Preliminary Establishment of the Indirect Immunofluorescence Assay for Detecting DSHDV in Paraffin-Embedded Tissues and Antigen Localization

【Objective】The objective of this study is to establish an indirect immunofluorescence assay (IFA) as a detection method of duck swollen head haemorrhagic disease virus (DSHDV) in paraffin-embedded tissue, subcellular antigen localization and dynamic distribution of DSHDV in vivo.【Method】DSHDV was purified by differential centrifugation and rabbit anti-DSHDV antibodies were obtained from rabbits vaccinated with purified viruses. Then the rabbit anti-DSHDV IgG was purified through DEAE-SephadexA-50 chromotography. IFA was established and used to detect DSHDV antigen in paraffin-embedded tissues of 28-day-old death ducks which experimentally infected with DSHDV and clinical death ducks. PAGE was used to analyze theDSHDV nucleic acid genome characteristics.【Result】The optimum conditions of IFA were as follows: Antigen was retrieved by microwave in 0.01 mol·L-1 citrate buffer solution (pH 6.0) for twenty minutes, and blocked with 10% horse serum at 37 ℃ for 30 min. Slices were incubated overnight at 4℃ with diluted primary antibody (1﹕50), and then incubated at 37℃ for 30 min with diluted FITC-labelled-secondary antibody (1﹕100) which contains 0.01% Evans blue. IFA was adopted in detection of DSHDV antigen in the liver of dead ducks. The results were positive in dead ducks infected with DSHDV and negative in dead ducks infected with duck plague virus, avian influenza virus (H5N1), duck viral hepatitis virus and Riemerella Anatipestifer. IFA was also abopted in detection of the virulent DSHDV antigen in different organs of artificially infected dead 28-day-old ducks, and the viral antigen was detected in the heart, liver, spleen, pancreas, lung, kidney, bursa of fabricius, esophagus, trachea, muscle, thymus, duodenum, jejunum, ileum, cecum and rectum, while viral antigens was not detected in the harderian gland, brain, skin and proventriculus. The antigens distributed mainly in the cytoplasm of the infected cells. The result of IFA detected the positive liver of virus isolation from the clinical dead ducks which were formaldehyde-fixed and stored for 1-6 years is positive. DSHDV genome was related to reovirus, had ten segements of dsRNA, showed "334" distribution pattern.【Conclusion】IFA is a sensitive and specific method for the detection of DSHDV in paraffin-embedded tissues. It is effective in studying the antigen locations of DSHDV. IFA can be applied in diagnosing and studying the distribution of the DSHDV in duck tissues. The DSHDV antigens distributed in the cytoplasm of the infected cells. The target organ that DSHDV mainly attacked are intestines, kidney, bursa of fabricius and thymus.