| Since April2010, a novel contagious disease in ducks and geese, with egg drop, feed uptake decline and nervous system signs,caused by a newly emerged virus has spread around Jiangsu, Zhejiang, Fujian, Anhui, Shandong, Henan and Hebei provinces. The study demonstrated that the pathogen causing the disease is a new flavivirus different from the existing data reported, tentatively called duck or goose flavivirus. The virus had100%infection,and morbidity rates and mortality rates was from5%to30%,causing breeding ducks and geese significant economic losses.In order to fully grasp the characteristics of the virus caused ducks, geese feed intake dropped, neurological symptoms, egg production rates and hatch rates significantly reduced since April2010, the experiments systematically have studied the biological characteristics of the flavivirus strain JS804in geeseseparated from this epidemic.The results showed that the virus belonged to a single-stranded RNA with an envelope,was approximately45-50nm in size,could proliferate in chick embryo,duck embryo,geese embryo,BHK-21cells and Vero cells, was no haemagglutination activity to the erythrocyte of chicken,duck,goose,and was sensitive to chloroform,ether,heat,acid and alkali.The purification of antigen from mouse brain using the sucrose acetone method could haemagglutinate the erythrocyte of goose, and the haemagglutination titer was51og22.The suckling mice were sensitive to the virus, and all of the mice by intracerebral injection with the virus were sick after seven days. Using RT-PCR method,it is found that the virus mainly multiply in the brain and liver of the challenging mice.The E Protein of flavivirus strain JS804in geese was used to inoculate BALB/c mice.After3times of vaccinations,the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0.After subcloning for3times, six positive hybridoma cell stains2A、3B4、3D9、3E9、3H11、5H11、were determined by ELISA, which ELISA titers of cell supernatants and ascites were10、10、102.103、102、102and103、103、104、106、105、104.The2A4and3B4belonged to IgA,while3E9and3H11belonged to IgG1.And3D9、5H11belonged to IgG2a, IgG2b respectively.All of the McAbs had k chain.The analysis of Western-blot and IFA indicated that the six strains of monoclonal antibody can react with the E Protein of flavivirus strain JS8O4in geese.3E9and3H11had neutralization titers against the flavivirus strain JS804in geese.In order to establish double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for detection of the flavivirus strain JS804in geese,polyclonal antibody against the flavivirus strain JS804in geese and monoclonal antibody against the E protein of the flavivirus strain JS804in geese were used as the capture antibody and detecting antibody,respectively.The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804in geese were1:3200and1:160in the check-board titration respectively, the reaction time of sample was1hour,and the optimal working dilution of HRP-labelled goat-anti-mouse IgG was1:10000. The positive standard value was0.247(OD450nm). The coefficient of variation of reproducibility was less than10%,and at least1.875μg/mL antigen could be detectable. The ELISA had no cross-reaction with NDV、AIV、IBV、IBDV、DHV and GPV. Twenty clinical samples were detected by the ELISA and RT-PCR with agreement rate of75%. The results revealed that the ELISA possessed good specificity and reproducibility,and higher sensitivity,indicating a suitable method for rapid detection of the flavivirus strain JS804in geese. |
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