The Isolation And Identification Of Duck Tembusu Virus And Its Pathological Study

An epidemic which was characterized by a rapid decline in egg production erupted in the south of China since April,2010.In 2011,it was named "Duck tembusu virus disease",this disease has a high infection rate and low mortality rate.there is a sharp decrease in egg production for laying ducks;for meat ducks and ducklings,the main symptoms of this disease is hard in standing and walking.It has brought great loss to Chinese economy.Duck tembusu virus is a member of flavivirus which consists of dengue virus,yellow fever virus,West Nile virus and the other virus endangering human health.Therefore,the duck tembusu virus has a chance to infect mammals and bring about sickness.There is no effective vaccine to prevent this virus,and its pathogenesis is still unknown.Therefore,it is necessary to study the gene structure and pathogenicity of duck tembusu virus.1.Isolation and identification of duck tembusu virus In this experiment,the duck tembusu virus was colleted from the incidence duck field of Hubei province.The infected ducks show creeps and water-like posture,also the egg production decreases from 50% to 0%.There were "perivascular cuffing" and "neuronophagia" in the cerebrum,which are typical symptoms of non-suppurative encephalitis,lymphocyte reduced in the spleen and granulosa cells separated from the basement membrane.The homogenate of the brain tissue from the natural infected duck was identified by RT-PCR.we inoculated the 10 days old duck embryo via capsule cavity and found that the infected duck embryos died with sever retardation and cutaneous haemorrhages,also,there are yellow patches in the liver.we infected DEF cells with the viral allantoic fluid and collect the virus-containing cell fluid after proliferation.To infect BHK cells,cell cytopathy was observed at 48 h after infection,BHK cells turned round and showed obvious cytopathic disease after infection for 72 h.The ELD50 of duck embryos was 10-4.42ELD50 / 0.3ml and the result of the virus plaque test was 4.72 × 107 pfu / m L.The result of Immunofluorescencetechnique showed that the virus appeared in the cytoplasm of BHK cells.The virus was unable to agglutinate the erythrocytes of chicken.The virion was round with envelop and its diameter was 40 ~ 60 nm.In summary,the isolated virus was identified as duck tembusu virus and named MC.2.Determination of the complete sequence of the virusRT-PCR was used to determine the complete sequence of the virus.The tested complete sequence was uploaded to Genbank and named MC.A phylogenetic tree of the polyprotein genes indicates that DTMUV strain MC is closely related(87.1% identity)to sitiawan virus while there is the lowest homology between DTMUV strain MC and yellow fever virus(53.1% identity).The complete sequence of MC was 10990 bp and the length of the unique open reading frame was 10278 bp which encodes 3425 amino acids.The protein cleavage site is similar to tembusu virus BYD1.It is composed of three structural proteins(PrM,C,E)and seven nonstructural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5).The number of Glycosylation site of MC strain was different from that of other tembusu virus.secondary structure of genomic noncoding region was predicted to be consistent with flavivirus through RNAfold.There is a large stem loop SLA and small stem loop SLB at the 5 'end of the non-coding sequence of MC strain and a loop 3'SL in the 3 'noncoding region.The conserved sequence of MC strain in the 3 'untranslated region was highly similar to other tembusu virus..Protein homology analysis found that the homology with other duck tembusu virus was above 97%.3.Pathogenicity of Duck Tembusu Virus on Duckling DucksThe duck temubusu virus strain MC inoculated 7 days old ducklings,7th day after infected,all were sacrificed,got the heart,liver,spleen,lung,kidney,cerebrum and other tissues from infected duckings and 4% formalin fixed tissues;the result of RT-PCR showed that duck tembusu virus existed in three ducks' cerebrum.The result of histopathological showed that three typical non-suppurative encephalitis,such as " perivascular cuffing" and "neuronophagia" found in the duck brain tissue of the virus.The lymphocytes of the splenic tissue were decreased.The result of immunohistochemical staining showed that the viruses were in the neuronal cells and the erythrocyte of cytoplasm of spleen and other tissues.