Pathogenesis of influenza A(H7N9) virus in aged non-human primates

The avian influenza A(H7N9) virus has caused high mortality in humans, especially in the elderly; however, little is known about the mechanistic basis for this. In this study, we employed non-human primates to evaluate the effect of aging on the pathogenicity of A(H7N9) virus. We observed that A(H7N9) virus infection of aged animals (defined as 20–26 years) caused more severe symptoms than infection of young animals (defined as 2 3 years). In aged animals, lung inflammation was weak and virus infection was sustained. Although cytokine and chemokine expression in the lungs of most aged animals was lower than that in the lungs of young animals, one aged animal showed dysregulated proinflammatory cytokine and chemokine production, resulting in it being euthanized. These results suggest that attenuated or dysregulated immune responses in aged animals are responsible for the severe symptoms observed among elderly patients infected with A(H7N9) virus. Total RNA was extracted from lung samples and blood by using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. RNA was labeledwith Cy3 dye with the QuickAmplabelingkit(Agilent Technologies), and hybridized to the Rhesus Macaque Gene Expression Microarray (Agilent Microarray Design Identification Number 026806; Agilent Technologies), as previously described [22]. Individual microarrays were performed for each lung sample that was collected from naïve and infected animals. Statistical analysis was performed using the LIMMA package. The log2of the intensity of each probe was background corrected and normalized between arrays (using the quantile method). Duplicate probes were averaged.

甲型H7N9禽流感病毒（Avian Influenza A(H7N9) Virus）可引发人类高病死率，老年群体尤甚，但目前学界对其致病的分子机制知之甚少。本研究采用非人灵长类动物（Non-human Primates），探究衰老对甲型H7N9禽流感病毒致病性的影响。研究观察到，老年动物（定义为20~26岁）感染该病毒后，症状较年轻动物（定义为2~3岁）更为严重。老年动物体内肺部炎症反应偏弱，但病毒感染持续存在。尽管多数老年动物肺部的细胞因子与趋化因子表达水平低于年轻动物，但有1只老年动物出现促炎细胞因子与趋化因子产生失调，最终予以安乐死。上述结果表明，老年动物体内免疫应答的减弱或失调，是感染甲型H7N9禽流感病毒的老年患者出现重症症状的原因。本研究参照制造商说明书，采用RNeasy Mini Kit（Qiagen）从肺部样本与血液中提取总RNA。采用QuickAmplabelingkit（Agilent Technologies）以Cy3染料对RNA进行标记，并参照既往研究[22]的方法，将标记后的RNA与食蟹猴基因表达微阵列（Rhesus Macaque Gene Expression Microarray，安捷伦微阵列设计识别编号：026806；安捷伦科技）进行杂交。针对来自未感染与感染动物的每一份肺部样本，均独立开展微阵列实验。统计学分析采用LIMMA软件包（LIMMA Package）完成。对每个探针的信号强度值进行log₂转换后，完成背景校正，并采用分位数法实现各微阵列间的归一化处理。对重复探针的信号值取平均值。