차세대 염기서열 분석(Next Generation Sequencing, NGS)은 대량의 병렬 데이터 생산으로 유전체의 염기서열 을 고속으로 분석하는 기술이며, 이 기술은 바이러스 유전체 분석에도 광범위하게 사용되고 있다. 하지만, 바이 러스의 전장 유전체가 100kb를 넘을 경우, 동일한 raw data라도 분석 방법 및 소프트웨어 그리고 매개변수 (parameter)에 따라 유전체의 크기와 구조가 다르게 결정된다. 따라서 유전체가 큰 바이러스 분석 시, 최적화된 NGS 분석 방법을 선택하는 것이 중요하다. 본 연구는 장수풍뎅이 누디바이러스(Oryctes rhinocerous nudivirus, 120kb) 유전체를 기반으로, 다양한 Assembly 소프트웨어(metaviralSPAdes, metaSPAdes, velvet, shovill, Geneious, megahit)를 사용하여, 최적화된 NGS 분석 방법을 고안하였다. Assembly 소프트웨어에 따라 바이러스 유전체 크기와 특징(Single Nucleotide Polymorphism, Insertion&Deletion, repetitive genomic variants)의 차이를 확인하였 다. Assembly 소프트웨어 간의 차이가 있는 염기서열은 Sanger sequencing을 통해 재확인하여, 참조 유전체 (reference sequence)를 구축하였다. 이 참조 유전체를 기반으로 가장 정확한 Assembly 소프트웨어와 parameter를 평가하였다. 본 연구는 분석 방법에 따라 달라지는 유전체의 특성을 이해하고, 바이러스 유전체를 정확하게 구축 하는 분석 파이프라인을 제공할 것으로 기대된다.

The purpose of this study was to determine the safe and effective method for preventing the occurrence of Oryctes rhinoceros Nudivirus in Allomyrina dichotoma on agricultural farms. There is a high demand for the use of A. dichotoma larvae in animal feed and as pet for educational purpose. Recently, we reported that OrNV is fatal virus diagnosed in A. dichotoma larvae in local farms in Korea. Mulberry leaves contain 1-deoxynojirimycin that represented anti-inflammatory, anti-virus, and anti-tumor effects. To prevent OrNV, we have fed the sawdust combined with 1% and 5% mulberry leaves powder to OrNV infected 2nd and 3rd stage larvae of A. dichotoma, and identified the mortality rate(%) during ten weeks. As a results, the 2nd stage larvae which were fed the sawdust combined with 5% mulberry leaves treatment recorded 60% mortality rate after ten weeks compared to the 100% mortality rate in the control. And the fatality rate of 3rd stage larvae which were fed 5% mulberry leaves treatment decreased 70% compared to the control. Therefore, application sawdust combined with mulberry leaves might be effective in the prevention and control of OrNV disease in A. dichotoma. Additionally, in the ten insect breading farms application test, OrNV virus disease have not appeared in the mulberry leaf powder treatment group.

Allomyrina dichotoma (order Coleoptera, family Scarabaeidae) is used for development of pharmaceuticals, pet or educational purposes and animal feedstuffs. The disease occurrence and distribution of Oryctes rhinoceros Nudivirus were investigated in Allomyrina dichotoma in Korea using PCR and analyzed the DNA seqeunces using BLAST(Basic Local Alignment Search Tool). The virus infected larvae were collected from 10 insect rearing farms in five different regions (Gyounggi, Chungbuk, Chungnam, Jeonnam, Daejeon). Frequency of OrNV virus infection appeared differently depending on the regions or rearing facilities (open field, vinyl house, indoor breeding system and etc.). The collected samples of Allomyrina dichotoma raised on open fields showed the highest possibilities of OrNV virus infection. The OrNV average infection rate of open fields rearing systems was 50.0%.

In 2015, we reported a viral disease extremely fatal to Allomyrina dichotoma larvae spread in the majority of the larva-rearing farms in Korea. Currently, the virus-infected larva is diagnosed by PCR-based amplification but this requires laboratory equipment and agarose gel electrophoresis. Loop-mediated isothermal amplification (LAMP), a highly sensitive DNA amplification method, uses DNA polymerase isolated from Bacillus stearothemophilus and a set of six primers. It has great potential for field use because DNA is amplified under single temperature and the overall reaction completes in 30 min without laboratory equipments. Here, we report the development of on-site diagnosis method for Korean horn beetle larva infected by Allomyrina dichotoma Nudivirus.