Adaptation And Pathogenicity Changes After Serial Passages In Chicken Embryo Of Duck Tembusu Virus

Since April2010, in China a novel duck disease, which was characterized by ovarian hemorrhage and decrease in egg production, was caused by Duck Tembusu virus (DTMUV). The disease broke out throughout the main duck-producing regions of China and caused serious economic loss. As an emerging disease, no effective vaccine against DTMUV infection in ducks was successfully used in clinical applications. So the study of the virus and development of an attenuated vaccine against DTMUV are particularly important.The DTMUV Du/CH/LSD/110128strain isolated and identified by our team was adapted and serially passaged by inoculating into the allantoic cavity of9-11-day-old specific pathogen-free (SPF) embryonated chicken eggs. At every20th passage starting with passage30(CF30), the allantoic fluid was examined for the presence of virus by the hemagglutination and RT-PCR and confirmed the virus titer by measuring the EID5o. The virulence virus could well adapt SPF chicken eggs and cause typical pathological lesions in embryos after serial passages.In order to study the attenuated and the pathogenicity changes to SPF ducklings of the different embryo-passaged derivatives, we divided ducklings into five groups (20birds in parental virus group and each15birds in other groups). SPF ducklings were inoculated with the parental strain, CF50, CF70, CF90and sterile allantoic fluid in the control group, respectively, by intracerebral application at7days of age. Three ducklings from each group were killed humanely at3,7,14,21and28days post-inoculation, respectively. And seventeen tissue samples were collected for virus detection by TaqMan Real-time RT-PCR. Based on these results, we can learn that the viral distribution in tissues and virus loads changed in the organization of ducklings inoculated with these viruses. The pathogenicity to SPF birds inoculated with embryo-passaged viruses was evaluated on the basis of morbidity, mortality, clinical symptoms and gross lesions compared with those after inoculation with the parental virus. In the light of the results, the virus could be detected in all the tissues. The viral loads in all tissues of each passage gradually decreased as time went on and the viral loads decreased in almost tissues of inoculated ducklings as the passage levels increased. Compared with the parental virus and other embryo-passaged viruses, the ducklings challenged with CF90showed no clinical signs and gross lesions.In order to research the immunogenicity of the different embryo-passaged derivatives, we divided ducklings into four groups (each10birds in parental virus group and negative control group and each8birds in other two groups). SPF ducklings were inoculated with the DTMUV Du/CFI/LSD/110128strain, CF70, CF90and sterile allantoic fluid in the control group, respectively, by intracerebral application at7days of age. All ducklings in groups were blooded humanely at5,10,15,20,25,30days post-inoculation. Serum antibodies specific for DTMUV were measured by indirect ELISA and western blotting tests. The ducklings showed seroconversion at5days after inoculated with different viruses, and antibody positive rate of the birds inoculated with CF90increased slowly and all the ducklings showed seroconversion at25days post-inoculation. The vaccination-challenge test showed Du/CH/LSD/110128CF90could provide effective protection to challenge with virulent virus. Based on these results, CF90was fully attenuated and retained the immunogenicity of the parental strain after serial passages, which may be a promising vaccine candidate strain against DTMUV.In order to study the genome changes in the process of virus adaptation in chicken eggs, we sequenced the complete genomes of embryo passaged-viruses, CF30, CF50, CF70and CF90. Compared the nucleotide sequences and the amino acid sequences of embryo passaged-viruses with those of parental virus showed that the genome of Du/CH/LSD/110128strain changed during serial passages in the chicken embryos. Fourteen nucleotide changes, which resulted in amino acid substitutions, were observed in the Capsid, prM, Envelope, NS1, NS3, NS4A, NS4B, and NS5proteins. And a72nt deletion was also observed in5nucleotides downstream of the stop codon region of the virus genomes after30passages. The amino acid mutations occurred in the NS1, NS3and NS4A might be responsible for adaptation in the chicken embryos, the viral replication and the pathogenicity changes to the ducklings of the Du/CH/LSD/110128strain.