兔出血症病毒（RHDV）是杯状病毒科的一员，不能在体外繁殖，阻碍了对其复制机制的研究进展。前期，我们构建了RHDV 复制子系统为探索RHDV 在细胞中的复制机理提供了一个平台。本研究借助该复制子系统，并利用两步亲和纯化，鉴定了与 RHDV 复制酶RdRp相关的宿主因子。我们发现宿主蛋白核仁素 (NCL) 与病毒RdRp 直接相互作用。同时，我们还发现 在RK-13细胞中，NCL 过表达显著增强RHDV的复制，而敲低NCL则严重破坏该病毒的复制。此外，我们还发现 NCL 与病毒非结构蛋白P16 和 P23 也存在直接相互作用。并且，敲低细胞内的NCL显著下调RdRp 与相关宿主因子的结合。这些结果表明宿主蛋白 NCL 对于 RHDV 复制是必不可少的，并且充当病毒复制酶和宿主蛋白之间的桥梁。
Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using twostep affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.
水生态系统中包含着极其丰富多样的病毒群，但目前对江河水中的病毒组成情况却知之甚少。本研究使用病毒宏基因组学方法探究了长江三角洲水域的病毒群特征，分析比较了6个采样点的病毒组。虽然各采样点病毒组在物种丰度上有细微差异，但总体上组成相似，均以有尾噬菌体目(Caudovirales)为主，并且淡水噬菌体种(Freshwater phage uvFW)也在各样本中普遍存在。位于南京的病毒群具有独特的组成特征，其中细小病毒科(Parvoviridae)的丰度较高。基于各病毒群特征基因的系统发育分析显示，有尾噬菌体目和CRESS-DNA病毒具有较高的遗传多样性。相反，微小噬菌体科(Microviridae)、细小病毒科(Parvoviridae)和核糖病毒域(Riboviria)的病毒相对保守。本研究首次揭示了大型江河生态系统中病毒群的组成结构及其多样性和保守性，有助于淡水资源的合理利用。
Viruses in aquatic ecosystems are characterized by extraordinary abundance and diversity. Thus far, there have been limited studies focused on viral communities in river water systems. Here, we investigated the virome of the Yangtze River Delta using viral metagenomic analysis. The compositions of viral communities from six sampling sites were analyzed and compared. By using library construction and next generation sequencing, contigs and singlet reads similar to viral sequences were classified into 17 viral families, including nine dsDNA viral families, four ssDNA viral families and four RNA viral families. Statistical analysis using Friedman test suggested that there was no significant difference among the six sampling sites (P > 0.05). The viromes in this study were all dominated by the order Caudovirales, and a group of Freshwater phage uvFW species were particularly prevalent among all the samples. The virome from Nanjing presented a unique pattern of viral community composition with a relatively high abundance of family Parvoviridae. Phylogenetic analyses based on virus hallmark genes showed that the Caudovirales order and CRESS-DNA viruses presented high genetic diversity, while viruses in the Microviridae and Parvoviridae families and the Riboviria realm were relatively conservative. Our study provided the first insight into viral community composition in large river ecosystem, revealing the diversity and stability of river water virome, contributing to the proper utilization of freshwater resource.
登革热患者恢复期血清抗体的衰减变化规律与感染风险及预后密切相关。机体感染登革病毒（dengue virus, DENV）后，体内的中和抗体随时间的推移逐渐下降至亚中和浓度时，极易因抗体依赖性增强效应（antibody dependent enhancement, ADE）促进重症登革的发生。本队列研究收集了云南省景洪市2013年登革热患者恢复期不同阶段的血清样本，探究DENV特异性抗体的动态变化规律，分析再次感染异血清型DENV发生ADE的风险。我们于2017年和2019年分别采集191份四年恢复期及99份六年恢复期血清样本。四年恢复期血清DENV特异性IgG阳性率为98.4%，而六年恢复期血清IgG阳性率下降至82.8%；同时中和抗体几何平均效价由1:155.35下降至1:46.66。在290个总体样本中，73名连续追踪患者同时参加了2017年和2019年回访。在连续追踪样本中，四年恢复期血清针对DENV-3中和抗体以及DENV-1、DENV-2和DENV-4交叉反应性抗体几何平均效价分别为1:167.70、1:13.80、1:18.54和1:45.26；两年后，其效价分别下降至1:53.18、1:10.30、1:14.60和1:8.17。在ADE风险分析中，随着恢复期延长，31-40岁和51-60岁年龄组连续追踪患者在2019年样本中针对DENV-4的ADE阳性人数增加，导致再次感染DENV-4发生ADE的风险增加，而再次感染DENV-1和DENV-2发生ADE的风险降低。本研究对登革高发区域重症登革的风险预测以及登革疫苗的相关研究提供了重要实验依据。
After dengue virus (DENV) infection, antibody-dependent enhancement (ADE) is easy to occur when the neutralizing antibody (NAb) gradually decreases to a sub-neutralizing concentration. In this cohort surveillance, we utilized sera samples collected from dengue fever patients at different convalescent phases in Jinghong City, to investigate the dynamic change rule of DENV-specific antibodies, and to analyze the risk of ADE caused by secondary infection with heterologous serotypes DENVs. For baseline serosurvey, 191 four-year and 99 six-year sera samples during convalescence were collected in 2017 and 2019, respectively. The positive rate of DENV-specific immunoglobulin G was 98.4% in 2017, which significantly decreased to 82.8% in 2019. The geometric mean titer (GMT) of NAb decreased from 1:155.35 to 1:46.66. Among 290 overall samples, 73 paired consecutive samples were used for follow-up serosurvey. In four-year sera, the GMTs of NAb against DENV3 and cross-reactive antibodies against DENV-1, DENV-2 and DENV-4 were 1:167.70, 1:13.80, 1:18.54 and 1:45.26, respectively, which decreased to 1:53.18, 1:10.30, 1:14.60 and 1:8.17 in six-year sera. In age-stratified analysis, due to the increasing number of ADE positive samples from 2017 to 2019 in 31–40 and 51–60 years groups, the risk of ADE in DENV-4 infection was positively associated with the extension of convalescent phase, and the odd ratio was higher than other groups. With the recovery period lengthened, the risk of secondary infection with DENV-1 and DENV-2 was reduced. Our results offer essential experimental data for risk prediction of severe dengue in hyper-endemic dengue areas, and provide crucial scientific insight for the development of effective dengue vaccines.
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