目的：上呼吸道病毒感染是嗅觉障碍最常见的原因之一，一般认为其由病毒损伤嗅觉通路引起。但是国内外目前并无上呼吸道感染后嗅觉障碍高危病毒筛选方法。本研究拟利用现有临床资源，采集上呼吸道感染后嗅觉障碍患者临床数据及生物学标本，设计上呼吸道常见病毒抗体检测芯片，对生物学标本进行病毒抗体检测，分析感染病毒同嗅觉障碍发生关联度，绘制致嗅觉障碍的高危上呼吸道病毒谱系。

方法：采集上呼吸道感染后嗅觉障碍患者（72例）及健康对照组（12例）详细的临床资料及随访资料，包括临床症状、嗅觉主观及客观评估、嗅球影像学评估、耳鼻喉客观查体、患者主观问卷调查及治疗方案。生物样本主要收集患者外周血样（提取 DNA、RNA及蛋白质）及嗅上皮组织。查阅已报道文献及数据库，设计上呼吸道感染相关病毒蛋白芯片。应用设计的病毒蛋白芯片对临床上确诊的上呼吸道感染后嗅觉障碍患者进行外周血特异性IgM抗体检测，筛选上呼吸道感染后嗅觉障碍患者高危致病病毒谱系，并通过ELISA试验及动物（小鼠）试验，验证蛋白芯片结果的可靠性。

结果：通过查阅文献，对目前已报道的同上呼吸道感染相关的病毒进行筛选集合，设计出包含目前上呼吸道感染相关的病毒芯片，芯片包括常见的呼吸道感染病毒：流行性感冒病毒（A 型及B 型）、副流感病毒（3,4 型）、呼吸道合胞病毒、冠状病毒（HCoV-229E、HCoV-OC43、COVID-19和Omicron(BA.4)）、腺病毒、柯萨奇病毒(A 组)、肠道病毒71型、疱疹病毒以及EB病毒。通过蛋白芯片对上呼吸道感染后嗅觉障碍患者（72例）及健康对照组（12例）进行外周血病毒特异性IgM抗体检测，其中上呼吸道感染后嗅觉障碍患者抗体阳性率最高的前3位是：柯萨奇病毒-A组16型抗体阳性率为73.6%，2019-nCoV阳性率为59.7%，B型流感病毒阳性率为48.6%，健康对照组中与之对应的阳性率分别为：50%、91.7%、41.7%。通过ELISA方法验证，检测血清中CV-A16 IgM 抗体水平，发现OD患者血清的CV-A16 IgM 抗体水平显著高于健康对照组，而CV-A16 IgG抗体水平OD患者组与健康对照组则无统计学差异。随机选取健康对照组和OD患者的血清检测CV-A16 中和抗体水平，发现 OD患者血清中的CV-A16 中和抗体显著高于健康对照组。在动物试验中，通过滴鼻攻毒的方式感染3周龄的IFNAR^-/- C57小鼠，成功建立了CV-A16的感染，CV-A16感染在嗅球中持续存在，且病毒载量维持在较高水平。对感染后第5天的IFNAR^-/- C57小鼠进行埋球试验，检测小鼠的嗅觉功能。相较于PBS组，CV-A16感染小鼠寻找食物的时间显著延长，提示CV-A16感染影响并降低了IFNAR^-/- C57小鼠的嗅觉功能。

结论：本研究通过临床队列分析与动物模型验证，首次系统揭示了柯萨奇病毒A16型（CV-A16）感染与上呼吸道感染后嗅觉障碍（OD）的因果关系，并明确了其致病特征与潜在机制。

Objective

Upper respiratory tract viral infection is one of the most common causes of olfactory disorders, accounting for 18%-36%, and is generally believed to be caused by viral damage to olfactory pathways. However, there is no high risk virus screening method for olfactory disorder after respiratory infection at home and abroad. This study intends to use existing clinical resources to collect clinical data and biological specimens of patients with olfactory disorder after upper respiratory tract infection, design common upper respiratory tract virus sequencing chips, perform viral sequencing on biological specimens, analyze the correlation between infected viruses and olfactory disorder, and draw the lineage of high-risk upper respiratory tract viruses causing olfactory disorder.

Method

Detailed clinical and follow-up data were collected from patients with olfactory impairment after upper respiratory tract infection (72 cases) and healthy controls (12 cases), including clinical symptoms, subjective and objective assessment of olfaction, olfactory bulb imaging assessment, objective ear, nose, and throat examination, subjective questionnaire survey of the patients, and treatment plan. Biological samples were collected from peripheral blood (DNA, RNA and protein) and olfactory epithelial tissue. A review of the reported literature and databases was conducted to design a viral microarray related to upper respiratory tract infections. The designed viral microarray was used to screen the peripheral blood of clinically diagnosed patients with olfactory dysfunction after upper respiratory tract infection for high-risk pathogenic viral profiles, and the reliability of the protein microarray results was verified by ELISA and animal testing.

Results

Through reviewing the literature, we screened a collection of currently reported viruses associated with upper respiratory tract infections, and designed a virus microarray that contains the current viruses associated with upper respiratory tract infections. The microarray includes common respiratory tract infections viruses: influenza viruses (type A and B), parainfluenza viruses (types 3 and 4), respiratory syncytial viruses, coronaviruses (SARS viruses, MERS viruses, and COVID-19), adenoviruses, coxsackieviruses (group A and B), enteroviruses, and rhinoviruses. (SARS, MERS and COVID-19), adenoviruses, coxsackieviruses (group A and B), enteroviruses and rhinoviruses. Peripheral blood viral antibodies were examined by microarray in patients with olfactory dysfunction after upper respiratory tract infection (72 patients) and healthy controls (12 patients), in which the top 3 highest antibody positivity rates in patients with olfactory dysfunction after upper respiratory tract infection were: coxsackievirus-group A type 16 antibody positivity of 73.6%, 2019-nCoV positivity of 59.7%, and influenza virus type B positivity of 48.6%, and the corresponding positivity rates in the healthy control group were: 50%, 91.7%, and 41.7%, respectively. The serum levels of CV-A16 IgM antibodies were verified by ELISA method, and it was found that the serum levels of CV-A16 IgM antibodies in OD patients were significantly higher than those in the healthy control group, whereas there was no statistically significant difference in the levels of CV-A16 IgG antibodies in the OD patient group and the healthy control group. Sera from healthy controls and OD patients were randomly selected for the detection of CV-A16 neutralizing antibodies, and it was found that the sera of OD patients contained significantly higher levels of CV-A16 neutralizing antibodies than those of healthy controls. In animal experiments, CV-A16 infection was successfully established by infecting 3-week-old IFNAR-/- C57 mice by nasal drip attack, and CV-A16 infection persisted in the olfactory bulb and the viral load was maintained at a high level. The buried ball test was performed on IFNAR-/- C57 mice on day 5 post-infection to test the olfactory function of the mice. Compared with the PBS group, CV-A16-infected mice took significantly longer time to search for food, suggesting that CV-A16 infection affected and reduced the olfactory function of IFNAR-/- C57 mice.

Conclusions

In this study, through clinical cohort analysis and animal model validation, we systematically revealed for the first time the causal relationship between coxsackievirus type A16 (CV-A16) infection and olfactory dysfunction (OD) after upper respiratory tract infections and clarified its pathogenic features and potential mechanisms.