Yu Han-Gang, Sizemore Gina, Smoot Katy, Perrotta Peter
Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV, USA.
EZCARE Walk-In Medical Center, Moorefield, WV, USA.
J Clin Transl Sci. 2021 Sep 14;5(1):e196. doi: 10.1017/cts.2021.856. eCollection 2021.
SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the virus passes from the upper respiratory tract to the myocardium. We hypothesized that SARS-CoV-2 is present in the blood of COVID-19 infected patients, spreading to other organs such as heart.
We targeted two viroporins, Orf3a and E, in SARS-CoV-2. Orf3a and E form non-voltage-gated ion channels. A combined fluorescence potassium ion assay with three channel modulators (4-aminopyridine, emodin-Orf3a channel blocker, and gliclazide-E channel blocker) was developed to detect SARS-CoV-2 Orf3a/E channel activity. In blood samples, we subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity.
In lentivirus-spiked samples, we detected significant channel activity of Orf3a/E based on increase in fluorescence induced by 4-aminopyridine, and this increase in fluorescence was inhibited by emodin and gliclazide. In 18 antigen/PCR-positive samples, our test results found 15 are positive, demonstrating 83.3% concordance. In 24 antigen/PCR-negative samples, our test results found 21 are negative, showing 87.5% concordance.
We developed a cell-free test that can detect Orf3a/E channel activity of SARS-CoV-2 in blood samples from COVID-19-infected individuals, confirming a hypothesis that the virus spreads to the heart via blood circulation.
在新冠肺炎患者的心脏中发现了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）。目前尚不清楚该病毒如何从上呼吸道传播至心肌。我们推测SARS-CoV-2存在于新冠肺炎感染患者的血液中，并扩散至心脏等其他器官。
我们针对SARS-CoV-2中的两种病毒孔蛋白，即开放阅读框3a（Orf3a）和E蛋白。Orf3a和E蛋白形成非电压门控离子通道。我们开发了一种结合荧光钾离子检测法与三种通道调节剂（4-氨基吡啶、大黄素——Orf3a通道阻滞剂和格列齐特——E蛋白通道阻滞剂）的方法，以检测SARS-CoV-2的Orf3a/E通道活性。在血液样本中，我们通过减去加入大黄素/格列齐特前后的荧光信号来检测Orf3a和E通道活性。
在慢病毒加样样本中，基于4-氨基吡啶诱导的荧光增加，我们检测到了Orf3a/E的显著通道活性，并且这种荧光增加被大黄素和格列齐特所抑制。在18份抗原/聚合酶链反应（PCR）阳性样本中，我们的检测结果发现15份为阳性，一致性为83.3%。在24份抗原/PCR阴性样本中，我们的检测结果发现21份为阴性，一致性为87.5%。
我们开发了一种无细胞检测方法，可检测新冠肺炎感染个体血液样本中SARS-CoV-2的Orf3a/E通道活性，证实了该病毒通过血液循环扩散至心脏的假说。
