The Role Of Sialic Acid Receptor In Neurotropism Of Newcastle Disease Virus

Newcastle disease?ND?is caused by avian paramyxovirus serotype 1?APMV-1?,also called Newcastle disease virus?NDV?,which belongs to genus Avulavirus of family Paramyxoviridae.NDV causes high morbidity,mortality and considerable economic losses in the poultry industry worldwide.Occurrence of ND must be reported according to the World Organization for Animal Health?OIE?.The virulence of NDV varies between different strains.The clinical signs,lesions and severity of ND in chickens depend on viral virulence,avian species,immune status and age.Based on its pathogenicity,NDV strains have been classified into three major groups: velogenic,mesogenic and lentogenic.The velogenic strains are further divided into velogenic viscerotropic?VVND?and velogenic neurotropic?VNND?.VNNDV infected chickens usually show neurologic signs such as torsion,head and neck tremor,circling,legs or wings paralysis and lead to a high mortality rate?approaching 90%?.Sialic acid?SA?as a NDV receptor is important for determining the host range and tissue tropism.However,role of SA receptor in NDV neurotropism is complete unknown.In this study,we investigated whether viral binding and replication of NDV are affected by SA receptor in chicken nervous system.It would provide a valuable clue for the mechanism of CNS diseases caused by NDV.The main results are following:1.The clinical and pathological changes of chicken brain tissues caused by NDV infection.Twenty-seven chickens were equally divided into three groups.Group 1 and group 2 were inoculated through intraocular-nasal routes with 0.2 mL per bird of PBS containing 106 EID₅₀ of virulent strain F48E9 and aviruelent strain LaSota,respectively.Group 3 was inoculated with PBS as a control.The SPF chickens infected F48E9 showed cough,dyspnea,asthma,loss of appetite,diarrhea,crop effusion,and severe neurological symptoms such as neck twist,no external irritation,unilateral leg paralysis and all died between 3 and 5 dpi.HE staining showed predominant microscopic lesions associated with virus replication in the brain consisted mostly of multifocal neuronal necrosis,nonsuppurative encephalitis characterized by lymphocytic perivascular cuffing,plump vascular endothelium,and multifocal astrogliosis in chicken brain infected by virulent strain F48E9.There were no abnormalities in clinical symptoms and pathological changes in LaSota infected group and control group.2.Replication of virulent and avirulent strains in chicken central nervous system?CNS?.The viral titers in the chicken CNS were detected at 1,3 and 5 dpi by TCID₅₀ and EID₅₀.The results showed that the virulent strain F48E9 could be efficiently replicated in the CNS and the viral replication in the cerebrum was more sensitive than in the cerebellum and spinal cord.,but there was no LaSota virus were detected.These data indicated that the neurotropism of the virulent and avirulent strains was significantly different.The avirulent virus is unable to replicate effectively in chicken CNS.However,avirulent LaSota strain was able to replicate in chicken primary neuronal cells,even though its replication was less efficient than that of the F48E9 strain.These results indicated incapacity of lentogenic strains replication in neural cells.3.Expression of SA receptor in chicken brain and primary neuronal cells.Immunohistochemistry and immunofluorescence staining with MAA for SA?2,3Gal and SNA for SA?2,6Gal were performed in the infected brain tissue and primary neuronal cells.The results showed that both SA receptors were detected in the brain tissue and neural cells of the infected group and the control group.The distribution of SA in different zones of the brain showed different,but there was no significant difference between the infected and non-infected groups.4.SA receptors are required for viral binding and replication.First,in order to investigate the capability of NDV binding to neural cells,chicken primary neuronal cells were incubated with the virulent and avirulent viruses at 4°C.After the cells were washed with PBS,the cell-bound viruses were harvested and titrated by the EID₅₀ method.Both F48E9 and LaSota viruses were capable of binding to the neuronal cells,and there were no significant differences in the cell-bound viral titers between both viruses,indicating that the virulent and avirulent strains had similar abilities in cell binding.The binding and replication ability of NDV were examined by enzymatic treatment.The results showed that the number of cells combined with the virus was significantly decreased?P <0.05?in all treatment groups compared to control group,and treated with Vibrio cholerae neuraminidase?VCNA?was more effective than those treated with ?2-3,6,8,9 neuraminidase A?NeuA?.The results of the viral replication were consistent with the binding reaction after treatment with neuraminidase,but the binding and replication ability after enzymatic treatment were not completely inhibited.In summary,SA?2,6Gal and SA?2,3Gal receptors are essential for the binding and replication of NDV to neuronal cells,and other molecules may be involved in this process.Taken together,the SA?2,6Gal and SA?2,3Gal receptors play a crucial role of viral binding and replication in chicken neural cells.