Replication Mechanism Of Chinese Sacbrood Virus In Eastern Honeybee,Apis Cerana Cerana And Its Control Strategy Base On RNA Interference

The Eastern honeybee,Apis cerana cerana,is the native honeybee species in China,which is distributed in the most of China.Chinese sacbrood is first identified in Guangdong China in 1972,which poses a serious threat for the population of A.c.cerana.SBV infecting Chinese honeybee was named Chinese sacbrood virus(CSBV).CSBV caused larvae death due to failure of pupation that leads to the formation of a sac-like shape,and adult bees without giving any outward symptoms.There are no methods or effected drugs to cure CSBV,mainly focusing on prevention.CSBV has also been known to exist in the Fujian province for nearly 40 years and has recently caused the death of individual bees and entire colonies.Despite the threat that CSBV poses to bee populations,few studies have focused on CSBV infection in Fujian province.Basic information,such as the complete viral genome sequence of the CSBV Fujian isolate is still unknown.And there are no reported on the function and replication mechanism of the proteins of CSBV.In this study,the complete genome sequence of CSBV-FZ was obtained by gene clone.GenBank accession number of CSBV-FZ is KM495267.The complete viral genome of CSBV-FZ contained a genome of approximately 8.8 kb,which encoding 2,848 amino acids.CSBV-FZ shared 90-97%nucleotide identity and 94-99%amino acid identity with other SBV isolates,respectively.To illustrate the genetic relationships between SBV isolates,a phylogenetic tree analysis was constructed by Clustal X 2.0 and MEGA 5.2 software.The neighbor-joining tree found three distinct groups of SBV isolates.The SBV isolates from India,which originated from the ^A.c.indica subspecies,were clustered together as the first distinct group.The Vietnam isolate(AcSBV-SBM2)from A.c.cerana,belonged to the second distinct group.The UK,Korea and China isolates were clustered together as the third group.In this group,the SBV-UK,AmSBV-Kor 19 and AmSBV-Kor21 isolates,originating form A.mellifera,were clustered together as one subgroup.The CSBV isolates and AcSBV-Korea isolate,originating from A.c.cerana,were clustered together to form the other subgroup.The phylogenetic results indicate that the genetic variation in SBV isolates are dependent on the host(honeybee species and subspecies)and geographic origin.We used the Baculovirus expression system and Yeast two-hybrid technology to study the relationship between the structural protein VP1 and non-structural protein RdRp.Firstly,polychonal antibodies against structural protein VP1 and non-structural protein RdRp of CSBV were prepared by prokaryotic expression system.The antibody specificity was detected with Western blot and ELISA.Secondly,the expressions of VP1 protein and RdRp protein in Sf9 cells were analyzed using immunofluorescent.The result showed that VP1 protein was distributed as vesicle in the cytoplasm.RdRp was distributed as inclusions.VP1 protein and RdRp protein were co-localization in the Sf9 cells,indicating that the existence of interaction between VP1 protein and RdRp protein.Yeast two-hybrid technology further demonstrated that VP1 protein and RdRp protein had specific interaction.These results suggest that the structural protein VP1 interact with nun-structural protein RdRp.To study the expression of VP1 protein and RdRp protein and replication in the cultured cells,the primary cell culture of A.cerana was established in vitro.The eggs of honeybees,collected 36-48 h after oviposition,were sterilized in 70%ethanol.The embryonic fragments were dissected from the eggs and then incubated with 0.25%trypsin in Tyrode's solution.The embryonic tissues were grown in Kimura's insect medium at 28?.By 12 h after preparation,tightly packed epithelium-like cells,approximately 8-10 ?m in diameter,had grown outward from the explants of embryonic tissue,forming a monolayer of primary cells.The monolayer continued to expand,forming large epithelium-like cell sheets in the culture flask within 3-4 days for CSBV infected.At 36 hpi,VP1 protein was distributed as filamentous or vesicular inclusions in the cytoplasm.RdRp protein was distributed as inclusions.VP1 protein and RdRp protein were co-localization in the cells,with the VP1 wrapped the RdRp.Viral particles,approximately 30 nm in diameter,had accumulated within vesicles or were arranged in a filamentous array in virus-infected cells at the same time with transmission electron microscopy.To study the proliferation of CSBV in cultured cells,we examined the expression level of viral cistrons from CSB V related genes using real-time quantitative RT-PCR(RT-qPCR)at different times after viral inoculation.The results showed that the expression levels of these viral genes in virus-associated cells increased rapidly from 12 to 48 hpi,reaching a maximum at 48 hpi.After 48 hpi,the expression level of these viral genes in virus-associated cells decreased rapidly.To suppress the replication of CSBV infected larvae in A.c.cerana using RNA interference.The methods of rearing honeybee larvae and viral inoculation under laboratory conditions were established.CSBV homologous specific dsRNAs(dsHelicase,dsProtease,dsRdRp and dsVP1)were synthesized using T7 RiboMAXTM Express RNAi System Kit.The dsRNA and CSBV were added into the food,dsGFP as control.The second instar larvae were first fed with food containing dsRNA.Twelve hours later,the larvae were then fed with food containing CSBV.The result showed that pupation rate significantly increased by feeding dsRdRp.The mRNA levels of CSBV were detected by RT-qPCR in dsRNA treated larvae.The result showed that CSBV gene expression level was significantly decreased by feeding dsRdRp,which indicating that dsRdRp can effectively inhibit the proliferation of CSBV.In order to apply dsRdRp to production practice,RdRp gene cloned into the L4440 vector,and transformed into E.coli strain HT115 to express dsRdRp.Bacteria contained dsRdRp were fed to CSBV-infected colonies for studying its effect on the prevention and treatment of CSBV.The results indicated that the number of sac-like larvae was significantly reduced.The sealed brood was significantly increased.The population of colony was potentially growth.Meanwhile,virus carried rate of offspring was significantly decreased.The gene expressive quantity of CSBV was also significantly decreased,which indicate that CSBV specific homologous dsRdRp can effectively inhibit viral replication to achieve therapeutic effect for CSBV.