Isolation, Pathogenicity And Cell Adaption Research Of Duck Tembusu Virus

At the end of2009, there was a disease which could lead to laying ducks ovarian bleeding,rapid declinereduction of egg quantity in part of Southern areas in China. In these areas, there areloads of duck-raising farm. The ducklings had the neurological symptoms, and some of them diedafter infection. This disease widespread during these period, covering Fujian, Zhejiang, Jiangxi,Guangdong, Guangxi, Anhui, Shandong, Henan, Hebei and Beijing district. It cost devastatingeconomic loss. Confirmed by the large number of experimental, the disease is caused by DuckTembusu virus.In this study, three Duck Tembusu viruses were isolated from the duck farm incident ofdisease of Shandong province. These three Duck Tembusu virus was named respectively afterDu/CH/LSD/110128, Du/CH/LSD/110130, Du/CH/LSD/101101. First of all, we analyzed thegenetic homology and constructed the phylogenetic trees, between these three virus and otherflavivirus representive strains, found that has a close relationship. We chose Du/CH/LSD/110128for further experiments. The virus genome is a single plus strand RNA containing an ORF whichconsists of10278nucleotides. The ORF encode a polyprotein composed of3425amino acids. Thegenomic coding sequence is structural protein C, PrM, E, and non-structural protein NS1, NS2A,NS2B, NS3, NS4A,2K, NS4B, NS5.We selected7-day-old duckings for poison attack experiment with Du/CH/LSD/110128strain,and initially chose three different inoculation approachs, intracerebral injection, legs intravenousinjection and the combination of muscle, eye-dropped and intranasal. The two groups withinfection in brain and leg vein show apparent neurological symptoms-head and neck drivebackward、ataxic gait、slow-reacting factor as well as the death, the incidence rate of100%, themortality rates were18.2%and5.3%, while muscle intranasal eye group was unaffected. Theresults showed that the intracerebral injection is the best way for the virus to culture the virus invivo, legs intravenous injection less effective. In addition, Chicks poison attack experiment usedintracerebral injection of7-day-old chikens, the incidence rate of30%, and death not occurred.Mature duck poison attack experiment used60-day-old ducks, chose tow different inoculationapproachs, intracerebral injection and legs intravenous injection, the incidence rate were50%and30%. This two ways are not caused death of SPF duck. It indicates that Duck Tembusu virus cause ducks incidence with age dependent.In addition, it was cultured in the Vero E6and BHK21Cell to investigate the adaption of cellswith Du/CH/LSD/110128duck embryo allantoic poison former generations. Results in Vero E6cells and BHK-21cells display Du/CH/LSD/110128stain could be cultured stably and passaged.Initially it showed apparent CPE, but after passage, the CPE disappeared.Because the virus of Flaviviridae could lead to zoonosis, Duck Tembusu virus may transportin natural situation and infect mammals. To investigate this point, we passagedDu/CH/LSD/110128in the brain of BALB/c mouse. Recently, we had4th continuous passageculture in mice. It shows that Duck Tembusu virus of mice arre not susceptible.