Small RNA profiling of extracellular vesicles secreted by A549 cells infected with West Nile virus and treated with interferon alpha. Small RNA profiling of extracellular vesicles secreted by A549 cells infected with West Nile virus and treated with interferon alpha

The goal of this study is to determine if infection with flavivirus (West Nile virus) and action of proinflamatory cytokine interferon alter incorporation of host RNAs into extracellular vesicles secreted by human cells. Overall design: Human lung carcinoma cells A545 were infected with Kunjin strain of West Nile virus at multiplicity of infection (MOI)=1 or incubated in the media containing 300u/ml IFN alpha 2a. Cells were then maintained in exosome free media for the next 24h. At 24 h post infection extracellular vesicles were isolated from culture fluids of infected or uninfected (Mock) cells using ExoQuick TC Exosomes isolation reagent. Enriched fractions of small RNAs were isolated from extacellular vesicles using mirVana miRNA isolation Kit (Ambion, USA) and used for library construction and RNA-Seq on Illumina HiSeq platform.

本研究旨在探究黄病毒（flavivirus，西尼罗河病毒（West Nile virus））感染与促炎细胞因子干扰素的作用，是否会改变宿主RNA向人类细胞分泌的细胞外囊泡（extracellular vesicles）中的掺入情况。实验设计概述：将人肺癌细胞A545以感染复数（multiplicity of infection, MOI）=1的比例感染西尼罗河病毒昆津毒株，或置于含有300U/ml干扰素α2a（IFN alpha 2a）的培养基中培养。随后将细胞更换为无外泌体（exosome）培养基继续培养24小时。感染后24小时，使用ExoQuick TC外泌体分离试剂（ExoQuick TC Exosomes isolation reagent）从感染、未感染（Mock）细胞的培养液中分离细胞外囊泡。采用mirVana miRNA分离试剂盒（Ambion公司，美国）从细胞外囊泡中富集小RNA组分，随后构建测序文库并在Illumina HiSeq平台上进行RNA测序（RNA-Seq）。