GN01 is a new antiviral medicine acting against Korean Sacbrood virus (KSBV) of honeybees. It contains 5 mg/mL of active ingredient, double stranded RNAs(dsRNA), that homologous to KSBV ribonucleic acid coding coat protein (VP1) of virus and inducing RNA interference (RNAi). RNA medicine is generally recognized as safe for rapid breakage by intrinsic ribonuclease and limited absorption from gastrointestinal tract. However, there were no data of repeat-dose toxicity in laboratory animals for dsRNA targeting SBV. This study was performed to investigate toxicity of GN01 in SD rats after weekly oral dosing for 28 days and to determine its no-observed-adverse-effect-level (NOAEL). Male and female SD rats were orally administered with GN01 at 0, 25, 50 and 100 mg/kg bw/day of dsRNA once per week for 28 days (total 5 administrations). The highest dose 100 mg/kg bw/day was determined based on the maximum volume injectable (20 mL/kg bw) via gavage. During treatment period, clinical signs, functional and sensory responses, body weights, food and water consumption, ophthalmological findings and urinalysis were investigated. After treatment period, hematological and clinical biochemistry tests and examination of necropsy findings, organ weights and histopathological lesions were performed. There were no significant differences between all test groups and vehicle control group in all measured parameters. Therefore, the NOAEL of GN01 was determined 100 mg/kg bw/day, the highest dose administered. In conclusion, repeated oral administration of GN01, a dsRNA medicinal product, is safe even at the maximum available dose in rats.

Sacbrood virus (SBV) is one of the most destructive honey bee virus. The virus causes failure to pupate and kills honey bee larvae. The infacted larvae`s color is change to brown. At the end, honey bee colony is destructed. Recently Korean Scabrood virus(KSBV) caused a great loss of Korean honey bee(Apis cerena) colonies for short period. Therefore, We need a highly rapid diagnosis method for rapid detection of KSBV. In this study, We need amicro-scale chip-based real-time PCR system (GeneChecker®). This system was developed for rapid, specific PCR based diagnosis. This system has uncommonly fast heating and cooling system. So We was able to detecting of KSBV in Apis cerena in short time. This system needs small reaction volume(total 10ul). This volume include SsoFast™ Evagreen Supermix and serially diluted cDNA templates showed a high sensitivity of 101copies.That machine can setting each PCR stage time. A specific detection primer set (KSBV-123-F/R) was used to amplify a unique 123bp DNA fragment. This PCR assays using serially diluted cDNA templates showed a high sensitivity of 101 copies. When applied to KSBV-positve samples, the result showed high specifity. The minimum diagnosis time was 9m 47s (30cycle). The amplied positive samples appear red fluorescent color. This novel detection method could be used a PCR-based diagnositic tool (GeneChecker®). The results showed high sensitivity and specifity in short time. And this diagnosis method is expected to be applied to rapidly detect various pathogens.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, Korean Sacbrood virus (KSBV) caused a great loss in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a novel micro PCR-based detection method, termed ultra-rapid real-time PCR (URRT-PCR) were applied for rapid detection for korean sacbrood virus (KSBV) from honeybees (Apis cerana) infected with SBV in Korea. The LAMP could be detect the virus in RT-LAMP reactions containing 102copies of pBX-KSBV within 30 min, which was 10 times more sensitive than a RT-PCR assay. The URRT-PCR showed high sensitivities which were able to detect 10 copies in the standard assays. In the application of URRT-PCR detection to an KSBV-infected honeybee, the shortest detection time was 10 min 12 sec, including reverse transcription. In addition, these methods could be distinguished between KSBV and other closely-related SBV strains, These rapid methods were rapid molecular-based diagnostic tools and useful tool for the rapid and sensitive diagnosis of KSBV infection of honeybees.

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.

Sacbood virus (SBV) is an infectious disease, resulting in failure to pupate and death and kSBV is a disease caused perish Apis cerana of 75% in South Korea. RNA dependent RNA Polymerase(RdRP) is one of polyprotein of viral genome and an enzyme that catalyzes the replication of RNA from an RNA templates and an essential protein encoded in the genomes of all RNA containing viruses with no DNA stages. In this study, recombinant construct with RdRP partial region of kSBV was used for sequence analysis to clarify about Korean SBV. As a result it could be determined that the virus develops by infection of Korean Apis cerana called kSBV. Also, we named Apis cerana-kSBV-region to the name of the unique region of gene that kSBV has. In comparison of the RdRP region of bee RNA virus on nucleotide sequence, its sequence from same species have less variability as well as each virus species has a certainty of RdRp region. It indicated that mutations of RdRP region of each virus species is able to be a useful indicator of honeybee virus detection.