Characterization Of Tissue Damage And Identification Of Infected Target Cells Caused By Novel Goose Astrovirus

The novel goose astrovirus（N-GAst V）is a discovered pathogen in recent years,which mainly causes gout in viscera and joints of sick goslings.After infection,goslings usually become sick at 5～20 days of age,and the mortality rate can be as high as 50%,which causes serious economic losses to goose industry in China.In order to further clarify tissue damage and antigen distribution and localization of tissues and organs in goslings infected with N-GAst V,we carried out the following studies:1.Development of immunohistochemistry for N-GAst VIn this study,hybridoma cells preserved in the laboratory were resuscitated,ascites were induced and purified in mice.WB and IFA identification showed that the ascites could specifically bind to ORF 2 protein of N-GAst V.Ascites were tested with a titer of 1:102,400.Using the ascites as the primary antibody,the optimal reaction conditions of the monoclonal antibody mediated immunohistochemical detection method were obtained through repeated exploration and optimization of several experimental conditions in the immunohistochemical procedure,such as antigen repair time,peroxidase blocking time,reaction conditions of the primary antibody,reaction conditions of the secondary antibody,and reaction time of the chromogenic substrate.That is,microwave repair for 7 min,incubation with 3%H2O2 for 20min,incubation with primary antibody diluted 1,000 times at 4℃overnight,incubation with secondary antibody diluted 500 times at 37℃for 1 h,and incubation with DAB wet box for8 min.Finally,a new immunohistochemical method for the detection of N-GAst V was established.2.Study on the characteristics of tissue damage of gosling caused by N-GAst VN-GAst V SDTZ strain was cultured from chicken embryo liver cells,which was isolated and preserved in the laboratory,the purity of N-GASTV was identified by RT-PCR,and TCID₅₀of the virus is 10^-4.9/0.1 m L.After a large number of proliferation,it was stored at-80℃for later use.120 1-day-old healthy goslings were randomly divided into three groups.Group A was injected with 0.3 m L of viral solution into leg muscles,group B was injected with 0.3 m L of viral solution orally,and group C was injected with 0.3 m L of normal saline into muscle as the control group,and observed continuously for 24 days.At 12 hpi,1 dpi,3dpi,5 dpi,7 dpi,9 dpi,12 dpi,15 dpi,18 dpi,21 dpi and 24 dpi,the body weight,morbidity and mortality of each group were recorded.Three animals in each group were killed randomly,the heart,liver,spleen,lung,kidney,bursa of fabricius,thymus,pancreas,glandular stomach and duodenum were dissected and fixed in 4%paraformaldehyde solution for 24 h,paraffin sections were prepared for HE staining and histopathological changes were observed.The results showed that the goslings in infected group showed depression of spirit,decreased feeding and other clinical symptoms,urate attachment and deposition on the surface of heart and liver,kidney enlargement,bleeding and ureter urate deposition.Compared with the control group,the weight of goslings in infected group increased slowly,especially in intramuscular infected group.HE staining results showed that the histological changes of the two groups of infected goslings were basically the same.The histological changes showed edema of cardiac muscle fibers,widening of interstitium,and infiltration of a few red blood cells and inflammatory cells.Hepatic cell steatosis and vacuolar degeneration;Kidney glomerulus swelling,part of the renal tubule cell necrosis,shedding;A sheath of lymphocytes is formed around the brain vessels.3.Identification of target cells infected with N-GAst VThe established immunohistochemical method was used to detect the antigen of N-GAst V.Immunohistochemical results showed that N-GAst V existed widely in cardiac cardiomyocytes.Hepatocytes of liver;Lymphocyte around the central artery of spleen and lymphocyte nodule in white medullary;Squamous and cubic epithelial cells of bronchi,respiratory capillaries and lining of pulmonary chambers;Epithelial cells of proximal convoluted tubules,distal convoluted tubules and collecting tubules of kidney;Lymphocytes in the medulla of fabricius follicles;Reticular cells in the medulla of thymus lobules;B cells and D cells in the islets of pancreas;Neurons of the brain and medulla;Adenosine cells and columnar epithelial cells of glandular gastric mucosa;Columnar cells and Panth cells of duodenal intestinal gland.The positive signal showed that N-GAst V was mainly located in cytoplasm.Positive signals could be detected in the heart,liver,lung and kidney in the two infected groups after 12 h of infection.Positive signals were detected in spleen and thymus of the intramuscular injection group 12 h after infection,while positive signals were detected in spleen and thymus of the oral administration group 1 d after infection.Positive signals were detected in glandular stomach and duodenum in oral group 12 h after infection,and positive signals were detected in intramuscular injection group 1 d after infection.The positive signals of pancreas,bursa of Fabricius,brain and medulla were observed at 1 d,3 d and 7 d after infection.At 21 d after infection,no positive signal was detected in bursa of Fabricius,pancreas,brain and medulla in both groups.At 24 d after infection,no positive signal was detected in thymus and spleen in both groups.The staining intensity from 7 to 12 days after infection of goslings was significantly higher than that of other days,and the staining intensity of kidney was significantly stronger than that of other tissues.In conclusion,the antigen localization of 1-day-old goslings infected by intramuscular injection and oral administration was carried out by using the established new immunohistochemical method of N-GAst V.The results showed that most tissues and organs were rapidly damaged at the early stage of infection,and the virus was most widely distributed in tissues from 7 d to 12 d after infection.Viruses are mainly distributed in the cytoplasm of all cell types.The staining intensity of goslings from 7 d to 12 d after infection was stronger than that of other days,and the staining intensity of kidney was stronger than that of other tissues.It is proved that N-GAst V has extensive tissue tropism,and the kidney is its main target organ,which provides a scientific basis for further elucidating the pathogenic mechanism of N-GAst V.