Isolation And Identification Of Duck Tembusu Virus CD Strain And Pathological Study On Ovarian Injury

Duck Tembusu virus disease is a duck infectious disease caused by duck Tembusu virus（DTMUV）infection,which can cause paralysis of ducklings and a significant drop in egg production in laying ducks.No effective therapeutic drugs have been developed for the disease which are based on vaccine prevention.DTMUV is an RNA virus with the characteristics of high genetic diversity,rapid evolution and diverse transmission modes wihch make it difficult to prevent and control by conventional means of epidemic prevention.Although there are many studies on Tembusu virus disease,its infection mechanism on the ovary is still unclear,especially whether the virus has a direct effect on the cells in the ovary,which needs to be further improved.This research mainly includes the following contents:1.Isolation and identification of DTMUVDiseased ducks came from an infected duck farm in Hubei Province.The naturally infected ducks had decreased egg production with neurological symptoms.The autopsy showed ovarian hemorrhage and follicle deformation.Hyperplasia,apoptosis or necrosis of follicular granulosa cells;a large number of vacuolar cells around the follicles;follicular stalk arterial hyperplasia,smooth muscle cell necrosis and inflammatory cell infiltration in the proliferative arteries.A strain of DTMUV was isolated from the ovary tissue of a diseased duck.Whole genome sequencing of the virus showed that the strain was on the same branch as the domestic isolates Y and GL strains.It was named duck Tembusu virus CD strains.This strain can grow on DEF cells and develop cytopathic effects 24 h after inoculation.2.Pathogenicity of DTMUV to ducks of different agesDTMUV-CD strain can infect ducks.After infection,the ducks showed decreased feed intake,depression,and lameness.Histopathological observation found that the ovarian follicles were deformed,and the eosinophilic granulosa cells were enhanced;the brain tissue showed non-suppurative encephalitis;sciatic nerve bundles Inflammatory cell infiltration,swelling and necrosis of nerve fibers;inflammatory cell infiltration in leg muscles.DTMUV-CD strain can infect egg-laying ducks,and the clinical symptoms showed that the egg production of the diseased ducks decreased.The autopsy found that the ovaries were atrophied,there were no mature follicles,and the growing follicles were hemorrhagic.Histopathological examination revealed the proliferation of germ epithelial cells and vacuolar cells on the ovarian surface of diseased ducks;the proliferation,vacuolization or necrosis of granulosa cells of growing follicles,follicular atresia;inflammatory cell infiltration of sciatic nerve peritoneum;ovarian immunohistochemical staining found that virus was located in the cytoplasm of the granulosa cells of the growing follicle.3.Proliferation of duck tembusu virus isolates on different cellsThe DTMUV-CD strain was inoculated into primary duck ovarian granulosa cells and theca cells.DTMUV could grow on both types of cells,and cytopathic changes occurred at 24 h and 36 h.The cells in the infected group became larger and rounder,with enhanced refractive index,and the degree of granulosa cell lesions was more serious than theca cells compared with control group.The results of immunofluorescence staining,real-time PCR and viral TCID₅₀showed that the viral load of granulosa cells was higher than that of theca cells.The above research results show that duck Tembusu virus disease is prevalent in Hubei,and it is found that this strain has the highest homology with the domestic duck Tembusu virus Y strain through evolutionary analysis and homology comparison.DTMUV can infect both breeding and laying ducks,and the damage is mainly manifested in the ovary and nervous system.DTMUV is located in the granulosa cells of growing follicles after infecting egg-laying ducks,causing damage to the granulosa cells,resulting in a large number of follicles atresia,resulting in a decline in egg production.