| The scallop Chlamys farreri (Jones & Preston, 1904), one of the most famous marine products, is widely farmed along northern coast of China. Since 1990s, China scallop farming industry has been going through a period of severe crisis due to the ongoing outbreaks of mass mortality. Supported by the Key Project of Chinese National fundamental Research and Development ("973" Programme), systematic investigations including epidemiological survey, as well as many experimental infection studies have been carried out by our research group since 2000. All results suggested that the causative agent is an unknown spherical virus. Based on the results obtained, the disease was named "acute virus necrobiotic disease (AVND)".For establishing a rapid, sensitive and reliable protocol to diagnose the viral disease, monoclonal antibodies (MAbs) against AVND virus were developed. The virus was isolate from the moribund scallops that were collected during mass mortalities. The virus was purified by sucrose density gradient centrifugation and then used as an antigen to immunize the mice of Balb/c strain. The spleen cells from the immunized mice were fused with NS-1 myeloma cells and the hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Finally, 4 stable MAbs (2B3, 3C8, 3G7 and 4C7) that strongly react with the AVND virus antigen both in ELISA and IFA were obtained. Additionally, the specificity of the MAbs to this virus was tested by immunogold electron microscopy (IBM) with 10 nm colloidal gold as a marker. The results demonstrate that all 4 MAbs recognized epitopes on the envelope of the virions. The immunoglobulin isotype of the MAbs was determined using a Mouse Monoclonal Antibody Isotyping Kit (Sigma) and the result showed that MAbs 2B3, 3C8, 3G7 and 4C7 were IgG 2b, IgG 1, IgG 2b and IgG 2b, respectively.In this study, a MAb-based indirect ELISA was established. It has a sensitivity of 5 ng viral proteins and was used for detection of the infection rate and intensities of the scallops (one year old), which were sampled during mid April to mid October of 2003. The resultsexhibit that the positive rate and intensities sharply rose from mid July to late August and reached the spike of the whole cultivation year in the late of mid July. Which corresponds perfectly with the mortality rate of farmed C.farreri during this period.Furthermore, Dot-Immunoblot as a quick and simple diagnosis protocol for field test was developed and its has a limit of 1 : 100 diluted infected tissue honogenates.Addtionally, the naturally diseased scallops Chlamys farreri, which were sampled during mass mortality in summer of 2003, were detected by means of histopathological and MAb-based immunofluorescence assay (IFA) . The results of histological examination demonstrated that a series of histopathological changes including cell swelling, basophilic increase, disorder, partial sloughing and excessive necrosis or sloughing were always observed in epithelia of many different organs, e.g. mantle, gills, stomach, intestine and kidney. Additionally, the result from the in situ detection of the AVND virus antigen by means of the MAb-based immunofluorescence assay showed that this pathological changes or lesions were perfectly coincident with the positive cells (fluorescencing cells). For example, where the positive cells were denser in some local area of epithelia, were the pathological lesions more serious. Which would reveal the roles of this virus in pathogenesis and would further confirm that the AVND virus is the main causative agent of mass mortalities among cultured scallop Chlamys farreri that were farmed in northern coast of China.In present study, the newly characterized MAbs may improve our ability to diagnose the infections caused by AVND virus and permit us for further investigations on the pathogenesis of this newly reported scallop etiologic virus. |
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