Laboratorio de Virología, Universidad El Bosque, Bogotá D.C., Colombia.
PLoS One. 2022 Apr 6;17(4):e0266450. doi: 10.1371/journal.pone.0266450. eCollection 2022.
Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. In 2013, the virus was introduced to the Americas and caused approximately 2.7 million cases of infection during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak motivated the characterization of an isolate from the Colombian outbreak, starting from analysis of the complete genome to the biological behavior in vitro. The full genome was retrieved using next-generation sequencing. The infective and replicative capacities were evaluated in HEK293T, Huh-7, and MRC-5 cell lines. The infection rates were determined by flow cytometry, and the cytopathic effect was assessed by a resazurin fluorescent metabolic assay. The viral yield was quantified using the virus plaque formation assay, while the viral proteins and genomic RNA kinetics were subsequently evaluated by western-blot and RT-qPCR. The COL7624 isolate clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 protein distinguished it from other Colombian sequences reported in 2014. After 48 h post infection (hpi), the three cell lines analyzed reached infection percentages exceeding 65%, generating a high load of infectious viral progeny. The infection kinetics indicated that the replication peak of this CHIKV isolate is around 24 hpi, although gRNA is detectable in the culture supernatant from 4 hpi onwards. The infection caused the overexpression of interferon and pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-8. The COL7624 CHIKV isolate exhibited a high infective and replicative capacity as well as activation of cellular immune responses, similar to isolates belonging to the other genotypes.
基孔肯雅病毒是一种节肢动物传播的病毒，可引起基孔肯雅热，这种疾病的特征是严重的肌肉和关节疼痛。2013 年，该病毒传入美洲，随后两年内导致约 270 万例感染。由于缺乏对暴发期间流行的病毒株的生物学行为的了解，促使从分析完整基因组到体外生物学行为对哥伦比亚暴发的分离株进行了表征。使用下一代测序技术检索了全基因组。在 HEK293T、Huh-7 和 MRC-5 细胞系中评估了感染性和复制能力。通过流式细胞术测定感染率，并通过 Resazurin 荧光代谢测定法评估细胞病变效应。使用病毒空斑形成测定法定量病毒产量，随后通过 Western blot 和 RT-qPCR 评估病毒蛋白和基因组 RNA 动力学。COL7624 分离株与亚洲美洲谱系中的其他美洲和加勒比序列聚类。E2 蛋白中的 T669A 取代将其与 2014 年报告的其他哥伦比亚序列区分开来。感染后 48 小时（hpi），分析的三种细胞系的感染率超过 65%，产生了大量感染性病毒后代。感染动力学表明，该 CHIKV 分离株的复制峰值约为 24 hpi，但 gRNA 可从 4 hpi 起在培养上清液中检测到。感染导致干扰素和促炎细胞因子（如 IL-1β、TNF-α 和 IL-8）的过度表达。COL7624 CHIKV 分离株表现出高感染性和复制能力以及细胞免疫反应的激活，类似于属于其他基因型的分离株。
