L1-52/55K蛋白定点变异与3型、7型腺病毒感染毒力相关性和机制研究

Acute respiratory disease infection outbreaks caused by human adenovirus types 3 and 7 (HAdV-3 and -7) were reported in China and other countries recently. Infants are more susceptible and often progressing to severe infections, even death, which is associated with HAdV-7. The mechanism and influencing factors of different infectious virulence between the two type viruses is still unclear. Our preliminary surveys on adenovirus molecular epidemiology and comparative genomics of HAdV-3 and -7 demonstrated that a significant variation within the region between amino acids 19 and 22 of L1-52/55K protein that closely related with infectious virulence was found. Therefore, based on systemic study of adenoviruses, we will first targeted editing the region between amino acids 19 and 22 of L1-52/55K protein to construct the mutant strains of HAdV-3 and -7 by the use of CRISPR/Cas9. Second, comparison and analysis of the dynamics of viral replication and virulence of clinical and mutant strains of HAdV-3 and -7 by the use of cellular model. Finally, by use of immunosuppressed mouse infection model for clinical and mutant strains of HAdV-3 and -7, the clinical symptoms and pneumonia histopathology, along with inflammatory corpuscle, cytokines and other immunological parameters will be statistically analyzed. Experiments mentioned above will validate the prevailing hypothesis that “targeted variation of L1-52/55K protein influence the infectious virulence of HAdV-3 and -7”. This research will further demonstrate the immune mechanism over the severe adenovirus infection of immunocompromised population and provide a new theoretical basis and a solid research foundation for protection and therapy of human adenovirus infection diseases.

近年来3、7型腺病毒引起的急性呼吸道感染在我国及世界上频频暴发，7型腺病毒更易引起婴幼儿重症感染，甚至死亡，而有关3、7型腺病毒感染毒力差异的影响因素和机制尚不清楚。本课题组在前期腺病毒分子流行病学和比较基因组学研究发现，3、7型腺病毒在与感染毒力密切相关的L1-52/55K蛋白19~22位点变异显著。因此，在腺病毒系统研究基础上，本课题①利用CRISPR/Cas9构建L1-52/55K蛋白定点变异的3、7型腺病毒突变株；②利用细胞模型分析3、7型临床分离株与突变株的病毒复制动力学和毒力变化；③通过小鼠模型感染3、7型临床分离株与突变株中引起小鼠体征、肺组织病理学，炎症细胞和细胞因子的表达水平等指标的变化与差异，验证“L1-52/55K蛋白定点变异影响3、7型腺病毒感染毒力变化”的假说，进一步揭示免疫力低下人群重症腺病毒感染与免疫机制，为腺病毒疾病的预防和治疗提供新的理论依据和研究基础。

在前期腺病毒分子流行病学和比较基因组学研究发现，3、7型腺病毒在与感染毒力密切相关的L1-52/55K蛋白19~22位点变异显著。为了探讨L1-52/55K蛋白定点变异是否影响3、7型腺病毒感染毒力变化，我们通过构建L1-52/55K蛋白定点变异的3、7型腺病毒突变株，利用细胞模型分析3、7型临床分离株与突变株的病毒复制动力学和毒力变化发现L1-52/55K蛋白19~22位点氨基酸的插入或缺失与3型、7型腺病毒感染毒力变化不大，其基因突变有可能是病毒进化的偶然事件，与病毒毒力变化相关性尚无定论。.另一方面，腺病毒感染机体免疫反应显示重症腺病毒感染者与降钙素原（PCT）和C-反应蛋白（CRP）升高密切相关，而7型腺病毒感染者降钙素原（PCT）升高水平显著高于3型腺病毒感染者，而且7型腺病毒感染者外周血白细胞（WBC）数量显著低于3型腺病毒感染者，具有统计学差异，这为进一步研究3型、7型腺病毒感染导致的表型差异提供重要线索，降钙素原（PCT）在7型腺病毒感染过程中扮演的重要角色有待深入探讨确定。