肠道病毒是引起手足口病的主要病原体，其中柯萨奇病毒 A16（CVA16）是近年导致大规模流行的主要毒株之一。CVA16 具有严格的宿主特异性，人类是目前已知的唯一自然宿主，这对研究其感染机制及疫苗开发造成了极大限制。已有研究表明，人类清道夫受体 B 类成员 2（hSCARB2）是 CVA16 主要功能受体，在病毒入侵宿主细胞的早期阶段发挥关键作用。因此，研究人员尝试开发携带hSCARB2 基因的小鼠模型，以克服 CVA16 的宿主限制性并模拟人类感染的病理过程，用于疫苗及抗病毒药物的评价。然而，现有小鼠模型在 CNS 感染的表型特征及病理机制方面仍存在不足，尚需进一步优化。

为了弥补目前小鼠模型存在的不足，本研究利用 CRISPR/Cas9 技术构建 hSCARB2  KI 小鼠，并系统性评估其对 CVA16 的易感性。实验结果表明，该模型在 10、21 及 30 日龄均可被 CVA16 感染，并表现出典型的 HFMD 病理特征。尤其在 10 日龄小鼠中，CVA16 感染可引起严重的 CNS 损伤，导致运动功能障碍、共济失调、下肢瘫痪甚至死亡。此外，病毒载量检测显示，感染小鼠的脑组织中 CVA16 复制水平显著升高，提示该模型在 CNS 感染研究中的潜在应用价值。进一步分析发现，CVA16 感染导致 KI 小鼠大脑和脊髓中出现广泛的神经元坏死及炎症细胞浸润，显示出较强的神经毒性且感染小鼠脑组织中的 IL-6、IL-12 及 TNF-α 水平显著升高。同时，免疫荧光分析进一步揭示，感染小鼠的脑组织中特别是大脑皮层的 GFAP 和 IBA1 的表达显著增强，表明星形胶质细胞和小胶质细胞均被过度激活，并主要集中于大脑皮层，且其分布模式与病毒载量呈高度一致性。这些结果表明，CVA16 可能通过诱导神经胶质细胞的过度激活，引发 CNS 内的局部炎症反应，从而加剧神经元损伤，导致严重的神经功能障碍，最终引发共济失调、肢体瘫痪，甚至死亡。

综上所述，本研究成功构建了 hSCARB2 KI 小鼠，并证实该模型可有效模拟 CVA16 诱导的 CNS 感染及病理损伤，为研究 CVA16 的致病机制提供了理想的动物模型，同时也为未来的疫苗和抗病毒药物研发提供了重要的实验基础。

Enteroviruses are the primary causative agents of hand, foot, and mouth disease (HFMD), among which coxsackievirus A16 (CVA16) has emerged as one of the predominant strains responsible for large-scale outbreaks in recent years. CVA16 exhibits strict host specificity, with humans being the only known natural host, which poses significant challenges for studying its infection mechanisms and developing effective vaccines. Previous studies have identified human scavenger receptor class B member 2 (hSCARB2) as the major functional receptor for CVA16, playing a critical role during the early stages of viral entry into host cells. To overcome the host restriction of CVA16 and to simulate the pathological process of human infection, researchers have attempted to develop transgenic mouse models expressing the hSCARB2 gene for use in vaccine and antiviral drug evaluation. However, current mouse models still exhibit limitations in replicating the phenotypic features and pathological mechanisms of central nervous system (CNS) infection, indicating a need for further optimization.

To address this gap, this study used CRISPR/Cas9 technology to generate hSCARB2 knock-in (KI) mice and systematically evaluated their susceptibility to CVA16. The results showed that the model could be infected with CVA16 at 10, 21, and 30 days of age, exhibiting typical pathological features of HFMD. Notably, 10-day-old mice developed severe CNS damage following infection, resulting in motor dysfunction, ataxia, hindlimb paralysis, and even death. Furthermore, viral load analysis revealed significantly elevated levels of CVA16 replication in the brain tissues of infected mice, suggesting the model’s potential value in CNS infection studies.

Further analysis indicated that CVA16 infection induced widespread neuronal necrosis and inflammatory cell infiltration in the brains and spinal cords of KI mice, demonstrating strong neurotoxicity. Infected brain tissues also showed significantly increased levels of IL-6, IL-12, and TNF-α. Immunofluorescence analysis revealed that expression of GFAP and IBA1 was markedly increased in the cerebral cortex, indicating excessive activation of astrocytes and microglia. These glial cells were predominantly distributed in the cortex, and their localization highly correlated with viral load. These findings suggest that CVA16 may exacerbate neuronal damage and lead to severe neurological dysfunction by triggering local inflammatory responses through excessive activation of glial cells, ultimately resulting in ataxia, limb paralysis, and death.

In conclusion, this study successfully generated hSCARB2 KI mice and confirmed that the model effectively simulates CVA16-induced CNS infection and pathological damage. This provides an ideal animal model for studying the pathogenic mechanisms of CVA16 and lays a critical experimental foundation for the future development of vaccines and antiviral therapies.