Steyer Andrej, Konte Tilen, Sagadin Martin, Kolenc Marko, Škoberne Andrej, Germ Julija, Dovč-Drnovšek Tadeja, Arnol Miha, Poljšak-Prijatelj Mateja
Faculty of Medicine, Institute of Microbiology and Immunology, University of Ljubljana, Ljubljana, Slovenia.
Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia.
Front Microbiol. 2018 Mar 2;9:371. doi: 10.3389/fmicb.2018.00371. eCollection 2018.
Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope "hot-spots" of modified and optimized norovirus-host interactions.
诺如病毒是急性胃肠炎的主要病因，可感染所有年龄组的人群。在免疫功能低下的患者中，诺如病毒感染可发展为慢性腹泻或出现病毒长期无症状排泄。实体器官移植受者中经常报告有慢性诺如病毒感染，并可见诺如病毒在宿主体内快速进化。在本报告中，我们描述了一例免疫功能低下患者的慢性诺如病毒感染病例，该患者接受了5年多的随访。本研究的目的是明确慢性感染的免疫功能低下宿主中诺如病毒的进化情况，并确定诺如病毒衣壳蛋白中可能的选择位点。在随访期间，收集了25份连续的粪便样本，其中9份被选用于生成覆盖病毒RNA依赖性RNA聚合酶（RdRp）和病毒衣壳蛋白（VP1）基因的扩增子。使用下一代测序技术对扩增子进行测序。定义了单核苷酸多态性，结果显示VP1基因组区域的突变率比RdRp基因组区域高近3倍（分别为每100个核苷酸7.9个和2.8个可变位点）。这表明病毒基因组中的突变并非随机积累，而是感染周期中突变选择的结果。使用ShoRAH软件，我们能够重建9个选定样本中出现的单倍型。对推导的氨基酸单倍型序列进行比对，并使用Datamonkey程序分析这些位置的选择压力。在VP1蛋白通常描述的A、B、C和D表位中，25个阳性选择位点中只有12个位于其中。确定了以前未描述过的新的阳性选择位点，这些位点可能反映了诺如病毒对最佳组织血型抗原结合的适应性，或诺如病毒抗原特性的改变。这些数据为诺如病毒的进化动力学提供了新的见解，并指出了诺如病毒与宿主相互作用修饰和优化的新的假定表位“热点”。
