Predicted results of EVB conformational epitopes.

Enterovirus B (EVB) is a common species of enterovirus, mainly consisting of Echovirus (Echo) and Coxsackievirus B (CVB). The population is generally susceptible to EVB, especially among children. Since the 21st century, EVB has been widely prevalent worldwide, and can cause serious diseases, such as viral meningitis, myocarditis, and neonatal sepsis. By using cryo-electron microscopy, the three-dimensional (3D) structures of EVB and their uncoating receptors (FcRn and CAR) have been determined, laying the foundation for the study of viral pathogenesis and therapeutic antibodies. A limited number of epitopes bound to neutralizing antibodies have also been determined. It is unclear whether additional epitopes are present or whether epitope mutations play a key role in molecular evolutionary history and epidemics, as in influenza and SARS-CoV-2. In the current study, the conformational epitopes of six representative EVB serotypes (E6, E11, E30, CVB1, CVB3 and CVB5) were systematically predicted by bioinformatics-based epitope prediction algorithm. We found that their epitopes were distributed into three clusters, where the VP1 BC loop, C-terminus and VP2 EF loop were the main regions of EVB epitopes. Among them, the VP1 BC loop and VP2 EF loop may be the key epitope regions that determined the use of the uncoating receptors. Further molecular evolution analysis based on the VP1 and genome sequences showed that the VP1 C-terminus and VP2 EF loop, as well as a potential “breathing epitope” VP1 N-terminus, were common mutation hotspot regions, suggesting that the emergence of evolutionary clades was driven by epitope mutations. Finally, footprints showed mutations were located on or near epitopes, while mutations on the receptor binding sites were rare. This suggested that EVB promotes viral epidemics by breaking the immune barrier through epitope mutations, but the mutations avoided the receptor binding sites. The bioinformatics study of EVB epitopes may provide important information for the monitoring and early warning of EVB epidemics and developing therapeutic antibodies.肠道病毒B型（Enterovirus B, EVB）是一类常见的肠道病毒，主要由埃可病毒（Echovirus, Echo）和柯萨奇病毒B型（Coxsackievirus B, CVB）组成。人群普遍对EVB易感，尤以儿童群体为甚。自21世纪以来，EVB在全球范围内广泛流行，可引发病毒性脑膜炎、心肌炎以及新生儿败血症等严重疾病。借助冷冻电镜（cryo-electron microscopy）技术，科研人员已解析出EVB及其脱壳受体（uncoating receptors）FcRn与CAR的三维（three-dimensional, 3D）结构，为病毒致病机制研究以及治疗性抗体开发奠定了基础。目前已确定少量与中和抗体（neutralizing antibodies）结合的表位（epitope）。目前尚不清楚是否存在其他未被发现的表位，亦不清楚表位突变是否如流感病毒与新型冠状病毒（SARS-CoV-2）一般，在病毒的分子进化历程与流行过程中发挥关键作用。本研究通过基于生物信息学的表位预测算法，对6种代表性EVB血清型（serotype）（E6、E11、E30、CVB1、CVB3及CVB5）的构象表位（conformational epitope）进行了系统性预测。研究发现，这些表位可分为3个簇，其中VP1 BC环、C末端以及VP2 EF环是EVB表位的主要区域。其中，VP1 BC环与VP2 EF环可能是决定病毒利用脱壳受体的关键表位区域。基于VP1序列与全基因组序列开展的进一步分子进化分析显示，VP1 C末端、VP2 EF环以及潜在的“呼吸表位”VP1 N末端均为常见的突变热点区域，提示进化分支（evolutionary clades）的出现由表位突变驱动。最终，突变足迹分析显示，突变位点均位于表位之上或其邻近区域，而受体结合位点（receptor binding site）上的突变则极为罕见。这表明EVB通过表位突变打破免疫屏障以推动病毒流行，同时这类突变规避了受体结合位点。本项针对EVB表位的生物信息学研究，可为EVB流行的监测预警以及治疗性抗体的开发提供重要参考依据。