Isolation And Identification Of Duck Tembusu Virus JS2010Strain And Establishment Of Indirect ELISA To Detect Antibodies Against Duck Tembusu Virus

In the spring of2010, a severe disease was found in several duck farms in east-central China, major clinic characters included a sudden decline of feed uptake accompanied by a heavy drop in egg production, ataxia of gait, indeed, a few of infected egg-laying ducks stoped laying eggs or died. So far, the disease has spread to most of the duck-producing regions in China including Shanghai Autonomous City, Jiangsu Province, Anhui Province, Shandong Province and Hebei Province and caused serious economic loss to the duck industry in China. It is of great significance to determine the pathogen of the disease and to establish its rapid diagnostic methods.Firstly, to isolate and identify the pathogen that caused disease in ducks, the samples of diseased tissue of the infected ducks from a duck farm in Jiangsu Province were homogenized. Then the filtered homogenates were inoculated into several10-day-old SPF chicken embryonated eggs (0.2mL/embryo) via the allantoic sac.The chick embryos died post-inoculation4-6days with the chick chorilallantoic membranes thickening and embryos swollen、hyperemia. The embryo allantoic fluid was collected and it was named JS2010strain which had an infectivity titer of103.68ELD50/0.2mL.We make fortether studise on the disease by hemagglutination test, virus culture, histopathological examination and nucleic acid identification. The results showed that the isolated virus had no ability to agglutinate1%erythrocytes including the chicken, duck, goose and swine. The virus could replicate in BHK-21cells but not in CEF or Vero, the virus fluid had an infectivity titer of105.33TCID50/0.2mL. The main expressions of CPE consisted in decreased cell diopter, rounding reduced cells, and cell exfoliation. The fatty degeneration of hepatocytes, intestinal bleeding and intestinal epithelial injury were found by pathologic observation. By RT-PCR and sequence analysis, the result showed that the isolated JS2010strain shared the highest similarity with DTMUV BYD-1(99%). The comprehensive analysis showed that the isolated virus strain was demonstrated to be Duck Tembusu virus.Secondly, the complete genomic sequences of DTMUV JS2010strain was analysed. The complete genomic sequences of DTMUV JS2010strain was amplified by RT-PCR using11pairs of primers designed according to the published sequence of DTMUV BYD-1strain(JF312912)deposited in GenBank. The resuts showed that the complete genome of DTMUV JS2010strain was composed of10990nucleotides in length, which included a10278nt single large open reading frame encoding a polyprotein. Sequence analysis showed that JS2010strain shared from99.2%to99.6%nucleotide homology with other DTMUV strains. The sequenced fragment of the E gene of DTMUV JS2010strain shared from87.7%to99.5%nucleotide identity with Tembusu virus, and phylogenetic analysis demonstrate that JS2010strain and BYD-1strain are in one lineage. All results showed that the JS2010strain shared the highest similarity with the Tembusu virus of Ntaya virus group.Then, the E gene was cloned and expressed to get the immunogenicity protein of DTMUV, which can be used to establish an indirect enzyme-linked immunosorbent assay diagnosis method. A pair of special primers according to the complete genome of DTMUV JS2010strain was designed to amplify E gene (1503), and E gene fragment was recombined to vector pET-32a(+) to construct recombinant plasmid pET-32a-E. The fusion protein E was obtained by transformed the recombinant plasmid into the expression competent cells Escherichia coli Rosetta, which was purified by Ni-NTA affinity chromatography column at last. The fusion protein E was analysed by SDS-PAGE and Western-blot, the concentration and purity of purified E which retained the natural protein activity was0.58mg/mL and95%. The result showed that the purified fusion proteins could react with duck serum containing antibody against DTMUV, and meet the expected requirement.Lastly, an indirect ELISA was developed based on the recombinant capsid protein E. Using the purified recombinant protein E as coating antigen, the reaction conditions of the indirect ELISA were optimized after many experiments, including2.9ug/mL coating antigen of the purified protein E,1:200dilution of testing serum and1:5000dilution of HRP conjugated anti-duck IgG. The critical value of positive and negative serum was0.361(OD450nm). Specificity、sensitivity and repeatability tests showed that the ELISA assay have a good specificity、sensitivity and repeatability. The establishment of indirect ELISA method was applicated to detect antibody of clinical chicken and duck serum from Jiangsu Province, Anhui Province and Shandong Province. The positive rate of chicken serum was24.42%(21/86)while the positive rate of duck serμm was32.64%(63/193).The result analysis confirmed that the established indirect ELISA method was effective which could be used to detect clinical vast samples.