血凝素RBD位点变异导致H5N1禽流感病毒毒力减弱的机制研究

It is on the top list of the research into the molecular determinants and the mechanism of the high pathogenicity of H5N1 avian influenza virus to control and prevent the virus epidemic. Previous studies by our team showed that, H5N1 avian influenza virus attenuated in virulence in mice after in vivo (mice) or in vitro (MDKC cells) passages, and by reverse genetic method,it showed that the E190G, K193E or G225E determined the attenuation, which was not associated with virus proliferation. On the other hand, the 3D structure model indicated that the three mutations led to local structure change significantly in RBD, as might caused the change in receptor binding. Recently, it was shown that there was a correlation between the receptor binding specificity of the virus and the virus tropism, cytokines induction by the virus. Therefore, in this project, we intends to further determine the Impact of above RBD variable sites on the receptor binding activity, on the virus dissemination in mice, on the pathological changes in mice and on the cytokines induction caused by the virus infection. Thus, we can clear the virulence attenuation mechanism caused by the three mutations and confirm the correlation between the virus receptor binding specificity and its pathogenicity, and it will expand the understanding of the molecular determinants and the mechanism of the virus high pathogenicity.

H5N1禽流感病毒高致病性的分子基础及致病机理是对该病毒防治研究中的重大问题。本课题组既往研究发现，H5N1禽流感经小鼠体内或MDCK体外传代后，对小鼠致病力显著降低，反向遗传学证实，RBD的E190G、K193E和G225E三个变异位点都与病毒毒力减弱有关，而且，病毒毒力减弱与病毒增殖能力无关。3D结构模型显示，这三个变异位点导致病毒RBD局部结构发生明显变化，可能引起病毒受体结合活性改变。最近研究发现，H5N1禽流感病毒受体结合特性影响病毒在体内播散和诱导细胞因子表达的能力。所以，本项目拟进一步开展上述RBD变异位点对受体结合活性、病毒在小鼠体内播散能力、引起组织病理变化及诱导细胞因子表达的影响，从而明确这三个变异位点导致病毒毒力减弱的机制，证实H5N1病毒受体结合特性与致病性的相关，有利于拓展对H5N1病毒高致病性的分子基础及其致病机理的认识。

本研究通过环境和宿主因素诱导A/Vietnam/1194/2004（H5N1）禽流感病毒产生突变，经蚀斑筛选纯化获得K193E、G225E和E190G三个HA的RBD突变。体外低温适应型RBD突变K193E对H5N1流感病毒受体结合影响较小，G225E突变能够显著增强病毒对α2,3型唾液酸受体的亲和力，E190G突变削弱病毒与α2,3型受体亲和力的同时略微增强病毒对α2,6型受体的亲和力。但这些突变并不能使病毒受体亲嗜性由α2,3型转变为α2,6型，故无法使H5N1病毒获得人间传播能力。体外低温适应型RBD突变K193E/G225E联合突变显著增强病毒在不同温度下（33°C、37°C和39°C）的体外复制能力。鼠肺适应型RBD突变E190G减弱病毒体外复制能力，且这种减弱不随温度变化而改变。这些结果表明，对流感病毒体外复制有重要影响的片段不仅仅局限于已经报道的PB2、PB1和NS1基因，RBD基因同样对流感病毒体外复制效率有显著影响。利用BALB/c小鼠模型检测突变病毒MLD50、体重变化和肺中病毒载量，结果证明，与野生型重组病毒rVN1194相比，RBD突变K193E/G225E和E190G显著减弱VN1194病毒致病性。.综上所述，K193E、G225E和E190G三个RBD突变对于H5N1流感病毒的影响和作用表明这三个突变无法使H5N1病毒突破宿主物种屏障。K193E/G225E双突变对病毒的体外增殖有显著增强作用。K193E/G225E和E190G突变能够显著减弱病毒在小鼠体内的致病性。本研究同时证明重组突变病毒rVN-K193E/G225E具有体外复制能力强、在小鼠体内毒力较弱的特点。此特点对于活病毒减毒疫苗的研究与开发具有重要意义，疫苗工业化生产中通过接种细胞增殖减毒活病毒疫苗，利用该突变有望打破减毒病毒体外增殖效率较低的瓶颈，提高减毒疫苗产量，本研究结论可为流感减毒疫苗设计提供理论支持。