Isolation And Identification Of A DTMUV, Mutation Induced By 5-FU And Its Pathogenicity Changes

Duck Tembusu virus disease, characterized by ovarian inflammation and egg production drop, is an infectious disease caused by the infection of Duck Tembusu virus?DTMUV?. This disease spread quickly and widely with high morbidity. Since its frist outbreak in 2010, it has inflicted huge economic losses in the duck industry in China.Until now, no commercial vaccine is available to prevent this disease. Therefore,development of a vaccine is of utmost importance.In this study, the samples were collected from a Muscovy duck farm, in which the laying Muscovy ducks had the disease of egg production unnormal drop, and inoculated into 11-day-old Muscovy embryos to isolate the pathogeny. A DTMUV was isolated and identified through RT-PCR and Immunofluorescence assay?IFA? and named ZJSBL01.The animal regression test showed that the affected Muscovy duck showed typical DTMUV disease symptom of egg production rate drop sharply and feed uptake loss.Ovarian follicles hyperaemia and spleen enlarge were observed at necropsy. The spleen contained high viral RNA copies detected by real-time PCR. The length complete genome of ZJSBL01 is 10,990 nucleotide, consisted of a single ORF encoding a 3425 aa polyprotein flanked by a 94 nt 5'-NTR and a 618 nt 3'UTR, respectively. The nucleotide sequence of complete genome of ZJSBL01 shared 97.4%-99.5% homologies with other Chinese waterfowl-origin Tembusu viruses and shared 88.7% identities with MM1775 strain isolated from mosquito. The phylogenetic relationships of the ORF of the ZJSBL01 with other flaviviruses were analyzed using MEGA5.05 software. The result showed that the isolate was clustered with the avian-origin Tembusu virus isolated in China, and genetic close with TMUV MM₁775 and Sitiawan virus, which indicated that ZJSBL01 belong to Ntaya group.The gene of domain III of envelop protein?EDIII? was amplified by RT-PCR andclone into expression vector p ET28 a. The combination vector was transfer into E.coli BL21?DE3? and then induced by IPTG to express EDIII protein. An indirect ELISA was established using EDIII as coating antigen. The reaction conditions were optimized including 4?g/m L EDIII as antigen coating 4h at 37 ?, 1:40 dilution of testing serum,3%BSA PBST blocking 2h at 37?, 1:1000 dilution of HRP conjugated anti-duck Ig G and substrate reaction 15 min with S/P cut-off value of 0.306.The ZJSBL01 Strain was passaged in DF-1 cells for 50 times and then the F50 passage were passaged in DF-1 cells supplementing 100 ?g/m L 5-FU to induce mutation for 20 times and gain F50-20 passage. The pathogenicity of ZJSBL01, F50 and F50-20 was evaluated in 7-day-old Shelducks. The results showed that the spleens of F50-20 were normal while ZJSBL01 and F50 group were enlarged at 3 days post-inoculation.When these viruses were inoculated into laying Muscovy ducks through intramuscular injection, the group of ZJSBL01 showed typical TMUV symptom of feed uptake decline,egg production drop, ovarian follicles hyperaemia and spleen enlarge. The group of F50-10 exhibited slight ovarian follicles and narrow fluctuation of egg production.Notably, egg production of F50-20 group did not decline with just slight spleen. The serum of F50-20 group was evaluated using ELISA. The result showed that the positive rate of antibodies against TUMV was 100% since 7 day-post-inoculate.Immune protection test showed that F50-20 can induce enough protection against the ZJSBL01. The feed uptake, egg production and ovarian of F50-20 immune group were normal when challenged with ZJSBL01. In conclusion, the F50-20 had been attenuated and contained excellent immunogenicity. ZJSBL01 F50-20 could use as a vaccine.The complete genome was sequenced. When compare to ZJSBL01, F50-20 has 28 nt mutations resulting in 10 aa mutation which distribute in E, NS1, NS3 and NS5 and other18 nt mutations are silent mutation. These mutations might relate to the pathogenicity.