Development of molecular methods for epidemiology of insect viruses

昆虫病毒流行病学分子方法的发展

• 批准号: 07556136
• 负责人: KOBAYASHI Masahiko
• 金额: $ 3.84万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556136/
• 关键词: insect viruses; molecular epidemiology; Bombyx mori; PCR; NPV; CPV; DNV; retrotransposon; 養蚕; レトロポゾン

We developed a diagnosis system for virus diseases in silkworms and wild insects based on comparative studies on DNA sequences in the virus and host insects. A forecast method of virus disease expansion in insect populations was established by using molecular epidemiological techniques.1.The polyhedrin genes and DNA helicase genes were cloned and sequences from Samia cynthia ricini nuclear polyhedrosis virus (SrNPV) and Antheraea yamamainuclear polyhedrosis virus (AyNPV).2.An immunological sensor was developed using an monoclonal antibody against Bombyx moricytoplasmic polyhedrosis virus (BmCPV) and a quartz oscilator to detect BmCPV rapidly and easily.3.Quantitative changes of two genomic DNAs in the Bombyx moridensonuclear virus type II (DNV2) were investigated in the midgut cells infected with DNV2, using the competitive PCR method.4.We developed a very simple method to detect all of known viruses in Bombyx morisimultaneously in one reaction. The condition of PCR and primer designs were optimized.5.We set a model rearing shelf containing healthy silkworm larvae and a few nuclear polyhedrosis ones. Expansion of the virus was monitored in the larvae, feces, diet, etc., on the shelf, using the PCR diagnosis system.6.Random amplified polymorphic DNAs (RAPDs) were found to be closely linked to the nonsusceptibility gene to DNV2 (nsd-2). A linkage map was constructed based on recombination values among the RAPDs and nsd-2.

在病毒和寄主昆虫DNA序列比较研究的基础上，建立了家蚕和野生昆虫病毒病诊断系统。本研究利用分子流行病学技术，建立了一种预测病毒病在昆虫群体中传播的方法：1.克隆了蓖麻蚕核型多角体病毒（Samia cynthia ricini nuclear polyhedrosis virus，SrNPV）和天蚕核型多角体病毒（Antheraea yamainuclear polyhedrosis virus，AyNPV）的多角体蛋白基因和DNA解旋酶基因，并进行了序列测定; 2.采用竞争性PCR方法，研究了家蚕中核病毒2型（DNV 2）感染中肠细胞后两种基因组DNA的定量变化。优化了PCR反应条件和引物设计。5.建立了含有健康蚕和少量核型多角体蚕的家蚕模型育架。在幼虫、粪便、饲料等中监测病毒的扩增，6.随机扩增多态性DNA（RAPDs）与DNV 2不敏感基因（nsd-2）紧密连锁。基于RAPD和nsd-2之间的重组值构建了连锁图。

项目成果

Abe H,Shimada T et al.: "Nucleotide sequence of the random amplified polymorphic DNA (RAPD) on the W chromosome of the silkworm,Bombyx mori (Lepidoptera : Bombycidae)." Applied Entomology and Zoology. 31. 633-637 (1996)

Abe H、Shimada T 等人：“家蚕 Bombyx mori（鳞翅目：Bombycidae）W 染色体上随机扩增多态性 DNA (RAPD) 的核苷酸序列。”

安部 広明・嶋田 透・小林正 彦: "カイコ濃核病ウイルスI型感受性遺伝子に連関するランダム増幅多型DNA(RAPD)" 日本蚕糸学雑誌. 64,3. 262-264 (1995)

Hiroaki Abe、Toru Shimada 和 Masahiko Kobayashi：“与蚕高核病毒 I 型易感基因相关的随机扩增多态性 DNA (RAPD)”日本血清学杂志 64,3 (1995)。

K. Hashido, et al.: "Favorite site for recombination of a defective non LTR retrotransposon, BMC1." J. Cell. Biochem.(in press). (1996)

K. Hashido 等人：“有缺陷的非 LTR 反转录转座子 BMC1 最喜欢的重组位点。”

阿部広明 その他: "カイコの系統保存へのDNA診断法の応用.-濃核病ウイルス1型感受性系統の保存-." 日本蚕糸学雑誌. 65巻(印刷中). (1996)

Hiroaki Abe 其他：“DNA 诊断方法在蚕品种保存中的应用。- 高核疾病病毒 1 型易感菌株的保存”，《日本血清学杂志》第 65 卷（出版中）。

S. Hoshizaki and T. Shimada: "PCR-based detection of Wolbachia, cytoplasmic incompatibility microorganisms, infected in natural papulations of Laodelphax striatellus (Homoptera: Delphacidae)" Insect Molecular Biology. vol.4.237-243 (1995)

S. Hoshizaki 和 T. Shimada：“基于 PCR 的沃尔巴克氏体检测，这是一种细胞质不相容的微生物，感染灰飞虱天然幼虫（同翅目：飞虱科）”昆虫分子生物学。