家蚕BmNPV沿气管体内传播的分子机制

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a specific pathogen to infect silkworm and usually gives rise to heavy losses to silk industry. This virus per os infects the epithelial cells of larval midgut of silkworm and finally causes systemic infection in vivo. We discovered that in the infected silkworm the BmNPVs binds with the larval tracheae and through which the viruses spread to various tissues or organs and subsequently causes systemic infection and finally makes the larvae died. This phenomenon lets us think two scientific questions, one is “How the BmNPVs bind with the larval tracheae ?”, and another is “How are the viruses pushed and move forward along with tracheae in vivo in silkworm ? ”. As the trachea mainly comprises of chitin and proteins, we are firstly planning to analyze (1) the possible binding between the virus and tracheae chitin. The viral envelope proteins containing chitin binding domain (ChtBD) encoded by the virus will be firstly predicted using bioinformations as target proteins for binding tracheae. The functions of these ChtBD domain will be identified by gene knock-out techniques through recombinant baculovirus construction and bioassay, and the structural basis responsible for the interaction between the ChtBD and chitin will be analyzed; (2) the possible binding between the virus and tracheae proteins. By using the yeast hybridization and Co-IP techniques we try to analyze the binding between viral proteins and tracheae proteins. Through the above two aspects, the binding mechanism between the BmNPV and tracheae should be demonstrated. Furthermore, recombinant virus with fluorescence tag will be constructed and used as tool to trace the virus movement in vivo, and confirm whether the viruses are pushed to reach the target tissues or organs by host actin like intracellular. The results from this study will answer the above two questions and make clear the molecular mechanism of BmNPV transmission in vivo.

家蚕核型多角体病毒是专一性感染家蚕的烈性病毒，该病毒通过经口感染家蚕中肠上皮细胞，然后引发全身感染。我们发现，该病毒在家蚕体内主要沿气管传播至家蚕的各个组织，最终实现病毒的全身性感染。我们提出两个科学问题，即“病毒是如何与气管结合的？”和“病毒是如何沿着气管扩散传播的？”。由于气管的主要成分是几丁质和少量蛋白质，所以，我们首先分析（1）病毒与气管几丁质的结合。分析含有几丁质结合域的病毒囊膜蛋白，利用基因敲除技术分析缺少这些功能域后病毒与气管结合的结果，找出与几丁质结合的关键病毒囊膜蛋白；（2）病毒与气管表面蛋白的结合。利用酵母双杂交等技术分析病毒囊膜蛋白与气管表面蛋白的结合。通过上述两个方面揭示病毒与家蚕气管结合的分子机制。进一步构建带有荧光标记的重组病毒，证实在家蚕体内病毒是否也借助于肌动蛋白的推动到达靶标组织。本研究的结果将基本阐明上述两个科学问题，揭示BmNPV体内传播的分子机理。

家蚕核型多角体病毒是专一性感染家蚕的烈性病毒，是造成蚕丝产业重大损失的主要病原。为了阐明该病毒是如何沿着气管扩散传播的分子机制，本研究利用构建带有荧光标记的重组病毒、蛋白质组学、酵母双杂交等技术、免疫荧光与激光共聚焦显微镜等技术等，分析了该病毒表面发挥作用的关键囊膜蛋白，分析了气管在病毒体内传播中的具体作用和分子机制。结果表明：（1）解析了家蚕气管的蛋白质组学，质谱鉴定出50种气管蛋白质，其中表皮蛋白质22种；（2）病理学和病毒基因表达分析等证明气管是病毒的易感组织。通过实时荧光定量PCR检测病毒部分特征基因的表达情况，发现病毒在气管组织内具有较高的复制水平，在气管内产生大量的用于水平传播的出芽型病毒粒子BV，有助于迅速传播，建立有效的系统性感染；同时研究还发现在气管内病毒产生较少的多角体，而在感染极晚期气管表面富集了大量的多角体。由此说明，家蚕气管在BmNPV感染过程中发挥了重要作用。进一步证明了气管是病毒的易感组织，也有力说明了BmNPV感染家蚕过程中的重要传播通道。（3）阐明了BmNPV在气管上传播的特征以及传播机制。利用免疫组化对BmNPV在气管上的感染进行了追踪，BmNPV感染在气管上呈现线性传播规律，即感染由成气管细胞逐渐向近气门端气管上皮细胞传播；（4）BmNPV感染后气管转录组学结果表明，感染后家蚕宿主能量代谢相关（糖代谢、脂代谢）、免疫应答激活基因存在大规模的下调，而基因的复制、蛋白质合成机器以及糖蛋白的生成显著上调。.本研究结果揭示了该病毒感染气管的特征，以及病毒利用气管进行体内传播的分子机制，证明了家蚕气管系统在杆状病毒的体内传播过程中发挥的重要作用，BmNPV沿气管、支气管以及微气管迅速传播至家蚕脂肪体、丝腺等各个组织，最终实现病毒的全身性感染，气管是病毒传播和扩散的重要“运输通道”。