人B组腺病毒纤毛蛋白与DSG2受体亲和力的差异及其对病毒致病力的影响研究

Human adenovirus type 3 (HAdV-B3) of B species is the most common type, however, Human adenovirus type 7 (HAdV-B7) and 14 (HAdV-B14), 55 (HAdV-B55) have more virulent, and tend to cause severe pneumonia even death among the adenoviruses causing acute respiratory infection. Recently it has been proved that all the four types detect Desmoglein 2 (DSG2) as receptor, not CAR or CD46 detected by other human adenovirus types. The amino acid similarities between fibres of HAdV-B7 and B14, B55 are up to 92%, but only 57-58% between HAdV-B7 and B3. We also found that HAdV-B7 and B14, B55 have higher infectivities to cells than HAdV-B3. Then there are two quenstions: Are the strong affinities between HAdV-B7, B14, B55 fibres with DSG2 asociated with severe infection? What are the critical amino acids of fibres of HAdV-B7, B14, B55 binding with DSG2? We have developed recombinant HAdV-B3, B7, B55 expressing fluorescent protein and the novel animal model infected with HAdV-B3. In this study, we compare the affinities of HAdV-B7, B14, B55 and B3 fibres for DSG2 with recombinant fibres and chimeric adenoviruses replaced with different fibres by affinity detection and efficiency analysis of infection. The critical epitopes of fibres binding DSG2 would be predicted by bioinformatics methods and be identified with mutational fibre proteins and adenoviruses with mutational fibre. Finally, the effection and mechanism of different adenovirus fibres on adenovirus pathogenicity will be studied using the cell model of tight junction and the Chinese tree shrew model . This research will not only contribute to a better understanding of the pathogenic mechanism of adenovirus, but also provide a theoretical basis on early warning of high pathogenic adenovirus, and the development of the novel adenovirus vectors, adenovirus vaccines or medicines.

在致急性呼吸道感染的腺病毒中，人B组3型最常见，而7、55、14型常引起重症感染。不同于其它型别腺病毒识别CAR或CD46等受体，这几种腺病毒均以纤毛蛋白结合受体桥粒芯蛋白2（DSG2）。我们发现人7、14、55型腺病毒纤毛蛋白高度同源，而与3型纤毛蛋白同源性较低；人7、55、14型腺病毒的感染能力较3型更强。人7、55、14型腺病毒的高致病力是否和纤毛蛋白有关？影响纤毛蛋白与DSG2结合的关键性氨基酸？我们已经制备了重组人3、7、55型腺病毒，本课题将表达野生型、突变型纤毛蛋白，构建纤毛蛋白置换及突变的的嵌合腺病毒等，比较人3、7、55型腺病毒纤毛蛋白与DSG2亲和力，鉴定与DSG2结合的关键性氨基酸，并利用紧密连接细胞模型和我们建立的树鼩感染模型，探讨纤毛蛋白对腺病毒致病和重症化的作用与机制，为高致病腺病毒预警、开发新型腺病毒载体、疫苗和药物提供理论基础。

本面上项目探讨人B组腺病毒纤毛蛋白差异与致病力强弱的相关性，按期按计划基本完成了任务：.纤毛蛋白表达。成功重组表达纯化得到10种型别腺病毒纤毛蛋白头knob，维持了自然的三聚体结构。.纤毛蛋白嵌合型腺病毒制备。成功制备基于Ad3的纤毛蛋白嵌合型腺病毒rAd3E-Fk7、rAd3E-Fk55和rAd3E-Fk5；及基于Ad5的rA5F3、rA5F7、rA5F55。rAd3E-Fk5可以感染金仓鼠细胞，纤毛蛋白是腺病毒毒力差异的重要因素。.纤毛蛋白表位。重组knob免疫诱导了高效价中和抗体，B组不同型别间存在广泛的交叉中和作用；制备了识别knob的广谱中和单抗，knob的F-G环上251KE252可能是与Ad7、Ad55、Ad11、Ad14p1受体结合的关键性氨基酸。Ad3 knob可以抑制rA5F3感染，但不能抑制rA5F7、rA5F55感染；而Ad55 knob可以抑制rA5F3、rA5F7、rA5F55感染，表明Ad3和Ad7、Ad55 knob的受体结合差异，可能与致病力有关。.纤毛蛋白与腺病毒致病。亲和力检测表明重组knob与hDSG2的亲和力Ad55>Ad7>Ad3，Ad5 knob与hDSG2没有结合，对细胞的感染力rA5F55>rA5F7>rA5F3，对树鼩的致病力Ad55>Ad3，对A549细胞噬斑大小Ad55>Ad7>Ad3，初步探讨了fiber与腺病毒致病力间的相关性。建立了极化培养上皮细胞模型，用Ad3、Ad55进行了感染实验，初步结果显示Ad55有更高的感染力，对紧密连接开放的影响暂未观察到差异。建立了B组人腺病毒感染树鼩模型，Ad55在树鼩体内有显著增殖并致典型肺炎。.腺病毒毒株分离及载体构建。分离鉴定多株人腺病毒临床分离株，完成全基因组测序和进化分析。发现Ad55基因组稳定，Ad4可以分为两个亚型4a和4p，4a为近几十年的主要流行株。建立了一种快速构建腺病毒载体的新方法，制备了Ad55-EGFP、Ad35-EGFP、Ad11-EGFP、Ad7-EGFP、Ad4-DsRed等多种型别腺病毒复制型载体，及一种新型示踪重组腺病毒，为腺病毒致病机制研究、药物筛选奠定基础。.本课题已共发表SCI论文12篇、中文期刊论文6篇；申请专利9项；培养研究生7人。经费使用规范，圆满完成了立项任务和目标。