| Since2009, an occurrence of a novel disease in China was caused by Duck Tembusu virus(DTMUV). The disease which affected duck industries in most areas of China, was characterized byovarian inflammation and decreased egg production. The disease had inflicted significant economiclosses in the duck industry in China. Up to now, no effective vaccine is available to prevent the infectionof disease. In order to guard against the disease, development of a valid attenuated vaccine is of utmostimportance.The strain Du/CH/LSD/110128was passaged by inoculation into the allantoic cavity of9-11-day-old specific pathogen-free (SPF) duck eggs. The strain Du/CH/LSD/110128can well adaptSPF duck embryos and caused typical pathological lesions in embryos.7-day-old SPF ducks were inoculated with the Du/CH/LSD/110128strain, passage levels50(DF50),75(DF75) and100(DF100) via intracranial inoculation, respectively. The pathogenicity to SPFducks was evaluated on the basis of the clinical symptoms and gross lesions. Meanwhile, tissue samplesof trachea, lung, liver, spleen, glandular stomach, duodenum, small intestine, large intestine, cecum,kidney, rectum, harderian gland, thymus, pancreas, cecal tonsils, bursa of fabricius, brain, marrow andovary were collected and their virus loads in these tissue samples were detected by TaqMan real-timePCR. The ducks infected with Du/CH/LSD/110128had obvious clinical symptoms and seriousneurological symptoms60hour post-infection and the morbidity and mortality of which was100%and17.1%respectively. The ducks infected with DF50had similar clinical symptoms with the ducksinfected with strain Du/CH/LSD/110128, with the morbidity100%and the mortality10.5%respectively.The ducks showed slight clinical symptoms after DF75infection, with morbidity33.3%and mortality4.2%respectively. In contrast, the ducks infected with DF100had no apparent clinical symptoms withmorbidity and mortality3.8%respectively. The detection of the virus loads revealed that DTMUV viruscan be detected from all tissue samples. The spleen, kidney, brain and ovary had high virus titers. Thevirus loads reduced gradually. The tissue samples with DF100infection had much lower virus titerscompared with those infected with other virus on14,21and28day post-infection.7-day-old SPF ducks were inoculated with the Du/CH/LSD/110128strain, passage levels75(DF75)and100(DF100) of strain Du/CH/LSD/110128via intracranial inoculation, respectively. The peripheralblood was collected through leg vein from the infected ducks at different time points. Recombination Eprotein and recombination NS1protein were expressed to establish ELISA method. Then we evaluatethe antibody level in serums collected from ducks infected with strain Du/CH/LSD/110128, DF75andDF100by ELISA. The results showed that the antibodies can be detected from5day after parental virus,DF75and DF100infection. The highest positive rate appeared at10to20day after parental virus, DF75and DF100infection. In addition, the serums collected from the ducks infected withDu/CH/LSD/110128had highest positive rate and the serums collected from the ducks infected withDF100virus had lowest positive rate.Immune protection test showed that DF75and DF100can induce enough protection against the Du/CH/LSD/110128. In a word, the results showed that DF100had been attenuated, and had excellentimmunogenicity. DF100could be used as vaccine.In order to study the genomic changes in the process of virus adaptation in duck embryo, the aminoacid sequences and the complete genomes sequence of Du/CH/LSD/110128, DF30, DF50, DF75andDF100viruses were compared. Nucleotide and amino acid changes, which were mainly located in Eprotein and NS1protein, were observed among the genomes of parent Du/CH/LSD/110128, DF30,DF50, DF75and DF100. These mutations might be accounted for the virulence decrease caused by duckembryo-passage. |
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