| Objective Chinese bee sacbrood virus was isolated and called CSBV-SXYL in the larvaes being infected with CSBV from Yulin,Shanxi Province,by the separation,purification and identification.Next,the biological characteristics of CSBV-SXYL was analyzed.At the same time,an applicable culture method for honeybee sourced cells was screened and established.After that,the cultured primary cells were inoculated with CSBV-SXYL,which lays the foundation for the research and training of bee virus.Methods By using differential centrifugation,CSBV-SXYL are isolated and purified and identified by RT-PCR method and electron microscope.At the same time,the specificity of CSBV-SXYL was verified by immune agar double diffusion using anti-CSBV standard positive serum.On the basis of this,the physicochemical properties of CSBV-SXYL were studied,such as sensitivity,acid resistance,heat resistance and so on.Ontology animal infection test will be conducted on the 2~3 days old larvaes by inoculating CSBV-SXYL.By observing and testing the infected larvae growth,clinical symptoms,pathological tissue section and HE staining etc.,CSBV-SXYL pathogenicity was to be found.Compared the Chinese bee larvae primary cells cultured in two different insects cell culture medium of grace and WH2,we can screen for the optimum medium of Chinese bee larvae primary cells cultured,and by cell viability comparison,we can also determine the concentration of fetal bovine serum(FBS)used for the culturing of Chinese bee larvae primary cells.Then,the cultured primary cells were inoculated with CSBV-SXYL,and the virus replication was detected with real-time quantitative RT-PCR.Results It is found that the diameter of virus partical is about 30 nm and looks like a ball with equal shaft icosahedron virus particles after the purification of CSBV-SXYL by negative staining observation through electron microscopy.By double diffusion test,CSBV-SXYL was able to react with anti-CSBV standard positive serum.RT-PCR amplification was performed and obtained the characteristic bands.By sequencing and BLAST,alignmented with the reference strains,CSBV-SXYL and CSBV-BJ nucleotide sequence homology is up to 97.2%.Physical and chemical properties analysis showed that CSBV-SXYL capsule membrane is not sensitive to ether and chloroform,which can still survive on the condition of p H3.0 but inactive till the temperature reaches 75 °C.After mixing CSBV-SXYL and basic larval diet(BLD)fed to 2~3 day Chinese bee larvae,after 3 days,larvae stopped ingestion,at 3-4 day,larvae discharged meconium,after 5 day,larvae appeared at the front bent,black body color,transparent head of clinical symptoms,such as infection after 7 days insect body filled with particles of liquid,presents the typical symptom of cystic bag.The pathological tissue slice observation,infection larva tissue between the epidemis and the demis hole formation,filles with liquid water and a series of pathological changes.The bee larvae primary cell culture medium filter showed that relative to WH2 culture medium,the growth of cells in the Grace medium large,round,transparent,neat,no particles,energy obviously higher than that of WH2 cultivation,and the medium containing 15% FBS Grace is more suitable for in bee larvge primary cell culture,It’s also observed that the inoculated CSBV virus could replicate by real-time quantitative RT-PCR method probe detectiond and proliferate by real-time quantitative RT-PCR method probe detectiond and proliferate rapidly along with the division of the host cells.Conclusions1 、 Steains Chinses bee Sacbrood virus was successfully isolated and named for CSBV-SXYL.2 、 CSBV-SXYL has a strong rtesistance to ether,chloroform and other organic solvents and enjoys high stability in acid condition,it can still survive under the condition of p H 3.0,but becomes inactive when the temperature is above 75 °C.3、The Chinses bee lavae primary cells grew well in Medium Grace with15% FBS,in which the replication of CSBV could proceed. |
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