보고서 정보
주관연구기관 |
농림수산검역검사본부 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2013-11 |
과제시작연도 |
2013 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
등록번호 |
TRKO201400001603 |
과제고유번호 |
1475007496 |
사업명 |
농림축산검역검사기술개발 |
DB 구축일자 |
2014-08-16
|
키워드 |
토종벌.사각벌통.개량벌통.꿀벌질병.Honey bee.Sacbrood virus.Phylogenetic analysis.South Korea.Apis cerana.Honey bee virus.Prevalence.Distribution.RT-PCR.Complete genome sequence.Iflavirus.Disease.Apis mellifera (A. mellifera).Apis cerana (A.cerana).Apis mellifera.Black queen cell virus.
|
초록
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◦ 세부과제명: 토종벌 개량벌통 위생사양 표준화 작성
- 토종벌 개량벌통 별 위생 사양 조건 비교시험
- 토종벌통과 개량벌통 별 위생 사양 조건 구축 및 안착화 비교
- 낭충봉아부패병 예비 치료후보물질로 제공한 약제에 대한 야외효능 효과 실험 수행
- 개량벌통으로의 위생 통갈이 기법과 안착화 연구
- 토종벌농가 중 개량벌통 사용 농가 실태 조사
- 국내 토종벌 종보존농가 중심으로 바이러스 진단 및 개량벌통 사용 권장
- 본부 내 개량벌통 통갈이 기법 고착화 실험
◦ 세부과제명: 토종벌 개량
◦ 세부과제명: 토종벌 개량벌통 위생사양 표준화 작성
- 토종벌 개량벌통 별 위생 사양 조건 비교시험
- 토종벌통과 개량벌통 별 위생 사양 조건 구축 및 안착화 비교
- 낭충봉아부패병 예비 치료후보물질로 제공한 약제에 대한 야외효능 효과 실험 수행
- 개량벌통으로의 위생 통갈이 기법과 안착화 연구
- 토종벌농가 중 개량벌통 사용 농가 실태 조사
- 국내 토종벌 종보존농가 중심으로 바이러스 진단 및 개량벌통 사용 권장
- 본부 내 개량벌통 통갈이 기법 고착화 실험
◦ 세부과제명: 토종벌 개량벌통 사양에 따른 꿀벌 질병 방제효과 조사
- 토종벌 사각벌통 꿀벌 질병 인공감염 시험
- 국내 토종벌 사각벌통 사용농가 병성감정실험
- 토종벌 기생파리감염증 발생 확인 및 진단법 개선
- 토종벌 개량벌통 꿀벌 질병 인공감염 시험
- 토종벌 사각벌통 및 개량벌통 꿀벌 질병 인공감염 치료효과 비교 실험.
Abstract
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Title (1st subtitle) : Genetic and phylogenetic analysis of South Korea SBV isolates infected in honey bee(Apis cerana)
Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infect
Title (1st subtitle) : Genetic and phylogenetic analysis of South Korea SBV isolates infected in honey bee(Apis cerana)
Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥ 98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains.
Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities.
This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.
Title (1st subtitle) : Prevalence and distribution of six bee viruses in Korean Apis cerana populations
The prevalence and distribution of six bee viruses was investigated in 527 Apis cerana samples which were collected from five provinces in South Korea. The most prevalent virus, black queen cell virus (BQCV), was present in 75.11% of 446 adult bee samples, followed by sacbrood virus (SBV) in 30.71%. Deformed wing virus (DWV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) were present at lower levels of 8.07%, 1.56%, and 0.44%, respectively. The most prevalent virus in 81 larvae samples was SBV, with an incidence of 60.49%, followed by BQCV in 48.14%, DWV in 6.17%, and KBV in 1.23% of samples. CBPV infection was not detected in larvae samples, and acute bee paralysis virus (ABPV) was not present in both larvae and adult bee. Simultaneous infections with up to four viruses were also identified. Of these, infections with SBV and BQCV were most frequent in 25.61% of samples. The distribution of these viruses varied considerably throughout the geographic regions investigated. The three provinces of Gyeongbuk, Jeonnam, and Chungbuk had the highest frequency of bee viruses.
Title (1st subtitle) : Analysis of the complete genome sequence of two Korean sacbrood viruses in the Honey bee, Apis mellifera
The complete genomic RNAs of two Korean sacbrood virus (SBV) strains, which infect the honey bee, Apis mellifera, were sequenced. The two sequences (AmSBV-Kor19, AmSBV-Kor21) were distinguished by the presence or absence of a PstI restriction site.
These strains comprised of 8784 bp and 8835 bp; contained a single large ORF (179-8707 and 179-8758) encoding 2843 and 2860 amino acids, respectively. Deduced amino acid sequences comparison with some insect viruses showed that regions of helicase, protease and RdRp domains; structural genes were located at the 5' end and non-structural genes at the 3' end. Multiple sequence alignment showed that AmSBV-Kor19 was missing a section between nucleotides 2311 and 2361 (present in SBV-UK and CSBV) but was similar to that of the Korean SBV strain that infects A. cerana (AcSBV-Kor). The differences in the AmSBV-Kor19 strain may be the result of the virus adapting to a different host.
Title (1st subtitle) : Occurrence and Prevalence of Honeybee Disease in Apis mellifera and Apis cerana in Korea
The occurrence and distribution of twelve bee diseases was investigated in Apis mellifera (A. mellifera) and Apis cerana (A. cerana) in Korea: american foulbrood (AFB), european foulbrood (EFB), chalkbrood, stonebrood, Nosema, sacbrood virus (SBV), acute bee paralysis virus (ABPV), deformed wing virus (DWV), black queen cell virus (BQCV), kashmir bee virus (KBV), chronic bee paralysis virus (CBPV) and Varroa destructor (V. destructor). Samples were collected from Korean apiaries located in different regions of the country in Novemver 2010-October 2011. V. destructor was the most prevalent disease in A. mellifera (identified in 75% of samples), followed by Nosema (46%), chalkbrood (39%), stonebrood (37%), BQCV (24%), DWV (22%), SBV (15%), KBV (13%), AFB (12%), and EFB (0.6%). SBV was identified in 64% of A. cerana samples and was especially dominant during the winter, when it was identified in 70% of samples. The results indicate that disease prevalence is associated with both susceptibility and season.
A. cerana was susceptible to SBV during hibernation but resistant to V. destructor, whereas A. mellifera was susceptible to V. destructor during the working period, which was associated with the spread of other viruses. This is the first paper reporting the detection of 12 distinct diseases in A. mellifera and A. cerana honeybees in Korea.
Title (1st subtitle) : Analysis of the complete genome sequence and capsid region of black queen cell viruses from infected honeybees (Apis mellifera) in Korea
Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsidregions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.
Title (1st subtitle) : Phylogenetic analysis of black queen cell virus genotypes in South Korea
The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isol ated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.
Title (1st subtitle) : Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea
Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region.
Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries
Title (1st subtitle) : Molecular characterization and phylogenetic analysis of deformed wing viruses isolated from South Korea
Deformed wing virus (DWV) is one of the most common viral infection in honeybees.
Phylogenetic trees were constructed for 16 partial nucleotide sequences of the structural polyprotein region and the RNA helicase region of South Korean DWVs. The sequences were compared with 10 previously reported DWV sequences from different countries and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus-1 (VDV-1). The phylogeny based on these two regions, the Korean DWV genomes were highly conserved with 95?100% identity, while they also shared 93?97% similarity with genotypes from other countries, although they formed a separate cluster. To investigate this phenomenon in more detail, the complete DWV genome sequences of Korea-1 and Korea-2 were determined and aligned with six previously reported complete DWV genome sequences from different countries, as well as KGV and VDV-1, and a phylogenetic tree was constructed. The two Korean DWVs shared 96.4% similarity.
Interestingly, the Korea-2 genome was more similar to the USA (96.5%) genome than the Korea-1. The Korean genotypes highly conserved with USA (96%) but low similarity with the United Kingdom3 (UK3) genome (89%). The end of the 5′ untranslated region (UTR), the start of the open reading frame (ORF) region, and the 3′ UTR were variable and contained several substitutions/transitions. This phenomenon may be explained by intramolecular recombination between the Korean and other DWV genotypes.
목차 Contents
- 총괄요약표 ... 1
- 연구과제 최종결과보고서 ... 3
- Ⅰ. 연구배경 및 목표 ... 3
- Ⅱ. 연구방법 및 수행전략 ... 5
- Ⅲ. 연구결과 ... 6
- Ⅳ. 연구결과요약 ... 24
- Ⅴ. 고 찰 ... 25
- Ⅵ. 연구결과 활용실적 및 계획 ... 25
- Ⅶ. 연구사업 추진상 문제점 및 건의사항 ... 27
- Ⅷ. 주요연구과제 변경사항 ... 28
- IX. 참고자료 ... 28
- Ⅹ. 영문초록 ... 28
- 끝페이지 ... 34
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