WHO WPRO Regional Polio Reference Laboratory and NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.
Department of the Laboratory, Guanghua Hospital of Traditional and Western Medicine, Changning District, Shanghai, People's Republic of China.
Virol J. 2019 Mar 29;16(1):40. doi: 10.1186/s12985-019-1151-7.
Enterovirus C96 (EV-C96) is a newly named type of enterovirus belonging to species C, and the prototype strain (BAN00-10488) was firstly isolated in 2000 from a stool specimen of a patient with acute flaccid paralysis in Bangladesh. In this study, we report the genomic and phenotypic characteristics of two EV-C96 strains isolated from individuals from the Tibet Autonomous Region of China.
Human rhabdomyosarcoma (RD), human laryngeal epidermoid carcinoma (HEp-2), and human cervical cancer (Hela) cells were infected with the Tibet EV-C96 strains, and enterovirus RNA in the cell culture was detected with a real time RT-PCR-based enterovirus screening method. The temperature sensitivity of Tibet EV-C96 strains were assayed on a monolayer of RD cells in 24-well plates. Full-length genome sequencing was performed by a 'primer-walking' strategy, and the evolutionary history of EV-C96 was studied by maximum likelihood analysis.
Strain 2005-T49 grew in all three kinds of cells, and it was not temperature sensitive. In contrast, none of the three cells produced CPE for strain 2012-94H. Phylogenetic analysis of the two Tibetan viruses, other EV-C96 strains, and EV-C prototypes showed that EV-C96 strains were grouped into three clusters (Cluster1-3) based on their VP1 sequences, which may represent three genotypes. Phylogenetic trees based on the P2 and P3 regions highlighted the difference between Chinese EV-C96 strains and the EV-C96 prototype strain BAN-10488. All Chinese strains formed a cluster separate from BAN-10488, which clustered with CV-A1/CV-A22/CV-A19.
There is genetic variability between EV-C96 strains which suggest that at least few genetic lineages co-exist and there has been some degree of circulation in different geographical regions for some time. Some recombination events must have occurred during EV-C96 evolution as EV-C96 isolates cluster with different EV-C prototype strains in phylogenetic trees in different genomic regions. However, recombination does not seem to have occurred frequently as EV-C96 isolates from different years and locations appear to cluster together in all genomic regions analysed. These findings expand the understanding of the characterization of EV-C96 and are meaningful for the surveillance of the virus.
肠道病毒 C96（EV-C96）是一种新命名的肠道病毒属 C 型病毒，其原型株（BAN00-10488）于 2000 年首次从孟加拉国急性弛缓性麻痹患者的粪便标本中分离得到。本研究报告了两株从中国西藏自治区个体中分离到的 EV-C96 病毒株的基因组和表型特征。
用人横纹肌肉瘤（RD）、人喉表皮样癌细胞（HEp-2）和人宫颈癌细胞（Hela）感染西藏 EV-C96 株，用基于实时 RT-PCR 的肠道病毒筛选方法检测细胞培养物中的肠道病毒 RNA。在 24 孔板的 RD 细胞单层上测定西藏 EV-C96 株的温度敏感性。采用“引物行走”策略进行全长基因组测序，并通过最大似然分析研究 EV-C96 的进化史。
株 2005-T49 在所有三种细胞中均生长，且无温度敏感性。相比之下，三种细胞均未产生对株 2012-94H 的细胞病变效应。对两株西藏病毒、其他 EV-C96 株和 EV-C 原型株的进化分析表明，根据 VP1 序列，EV-C96 株分为三个簇（簇 1-3），可能代表三个基因型。基于 P2 和 P3 区的系统进化树突出了中国 EV-C96 株与 BAN-10488 原型株之间的差异。所有中国株均形成与 BAN-10488 分离的簇，该簇与 CV-A1/CV-A22/CV-A19 聚类。
EV-C96 株之间存在遗传变异性，这表明至少存在几种遗传谱系，并在不同地理区域存在一定程度的循环。EV-C96 进化过程中一定发生了某些重组事件，因为 EV-C96 分离株在不同基因组区域的系统进化树中与不同的 EV-C 原型株聚类。然而，由于 EV-C96 分离株在所有分析的基因组区域似乎聚集在一起，重组似乎并不频繁。这些发现扩展了对 EV-C96 特征的认识，对病毒的监测具有重要意义。
