Biological Characteristics And Histological Location Of A Flavivirus Strain BZ In Duck

Since April2010, a novel contagious disease in many egg-laying ducks and ducklings,with egg drop, feed uptake declineand nervous system signs.Through the clinical andhistopathological profile for observation, combined with the pathogen isolation andidentification, serologic testing and molecular sequencing, it was confirmed that egg-layingducks and ducklings were harmed by the same kind of pathogen, the pathogen DuckFlavivirus is a new member of flavivirus. There is no specific treatment to DFV, because oflacking of effective prevention, the disease has been widely spread in our country especiallyZhejiang, Jiangsu, Anhui, Shandong. So far it has caused severe economic loss of duckindustry in China.1. Isolataion，Identification and Biological Characteristics of Duck Flavivirus virus BZstrainA new virus isolated from laying cherry duck flocks with typical eggs drop symptomswas named as BZ. The serum tests indicated that there were no reactions against avianinfluenza, Newcastle disease, duck enteritis virus. A pair of primers was designed andsynthesized according to the NS1gene sequence of Bagaza virus and a reverse transcriptasepolymerase chain reaction method was developed for strain BZ.Some biologicalcharacteristics of a newly isolated strain （BZ） of duck flavivirus were evaluated. The resultssuggested that the virus belonged to a single-stranded RNA with an envelope and wassensitive to chloroform and deoxycholate treatment. It was unstable at lower pH and could beinactivated at56℃for15min.The virus could replicate well not only in10-day old SPF chickembryos and duck embryos but also in duck embryo fibroblast monolayers with cytopathiceffect（CPE）,which there was no hemagglutinating activity to erythrocytes of chicken, duck,goose and pigeon et al. The Virus titer of chorioallantoic membrane was higher than any ofallantoic fluid and embryos by yolk sac inoculation.2. Development and application of real-time fluorescent quantitative RT-PCR assay forduck flavivirus virusA real-time fluorescent quantitative RT-PCR method was developed for the detection ofDuck flavivirus （DFV）. The specific primers and Taqman probe were designed and synthesized according to the NS1gene sequence of duck flavivirus. The standard curve（Y=-3.39X+43.18,r=0.973） was plotted based on the linear relationship between the amountof plasmid DNA and cycle threshold （Ct）. This method was specific only for Duck flavivirusvirus（DFV） but for Duck hepatitis virus，Avian influenza virus，Newcastle disease virus，Adenovirus，Duck plague virus and Porcine Japanese encephalitis virus. The detection limitreached1.9TCID50of the virus under the optimized conditions. Tissues in five ducksartificial infected for86hours with DFV were tested positive by this method, without parts ofcecal tonsils. And the positive detection rate of the method was consistent with routine virusisolation method.These results indicated that the real-time RT-PCR approach provided apowerful diagnostic tool with high sensitivity and specificity for the identification andquantitation of DFV and the whole process of the test could be completed within3hours.3. The comparative study and histologic positioning of DFV in duckling infectedartificially with different ways.Two handred of ducklings2day old were inoculated with DFV(10^5.9TCID₅₀/0.1mL) viathe brain,the neck subcutaneous, the eye-dropped and intranasal routes.The establishedfluorescence quantitative RT-polymerase chain reaction （PCR） was applicated to detect thedynamic distribution of DFV in ducklings, the results showed that: the dynamic distributionwere different among groups.The DFV RNA of the brain infected group was detected inbrain,spleen and cecal tonsils,the neck subcutaneous was detected in spleen, the eye-droppedand intranasal group was detected in spleen, tracheal,cecal tonsils and bursa. The quantity ofDFV RNA, the brain infected group was supreme, the eye-dropped and intranasal infectedgroup was minimum in the begining; The virus distribution of diverse tissues and organs isdifferent in the same inoculation way. The quantity of virus in all tissues is low relatively after84h.The dynamic distribution of the other two groups agreed with the brain infected group,but relatively slowly. The quantity of virus in heart, liver, spleen, lung, kidney and brain ishigher, in pancreatic, duodenal, bursa, trachea and cecum tonsils is relatively low in thesethree different infection ways. This study provides important test datas to elucidate thepathogenesis mechanism of DFV.