Deep sequencing of the mouse lung transcriptome reveals distinct long non-coding RNAs expression associated with the high virulence of H5N1 avian influenza virus in mice

Long non-coding RNAs (lncRNAs) play multiple key regulatory roles in various biological processes. However, their function in influenza A virus (IAV) pathogenicity remains largely unexplored. Here, using next generation sequencing, we systemically compared the whole-transcriptome response of the mouse lung infected with either the highly pathogenic (A/Chicken/Jiangsu/k0402/2010, CK10) or the nonpathogenic (A/Goose/Jiangsu/k0403/2010, GS10) H5N1 virus. A total of 126 significantly differentially expressed (SDE) lncRNAs from three replicates were identified to be associated with the high virulence of CK10, whereas 94 SDE lncRNAs were related with GS10. Functional category analysis suggested that the SDE lncRNAs-coexpressed mRNAs regulated by CK10 were highly related with aberrant and uncontrolled inflammatory responses. Further canonical pathway analysis also confirmed that these targets were highly enriched for inflammatory-related pathways. Moreover, 9 lncRNAs and 17 lncRNAs-coexpressed mRNAs associated with a large number of targeted genes were successfully verified by qRT-PCR. One targeted lncRNA (NONMMUT011061) that was markedly activated and correlated with a great number of mRNAs was selected for further in-depth analysis, including predication of transcription factors, potential interacting proteins, genomic location, coding ability and construction of the secondary structure. More importantly, NONMMUT011061 was also distinctively stimulated during the highly pathogenic H5N8 virus infection in mice, suggesting a potential universal role of NONMMUT011061 in the pathogenesis of different H5 IAV. Altogether, these results provide a subset of lncRNAs that might play important roles in the pathogenesis of influenza virus and add the lncRNAs to the vast repertoire of host factors utilized by IAV for infection and persistence.

长链非编码RNA（long non-coding RNAs，lncRNAs）在多种生物学过程中发挥多重关键调控作用。然而，其在甲型流感病毒（influenza A virus，IAV）致病过程中的功能仍未得到充分探索。本研究采用下一代测序技术，系统性比较了感染高致病性（A/Chicken/Jiangsu/k0402/2010，CK10）与非致病性（A/Goose/Jiangsu/k0403/2010，GS10）H5N1病毒的小鼠肺部的全转录组应答反应。从三次生物学重复样本中，本研究共鉴定出126个与CK10高致病性相关的显著差异表达（significantly differentially expressed，SDE）lncRNAs，另有94个SDE lncRNAs与GS10感染相关。功能类别分析显示，CK10调控的、与SDE lncRNAs共表达的mRNA与异常失控的炎症反应高度相关。进一步的经典通路分析亦证实，这些靶标显著富集于炎症相关通路。此外，本研究通过实时定量反转录PCR（quantitative real-time reverse transcription PCR，qRT-PCR）成功验证了9个lncRNAs及17个与大量靶基因相关的lncRNAs共表达mRNA。本研究选取了一个显著激活且与大量mRNA相关的靶标lncRNA（NONMMUT011061）开展深入分析，内容涵盖转录因子预测、潜在互作蛋白分析、基因组定位、编码能力预测以及二级结构构建。更为重要的是，在感染高致病性H5N8病毒的小鼠体内，NONMMUT011061同样被显著激活，这表明NONMMUT011061在不同H5亚型IAV的致病过程中可能具有普遍调控作用。综上，本研究鉴定出了一批可能在流感病毒致病过程中发挥重要作用的lncRNAs，同时将lncRNAs纳入了IAV用以感染与持续增殖的宿主因子庞大库中。