To the Editor,

Post-viral fatigue after SARS-CoV-2 infection is a major concern in the ongoing COVID-19 pandemic. Up to 10% of patients develop Long-COVID,¹ a syndrome defined by the National Institute for Health and Care Excellence as post-infectious long-term symptoms for more than 12 weeks, which cannot be explained by alternative pathologies.¹ While some Long-COVID symptoms, such as the shortness of breath or chest pain, might be associated with organ damage, the origin of post-COVID fatigue, including debilitating fatigue and impaired memory and concentration, remains unclear. Several viruses were associated with post-viral fatigue, such as Epstein–Barr-Virus (EBV) or influenza viruses. Also, myalgic encephalomyelitis/chronic fatigue syndrome, a WHO defined independent disease, may develop, amongst other triggers, after viral infections. However, the mechanisms leading to post-viral fatigue syndrome in overall and especially in Long-COVID are unresolved.² So far, a higher frequency of EBV viremia was observed only in acute severe COVID-19 infections.³

In the present prospective study, we analysed whether Long-COVID fatigue may be triggered by SARS-CoV-2 persistence in the gastrointestinal or respiratory tract after acute disease,² or by COVID-19-associated reactivation of EBV.

We tested for SARS-CoV-2 RNA in stool and throat washings, and for EBV DNA in stool, throat washings and blood by real-time PCR (EBV) and real-time RT-PCR (SARS-CoV-2) as described before.^{4, 5} The experimental procedures were performed with permission of the human ethics committee of the Medical University of Vienna (vote number: 2281/2020). All patients provided written informed consent. Samples were collected between 74 and 471 days (median: 235 days) after beginning of acute SARS-CoV-2 infection in 30 Long-COVID patients characterized by persistent fatigue, post-exertional malaise (PEM), autonomic dysfunction and/or orthostatic intolerance. Twenty age- and sex-matched patients, who have fully recovered after the SARS-CoV-2 infection, were recruited for control purposes. Samples were collected between 106 and 571 days (median: 275 days) after beginning of acute SARS-CoV-2 infection. All patients had mild infections, were not hospitalized and were infected between spring 2020 and autumn 2021. We further analysed virus-specific antibodies including SARS-CoV-2 IgA and IgG with commercial ELISA assays (Euroimmun). As Long-COVID patients showed a higher frequency of positive titres for specific EBV antibodies compared with controls in earlier studies,⁶ the patients' EBV IgM and IgG were tested by a commercial microarray (ViraMed). Demographic data as well as inclusion and exclusion criteria are depicted in Table S1. More detailed information on the methodological procedures is listed in the Supporting Information.

At time point of sampling, SARS-CoV-2 RNA was detectable neither in throat washing nor stool in any of the study participants by real-time RT-PCR. SARS-CoV-2 antibody titres (IgA and IgG) did not differ between the cohorts (Figure S1). Most study participants had been vaccinated after COVID-19 disease prior to sampling (24/30 in Long-COVID patients, 17/20 in controls). EBV real-time PCR was negative in all blood or stool samples. However, EBV DNA was detected in throat washings in 15/30 (50%) of Long-COVID patients while only in 4/20 (20%) of non-Long-COVID patients who had recovered from their SARS-CoV-2 infection (p = .0411). EBV load levels were not significantly different between the two cohorts in EBV-specific real-time PCR positive samples (Figure 1). All patients of both groups, except one patient in the fully convalescent COVID-19 group (SARS-CoV-2 – LC), had past EBV infections, as confirmed by EBV IgG seropositive and IgM seronegative status at the time of investigation, thus, the EBV replication observed was due to EBV reactivation rather than primary infection. EBV specific antibody titres as assessed by microarray in blood were not different between the groups (Figure S2).

Taken together, SARS-CoV-2 persistence could not be detected in our study participants up to 10 weeks after infection, but EBV reactivation in the throat was more common in patients with Long-COVID fatigue, also months after acute SARS-CoV-2 infection, compared with convalescent SARS-CoV-2 patients. This suggests that EBV replication may be a co-factor in a sub-group of patients developing Long-COVID fatigue. However, contributing factors, such as HLA-subtype depending on the answers to latent EBV infection, have not been evaluated in this study and represent limitations. Earlier reports of differences in the EBV antibody profile of Long-COVID patients⁶ could not be confirmed, possibly due to the small sample size analysed in our study. Further and larger studies are needed to clarify the impact and mechanism of EBV-associated Long-COVID fatigue.

致编辑，

SARS-CoV-2 感染后的病毒后疲劳是持续的 COVID-19 大流行中的一个主要问题。高达 10% 的患者会发展为长期 COVID，¹国家健康与护理卓越研究所将这种综合征定义为感染后长期症状超过 12 周，无法用其他病理学解释。^1个虽然呼吸急促或胸痛等一些长期 COVID 症状可能与器官损伤有关，但 COVID 后疲劳的起源，包括使人虚弱的疲劳以及记忆力和注意力受损，仍不清楚。有几种病毒与感染后疲劳有关，例如爱泼斯坦-巴尔病毒 (EBV) 或流感病毒。此外，肌痛性脑脊髓炎/慢性疲劳综合症是一种 WHO 定义的独立疾病，在病毒感染后可能会发展为其他诱因。然而，总体上导致病毒后疲劳综合症的机制，尤其是在长期 COVID 中，尚未得到解决。²到目前为止，仅在急性重症 COVID-19 感染中观察到更高频率的 EBV 病毒血症。^3个

^{在本前瞻性研究中，我们分析了长期 COVID 疲劳是否可能由 SARS-CoV-2 在急性疾病后在胃肠道或呼吸道中持续存在2}或由 COVID-19 相关的 EBV 再激活引发。

我们通过实时 PCR (EBV) 和实时 RT-PCR (SARS-CoV-2) 检测了粪便和咽喉清洗液中的 SARS-CoV-2 RNA，以及粪便、咽喉清洗液和血液中的 EBV DNA，如前所述前。^{4, 5}实验程序是在维也纳医科大学人类伦理委员会（投票编号：2281/2020）的许可下进行的。所有患者都提供了书面知情同意书。在 30 名长期 COVID 患者开始急性 SARS-CoV-2 感染后 74 至 471 天（中位数：235 天）收集样本，其特征为持续疲劳、劳累后不适 (PEM)、自主神经功能障碍和/或直立性不耐受. 出于对照目的，招募了 20 名年龄和性别匹配的患者，他们在 SARS-CoV-2 感染后完全康复。样本是在急性 SARS-CoV-2 感染开始后的 106 到 571 天（中位数：275 天）之间收集的。所有患者均为轻度感染，未住院，均在 2020 年春季至 2021 年秋季之间感染。我们使用商业 ELISA 测定法 (Euroimmun) 进一步分析了病毒特异性抗体，包括 SARS-CoV-2 IgA 和 IgG。由于与早期研究中的对照组相比，长期 COVID 患者表现出更高频率的特定 EBV 抗体阳性滴度，⁶患者的 EBV IgM 和 IgG 通过商业微阵列 (ViraMed) 进行检测。表 S1 中描述了人口统计数据以及纳入和排除标准。有关方法程序的更多详细信息列在支持信息中。

在取样的时间点，通过实时 RT-PCR，在任何研究参与者的咽喉冲洗液和粪便中均未检测到 SARS-CoV-2 RNA。SARS-CoV-2 抗体滴度（IgA 和 IgG）在队列之间没有差异（图 S1）。大多数研究参与者在采样前已接种 COVID-19 病后疫苗（长期 COVID 患者为 24/30，对照组为 17/20）。所有血液或粪便样本中的 EBV 实时 PCR 均为阴性。然而，在 15/30 (50%) 的长期 COVID 患者的咽喉冲洗液中检测到 EBV DNA，而只有 4/20 (20%) 的非长期 COVID 患者从 SARS-CoV-2 感染中恢复过来( p =.0411）。在 EBV 特异性实时 PCR 阳性样本中，两个队列之间的 EBV 载量水平没有显着差异（图 1）。两组的所有患者，除了完全康复的 COVID-19 组（SARS-CoV-2 – LC）中的一名患者外，都曾感染过 EBV，这在调查时由 EBV IgG 血清阳性和 IgM 血清阴性状态证实，因此，观察到的 EBV 复制是由于 EBV 再激活而不是原发感染。通过血液中的微阵列评估的 EBV 特异性抗体滴度在各组之间没有差异（图 S2）。

综上所述，我们的研究参与者在感染后长达 10 周内无法检测到 SARS-CoV-2 的持续存在，但喉咙中的 EBV 再激活在长期 COVID 疲劳患者中更为常见，在急性 SARS-CoV-2 感染后数月也是如此与恢复期 SARS-CoV-2 患者相比，感染率更高。这表明 EBV 复制可能是发生长期 COVID 疲劳的患者亚组的辅助因素。然而，促成因素，例如取决于潜伏 EBV 感染的答案的 HLA 亚型，在本研究中尚未得到评估并且存在局限性。关于长期 COVID 患者 EBV 抗体谱差异的早期报告⁶无法确认，可能是由于我们研究中分析的样本量较小。需要进一步和更大规模的研究来阐明 EBV 相关的 Long-COVID 疲劳的影响和机制。