Attenuation Of Duck Tembusu Virus By Serial Passage In Chicken Embryo Fibroblasts

Since April2010, the outbreaks of Duck Tembusu virus（DTMUV）disease characterized withrapid-egg-drop had resulted in a massive economic loss in the major duck-raising regions of China. Thedisease has been epidemic in most areas of China. To date, there is not available vaccine for preventionof the disease.Previously we isolated DTMUV FX2010strain from sick shelducks with rapid-egg-drop during theearly stages of the infectious disease outbreaks. To obtain low-virulent DTMUV variants as vaccinecandidate and then find some key residues for virulence attenuation the serial passage of the FX2010virus in chicken embryo fibroblasts (CEF) was performed in this study. Passage of viruses yieldedsignificant cytopathic effect （CPE） after the3rd passage and high passages variant could yield CPEduring24-36hours. These results suggested that the DTMUV had gradually adapted to the CEF. Thefinal variant,180th-passage of DTMUV FX2010, FX2010-180P, was obtained by purification5timesusing limited dilution method and2passages in CEF after173passages. The virulence of FX2010-180Pin SPF chicken embryos was reduced compared to FX2010.To eveluate the virulence differences between FX2010-180P and FX2010several duck infectionexperiments were performed.(1)21-old-day shelducks were inoculated intranasally (i.n.) withFX2010-180P and FX2010respectively;(2)21-old-day shelducks were inoculated intramuscularly (i.m.)with FX2010-180P and FX2010respectively;(3)180-old-day laying shelducks were inoculated i.m.with FX2010-180P and FX2010respectively;(4)12-week-day shelducks were inoculated i.m. or i.n.with FX2010-180P and FX2010respectively. In the first to third animal experiment, clinical fidings,pathology and virus replication in the inoculated-ducks were used to evaluate the pathogenicity of thevirus. In the fourth animal experiment, to evaluate viremia in ducks inoculated with FX2010andFX2010-180P, serum were collected at1-7day post-inoculation (dpi), and used to detect viral geneticmaterials by real-time PCR. In the first animal infection experiment, no any obvious clinical signs andpathological changes were found in the ducks inoculated i.n. with FX2010-180P and no virus wasdetected in some organs. These results suggested that serial passage of DTMUV in CEF reduced theinvasion ability by respiratory tract in ducks. Therefore, in the second animal experiment, the duckswere inoculated i.m. to evaluate the virulence of FX2010-180P. On the one hand, similar to the resultsof the first animal experiment, no any obvious clinical signs and pathological changes were found in theducks inoculated i.m. with FX2010-180P, but on the other hand low titers virus was detected only in thespleens of some ducks inoculated i.m. with FX2010-180P. The above results indicated thatFX2010-180P was low virulence for the ducklings, and could infect animals by intramuscular injection.To evaluate the virulence of FX2010-180P in laying shelducks the third animal infection experimentwas performed. Similar to the results of the second animal experiment, no any obvious clinical signsand pathological changes were found in the ducks and low titers virus was detected only in the spleensof some laying ducks inoculated i.m. with FX2010-180P. The results of the fourth animal experiment showed that no detecTab.le viremia in ducks inoculated with FX2010-180P at1-7dpi. In summary, thisstudy showed that FX2010-180P has several distinct features that were different from its parent virusFX2010, which include (i) reduced virulence in SPF chicken embryos;(ii) reduced invasion ability byrespiratory tract;(iii) no any obvious clinical signs and pathological lesions in inoculated-ducks (iv)detecTab.le lower titers virus and limited virus distribution in vivo;(v) no detecTab.le viremia ininoculated-ducks. The above features of FX2010-180P might make it become an excellent vaccinecandidate.The host-specific adaption and pathogenicity of flavivirus may be associated with geneticmodifications. To identify genetic changes of FX2010-180P the complete viral genome was sequenced.The results showed that the nucleotide sequence of FX2010-180P and its parental FX2010showed34nucleotide changes in the whole genome. However, out of these34nucleotides changes only20changesresulted in19amino acid change, and12nucleotide changes did not result in amino acid change. Inaddition,2nucleotide changes were found in the3’ noncoding region (NCR). Any one of the19aminoacids mutations in the structural proteins and non-structural proteins might affect the virulence ofDTMUV since the level of virulence of falvivirus was affected not only by the number of mutations butalso by their specific combination.