Isolation, Identification And Genetic Variation Of Duck Tembusu Virus In Guangxi And Indirect Elisa Based On Protein E

Duck tembusu virus (DTMUV) is an emerging agent that causes a severe disease in ducks. TMUV can cause the infected layer and breeder ducks decreased egg production and has caused significant economic losses in the Chinese duck industry. In this study, the clinical cases of the disease occurred in Guangxi in recent years was diagnosed and the relevant research of the etiologic agent was performed. The concrete contents and results of the study are as follows:Firstly, isolation, identification and phylogenetic analysis of Tembusu virusIn this study the clinical samples of suspected DTMUV infection in duck farm of Guangxi province from 2012 to 2015 were detected by RT-PCR, then the positive clinical samples were used for virus isolation and identification. Four isolates of the DTMUV were isolated and designated GX120915, GX130330, GX150829, GX151002, respectively. The isolates were purified by three rounds of plaque purification on the BHK-21 cells. The purified isolates were used for further study including whole genome sequencing, genetic variation analysis, molecular biological characteristics, ELD50 determination and animal experiment. The results showed that the full-length genome sequence of isolate GX120915, GX130330, GX150829 and GX151002 was 10991nt, 10991nt,10990nt and 10992nt, respectively, including a single open reading frame (ORF) that encoded a polypeptide of 3,425 amino acids (aa), and the 5’and 3’untranslated regions (UTR). Compared to the genome sequences of previously isolated TMUVs published in NCBI, there was above 96% simility at the nucleotide level, and above 98% simility at the amino acid level. The GX150829 isolate was more close to the other strains in different areas, they were clustaled into the same big clade, the isolates of GX120915, GX130330 and GX151002 with GX2013H; CQW1 isolate from Chongqin formed a small branch in the big branch with regional feature. No hemagglutination activity was observed with the isolates in allantoic fluid and ELD50 of the virus of the 3rd passage are 10-4.32-10-4.68/0.2 mL. The isolates exhibited high virulence in 7-day-old Jingxi ducks, the morbidity rates were 60%-80%, the mortality rates were 20%-40%. The clinical feature of sick ducks inoculated by the DTMUV isolates was identical to that of naturally infected ducks.Secondly, prokaryotic expression of E gene and identification of the recombinant proteinBased on the sequence of E gene of DTMUV GX 120915 strain, a pair of specific primer were designed.1194 bp E gene fragment was amplified by PCR and cloned into pMD18-T vector. After sequence verification, the fragment was subsequently cloned into prokaryotic vector pET-32a. After identification, the plasmid were transformed respectively into Escherichia coli BL21, the recombinant protein was expressed by IPTG induction, purified by NI-NTA agarose affinity column, and renaturated by different concentrations of urea. The SDS-PAGE and Western blot analysis of expressed protein demonstrated that the recombinant E protein of 53kDa was highly expressed and had excellent immunogenicity.Thirdly, developped indirect ELISA based on protein E from DTMUVAn indirect ELISA was developped for rapid detection of antibodies against DTMUV using the purified recombinant protein as coating antigen.The reaction conditions were optimized and the cut off-value judging negative or positive was 0.354(OD450nm).The developped method was used to detect positive serum samples to conventional duck viral diseases for assess the specificity. The results were below the cut off-value and showed that there were no cross-reaction to the sera against the DTMUV. The coefficients of variation of the intro-batch and inter-batch duplicability tests were below 10%.The sensitivity was 1:6400. The resluts demonstrated that the developped method was stable and the sensitivity was high.In summary, four TMUV isolates were isolated and identified in our study. The isolates have a strong pathogenicity in Jingxi ducks. The isolates were purified by plaque purification, and their complete genomes were sequenced and analyzed. Prokaryotic expression of E gene and identification of the recombinant protein were performed successfully and had the recombinant E protein with excellent immunogenicity. A indrect ELISA method was developped to detect the neutralizing antibodies against DTMUV with strong specificity, high sensitivity, good stability. The method was suitable for detection of clinical serum samples.