RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pest whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Since small dsRNAs are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. In this study, Bacillus thuringiensis (Bt) based dsRNA production platform was established under control of sporulation-dependent promoter and vp1 gene of Sacbrood virus (SBV) was introduced. The dsRNA against vp1 gene produced from the Bt suppressed the replication of SBV. In addition, the dsRNA was assembled into inulin coated-nanoparticle to increase stability of dsRNA in environment.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.

South Korea has over 38 millions of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (99%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Symptoms of KSBV include the rapid transmission of larval stage honeybees (A. cerana), many dead larvae found in the bottom of hive and comb. Honeybees (A. cerana) are a very important species because they provide a number of pollination services for various ecosystems in some provinces (ex. jeon-nam, jeon-buk province). They are also extremely important organisms within human society, both agriculturally and economically. The fact that a direct cause has been determined suggests that colony collapse is a complex problem with a combination of natural and anthropogenic factors. Possible instigators of colony collapse include: wax moth, viral and fungal diseases, increased population, decreased genetic diversity, climate changing and a variety of other factors. The interaction among these potential causes may be resulting in immunity loss for honeybees and the increased likelihood of collapse.

Sacbrood virus (SBV) is one of the most fatal pathogens against Asian honeybee, Apis cerana. This virus cause failure of the insect larvae to pupate and death of the adult insects. This study has analyzed the host genes affected by viral infection, by comparing the expression level of host transcripts infected with or without SBV. As a first step, we sequenced the cDNA libraries of Asian honeybee by using illumina RNA sequencing. The sequences were de novo assembled to acquire honeybee transcriptome sequences. The transcriptome was annotated by the sequence comparison to known protein sequences by BLASTX and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database with functional categories and description. By mapping the RNA-seq data to de novo assembled transcripts, we characterized the differentially expressed transcripts between SBV-infected and non-infected Asian honeybee.

Most Korean beekeepers have moved from south to north of Korea to collect nectar from black locust (Robinia pseudoacacia) flowers for 2 months. This provided a valuable opportunity to sample bees originating from diverse areas in one location. We initiated a survey of honeybee (Apis mellifera) colonies on the blooming period of Acacia to determine the prevalence of Nosema apis and black queen cell virus (BQCV) in 2013. Nosema causes significant losses in population size of honeybees. Sixteenth hives were sampled for this study. Bees were collected on the 4th and 13th of May, 2013. Nosema spore counts ranged from zero to 1,948,333 spores per bee. The average number of nosema spores per bee was calculated to be 450,000. Approximately 94% of the apiaries examined were infected with nosema, based on the presence of spores in the flowering period of Acacia. Also nosema is thought to be associated with black queen cell virus. RT-PCR analysis shows that BQCV infection rate was 100%. This indicates that nosema and BQCV is the predominant species affecting honeybee colonies.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of KSBV in cell cultures. The growth of KSBV was demonstrated by RT-PCR, quantitative real-time PCR, TEM and nucleotide sequence analysis. The results demonstrated that SBVshowed the replication signals in mammalian cell lines, including Vero cells without any signs of cytopathic effect (CPE). The results of RT-PCR, quantitative real-time PCR and in vivo infection with KSBV were also indicated the replication. Phylogenetic tree analysis shows our sequence included in distinct group with other SBV strains from China and Korea. It clearly showed the differenciation between field strain and attenuated strain through cell culture. The results of present study demonstrated for the first time that SBV like other animal viruses could be adapted and attenuated in cells through the sequential passages. The sequential adaptation through cell culture could result in discrepancy of pathogenicity of virus and morphological characterization. For this reason, the present results indicated that the cell adapted SBV could be a valuable tool to study the general properties of this emerging virus, including pathogenicity in the future.

Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.