Genomic Sequence Analysis And Pathogenicity Of The Duck Tembusu Virus

During investigations into the outbreak of duck viral infection in 2010 in China,with a severe drop in egg production,has become one of the main diseases endangering the duck industry in our country.Pathogenies production after onset characterized by persistent low rate,combined with secondary infection,death rate can reach 15%-20%.This study collected from some duck farms in shandong sterile temple suspected virus?Tembusu virus,TMUV?symptoms of infection disease ducks,carried out the following research: 1,according to conservative sequence specific primers,designed with RT-PCR technique,through the method of virus isolation,identification,get DTMUV;2 through dynamic pathology observation,has conducted the virus to 200 days of age cherry valley of pathogenies pathogenic research,focused on ovarian organ lesions significantly,clear the virus infection of pathogenies ovaries to produce time,lesion degree of pathological changes,and the relationship with viral load;3 What is more,the complete genome of this virus was sequenced and analyzed by design on each TMUV cDNA full-length primers.Isolation and identification of virus: The tissue samples were undergone excision,homogenization,filtering,and then inoculated into allantoic cavites of 9 day-old duck embryonated eggs.After three times passage,the duck embryonated eggs infected by this virus were died within 4 days and were observed severely swollen and hemorrhage.Similar to other flavivirus,it was a RNA virus,what is more,the identification results suggested that it was an enveloped virus and were sensitive to ether,chloroform.To collect the allantoic fluid by RT-PCR detection,several common source pathogens such as Duck pestilence Duck virus?Duck plague virus comes,DPV?,Duck wreath virus?Duck circovirus,DuCV?and Duck Hepatitis B virus?Duck Hepatitis B virus comes,DHBV?,Duck source of Avian Influenza virus?Avian state-run Influenza virus get,AIV?test were negative.According to TMUV-WR strain NS3-NS4 fragment conservative sequence design of specific primers amplified by rt-pcr method was suspected to strip?300 bp?,found that after sequencing,comparing with the published yellow virus flavivirus genus grace he virus IPDIA isolates nucleotides?GenBankNO: NC₀18705?similarity was 75.3%,compared with the domestic has released TMUV separation strains,with the highest DK/TH/CU-1strains?GenBankNO:KR061333.1?similarity,at 95%,with GX/1/12?GenBankNO:KP719117.1?the lowest similarity,1 strain of 91%.Successful one is the result of the shandong region of duck temple Sue virus,named SDT1 and scientific basis for further research.SDT1 isolates the whole genome sequence amplification and genetic evolution analysis: through the design on each TMUV cDNA full-length primers,segmented amplification,sequencing,joining together the isolates full-length genome sequence.Whole genome sequence analysis results show that SDT1 isolates the 10992 nt,contains an open reading frame?ORF?,encoding a length of 2625 aa more protein,prediction of how the protein-cutting site consistent with other members of TMUV,coding 10 functional protein.Huang on behalf of the members and temple Sue virus isolates of the evolutionary tree analysis table isolates belong to yellow virus grace he and members of the family,SDT1 isolates with other members of TMUV in genome-wide nucleotide and amino acid similarity between 95% and 98%,respectively,98.4% to 99.6%.Compared with other domestic isolates,SDT1 isolates with other isolates the average evolutionary distance?0.0299?is higher than other strains?0.0163?,the average evolution of distance between enriched temple Sue duck virus library.SDT1 strains of egg production of 200 days of age of cherry valley duck pathogenic test: select only 200 days of age 30 eggs of cherry valley duck regression animal experiment was carried out,before experiment,RT-PCR method to eliminate the duck temple Sue natural infection virus,including 20 ml?TCID50?at 0.5 each dose of vaccine by chest muscles duck temple Sue virus as poison attack group,10 points cage only by injection amount of PBS solution as the negative control group.Found that infected after attacking a significant loss of appetite,not willing to walk,listlessness,ataxia,death is opisthotonos and nerve symptoms.Poison attack group all disease,the incidence is 100%;The control group no significant clinical symptoms.Rt-pcr results found,can from infected duck blood,ovaries,liver,brain tissue DTMUV detected.Pathological histology observation results show that the follicle hyperemia,hemorrhage,follicular granulosa cell necrosis,ovaries licking leukocyte infiltration;Liver hemorrhage,congestion,a large number of liver cell degeneration,necrosis,hepatic lobule structure disorder,at the same time,there exist more new liver cells.Through continuous pathology observation,found that the most significant ovarian pathological changes,so we conducted the duck temple Sue virus infection in different stage of ovarian lesions and viral load correlation research.