鱼类淋巴囊肿病毒易感淋巴细胞的鉴定及其受体介导病毒入侵途径研究

In previous researches which is supported by the National Natural Science Foundation of China under the grant No. 31072232, we have identified two cellular receptor proteins with molecular weight of 27.8kDa (27.8R) and 37.6kDa (37.6R) involved in lymphcystis disease virus (LCDV) infection from flounder (Paralichthys olivaceus) gill (FG) cells and analyzed their gene sequences, found a viral attachment protein with a molecular weight of 32kDa (LCDV-VAP32) encoded by the ORF038 gene of LCDV-C, and proved that the interaction between LCDV-VAP32 and the 27.8R can mediate LCDV infection of FG cells, moreover, we developed monoclonal (polyclonal) antibodies against the 27.8R, 37.6R and LCDV-VAP32 protein respectively, and found that LCDV can infect peripheral blood leukocyte which is preliminarily identified as the lymphocytes, but it is unknown in terms of the lymphocyte subpopulation. Based on these results, this project is planning to identify the subtype of the LCDV-infected lymphocytes using the developed anti-flounder IgM, anti-IgT, anti-IgD monoclonal antibodies which specifically recognize the B lymphocytes, and anti-CD3, anti-CD4 and anti-CD8 antibodies which specifically recognize the T cells and Th1/Th2 subpopulations, localize the 27.8R/37.6R on the lymphocytes to clarify whether 27.8R/37.6R is expressed on B or T cells, and then analysis the dynamics of LCDV multiplication in susceptible lymphocytes; After LCDV infection and inactivated LCDV immunization of healthy flounder, the levels of IgM and IgT in serum and skin/gut/gill mucus and cytokines in serum, the variations of surface membrane immunoglobulin (SmIg+) B cells and CD 3+/CD4+/CD8+ T cells, as well as the dynamic expression of the main response-related genes in lymphocytes, will be analyzed. Following the studies about the interaction of LCDV attachment proteins and the cellular receptors on susceptible lymphocytes, as well as their binding sites and functional domains, the main internalization mechanism of LCDV will be explored by biochemical assays with inhibitors of clathrin-dependent endocytosis, caveola-mediated endocytosis or macropinocytosis, and the function of 27.8R/37.6R in mediating LCDV entry via endocytosis pathway will be investigated to better understand the molecular mechanism of receptor-mediated LCDV entry into lymphocytes. The predicted results of this project will benefit the exploring of molecular mechanism of LCDV entry, as well development of vaccine and novel drugs for LCDV prevention in fish.

课题组前一个基金项目（31072232）在牙鲆鳃细胞上鉴定出淋巴囊肿病毒(LCDV)的两个细胞受体（27.8R/37.6R），获得受体基因序列，找到一个与27.8R受体互作的病毒黏附蛋白，初步确定LCDV能够感染外周血淋巴细胞。本项目拟在此基础上，鉴定LCDV易感淋巴细胞亚群；在易感淋巴细胞上定位27.8R/37.6R受体，测定LCDV增殖动态；研究LCDV感染后不同淋巴细胞亚群数量变化动态，测定血清/黏液抗体和细胞因子水平及主要免疫基因应答差异，探明LCDV感染后不同淋巴细胞数量和免疫功能变化；研究LCDV黏附蛋白与易感淋巴细胞表面受体的相互作用及关键结合位点和结构域，解析27.8R/37.6R受体介导LCDV进入淋巴细胞的内吞途径，探索受体介导LCDV感染淋巴细胞的分子机制。为阐明LCDV侵染细胞分子机制及研发LCDV疫苗和新型治疗药物提供资料。

鱼类淋巴囊肿病是淋巴囊肿病毒(LCDV)引起的一种慢性病毒病，世界范围内广泛流行，损失巨大。但目前对LCDV侵染宿主细胞分子机制知之甚少。本基金项目（31872599）对前期发现的牙鲆LCDV的27.8kDa受体蛋白（27.8R）进行了鉴定，确定其由牙鲆阴离子通道蛋白2（VDAC2）和活化的蛋白激酶C受体1（RACK1）组成，且VDAC2和RACK1是介导LCDV入侵细胞的功能受体。研究了LCDV在VDAC2和RACK1受体蛋白介导下的入胞途径，发现LCDV入胞依赖胆固醇、发动蛋白和微管，采用窖蛋白/脂筏内吞途径入胞，不依赖网格蛋白和巨胞饮作用。鉴定了LCDV易感淋巴细胞及其LCDV受体分布，发现LCDV感染后血细胞内LCDV拷贝数和受体表达量随时间上升，而白细胞呈LCDV和27.8R阳性；鉴定出IgM+ B细胞为LCDV易感细胞，27.8R参与LCDV对易感淋巴细胞的侵染过程。研究了LCDV在易感淋巴细胞中的增殖动态，发现LCDV体内和体外感染后白细胞内病毒拷贝数呈先上升后下降趋势，在透射电镜下观察到处于不同发育阶段的病毒颗粒。分析了体内感染后外周血、脾脏、肾脏中LCDV+白细胞和LCDV+ B淋巴细胞数量变化，发现各细胞均呈先上升后下降趋势，变化规律与白细胞中病毒拷贝数基本一致。研究了LCDV感染/疫苗免疫牙鲆后CD4-1+ T、CD4-2+ T、CD8+ T、IgM+ B淋巴细胞数量以及血清总抗体、特异性抗体水平变化，发现感染组与免疫组具有显著应答差异，LCDV感染对T/B细胞产生损伤，并降低了B细胞的免疫功能；检测了15种免疫相关基因的表达变化，发现各基因均出现不同程度的上调表达，感染组基因表达较免疫组更为剧烈。鉴定了LCDV 32kDa黏附蛋白与VDAC2/RACK1受体蛋白互作结构域，确定32kDa黏附蛋白13-122aa与受体RACK1的135-220aa及VACD2的172-238aa之间、黏附蛋白240-291aa与受体RACK1135-220aa之间存在互作。转录组分析了牙鲆淋巴囊肿形成的机制，揭示了与囊肿形成相关的通路和差异表达基因，为阐明牙鲆淋巴囊肿形成的机制提供了新突破。研究结果不仅有助于对LCDV侵染细胞分子机制的阐明，拓展对虹彩病毒致病机制的认识，还有助于找到LCDV疫苗和新型治疗药物设计的潜在靶点，具有重要的科学意义。