| In2010, an outbreak of shelduck infectious disease, caused by unknown pathogen, resulted inretarded growth, egg production decline and death. In the begaining, the outbreaks happened in Jiangsu,Zhejiang, and Shanghai. Then, spread rapidly to all of the southeast provinces of China, includingShandong, Beijing, Hebei, Fujian, Anhui, Guangdong, Guangxi and so on. The disease continued totransmit until winter, all the ducks raised in China were under a huge threat by this unknown pathogen.Epizootic investigation suggested that most susceptible animals to this pathogen were shelducks, theinfection rate and morbidity of shelducks was as high as100%and mortality varied from5%to30%.To identify this new virus, a homogenate of the spleens of sick shelducks raised in Fengxiandistrict, Shanghai, was filtered through0.22um filters, and inoculated into the allantoic sacs of9-day-old SPF embryonated chicken eggs. All of the embryos of the inoculated eggs died2–6dayspost-inoculation and were severely swollen with edema. The supernatant of the homogenate of theembryos had an infectivity titer of105.5ELD50/mL.To identify the taxonomic placement of the virus, the purified causative agent which were treatwith sucrose density gradient centrifugation was examined by electron microscopy. Spherical andenveloped particles with a diameter of45-50nm. To define the nucleic acid type of FX2010, RNA andDNA were extracted from purified FX2010Upon treatment of the nucleic acid extracted from FX2010with RNase but not DNase, the RNA band was no longer detected, the result show that this virus wasRNA virus. At the same time, an FX2010cDNA library was made and plasmids extracted from thebacterial clones picked randomly from the cDNA library were directly sequenced. Two gene sequenceswere obtained, Sequence analysis showed that these genes of FX2010shared88.3%and88.0%nucleotide homology with those of the Tembusu virus Mm1775strain, respectively. In cross serumneutralization tests between FX2010and JEV, we found no cross-reactivity with heterologousantibodies; In cross serum neutralizing tests between FX2010and Tembusu virus, an antiserum againstthe partial E protein of Tembusu virus Mm1775strain neutralized FX2010at a serum dilution of1:90.We designated this virus Tembusu virus Fengxian2010（FX2010）.Studies have shown that the isolate were sensitive to ether, Chloroform, but it has nohaemagglutination activity. The virus could grow in duck embryo fibroblasts and caused CPE, as thepassage times increase, CPE became more regular. Aslo it can grew in Vero cells without any CPEs.After infected six shelducks with FX2010by inoculating them i.n. or i.m., we can isolate high titerof virus from kidney, brain, spleen, lung, pancreas, trachea, blood of the infected ducks, but low titer ofvirus from liver. After observed the tissue and organs of the infected ducks, we found the infected duckswere moderate necrosis of brain tissue, severe nephrosis, pneumorrhagia, severe necrosis of the spleen.Immunohistochemistry show that several tissues had virus.In order to understand the molecular genetics information of FX2010, several overlapping cDNAclones, covering the entire genome of FX2010strain, were obtained by primer walking RT-PCR. Thesequences of3’ and5’ termini of the viral genome were amplified by3’ and5’ RACE. Sequences complied from these clones show that FX2010strain is10991nt long, and encode ten proteins, Weanalyzed the complete genome sequence of FX2010strain with bioinformatics methods. Phylogeneticanalysis of genome sequences among FX2010strain, and other members of family flaviviridaedemonstrate that FX2010strain, BYDV are in one lineage, and both were belong Tembusu virus. Wealso predicted cleavage sites and N-linked potential glycosylation sites for the polyproteins of FX2010strain, the result show that the cleavage sites had certain conservation. |
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