes.But,there was no obvious clinical symptoms in chickens infected with the avirulent strain LaSota.These results suggested that the invasion of neurotropic virulent virus into the CNS system is the key for causing the neurological symptoms in chickens.2.The effect of NDV infection on the permeability of chicken brains.4 weeks old SPF chickens were infected with virulent and avirulent viruses,as well as a PBS control group.The fluorescein sodium(NaF)was used as a tracer to test the permeability of brains.According to tracer infiltration in brain tissues,the clinical symptoms and the infiltration of tracer NaF were detected in the virulent F48E9 infection group,but not in the avirulent La Sota and the control groups.These results suggested that the virulent virus changed the permeability of the chicken brains,while the avirulent had no effect on the permeability.3.Establishment of chicken BBB model in vitro.The brains of 16 to 18 day-old chicken embryos were collected and separated the primary by using Percoll gradient centrifugation.The endothelial cells were identified by an specific antibody against the CD31.The cells were cultured in Transwell plate to establish BBB model in vitro.4.The effect of NDV infection on BBB permeability of chickens.The viruelnt strain F48E9 can be effectively replicated in the chicken BMECs of the BBB model,but the avirulent strain LaSota can not.The NaF tracer experiments showed that the virulent strain infection had a large NaF overflow,while the infected avirulent strain had almost no NaF overflow.These results indicated that only virulent virus was able to replicate effectively and could destroy BBB integrity in chicken BMECs.Using Dynasore,an endocytosis inhibitor,the NaF overflow was significantly lower in the infected with virulent strain than that of then avirulent strains and untreated control group at 12h.This result showed that Dynasore had inhibitory effect on the early stage of BBB permeability.5.The inhibitory role of Dynasore in the NDV infected chickens.The infected SPF chickens with virulent NDV after treatment with different dose of Dynasore showed clinical symptoms and death.Low level of anti-NDV antibody(HI=2~2to 2~4)commercial chickens before and after the virulent strain infection of strong strain administrated with Dynasore showed that the chickens with mild neurological symptoms and no deaths occurred before the treatment.However,The chickens were death with severe clinical symptoms at treatment with Dynasore after infection.These results indicated that Dynasore has an preventive protection in the NDV infected chickens with a low level of antibody.In conclusion,NDV virulent strain can enhance the permeability of chicken brain and BBB model in vitro.Dynasore can suppress the change of BBB permeability at early stage of virulent strain infection,and it has an preventive protection in the NDV infected chickens with a low level of antibody(HI=2~2to 2~4).The synergy between the Dynasore and antibody remains to be studied.This study provides a basic information for the mechanism of virus invasion to CNS and the development of drugs for the prevention and treatment of NDV. |
