Table 9_Integrative study of chicken lung transcriptome to understand the host immune response during Newcastle disease virus challenge.xlsx

IntroductionNewcastle disease is one of the significant issues in the poultry industry, having catastrophic effects worldwide. The lung is one of the essential organs which harbours Bronchus-associated lymphoid tissue and plays a vital role in the immune response. Leghorn and Fayoumi breeds are known to have differences in resistance to Newcastle disease. Along with genes and long non-coding RNAs (lncRNAs) are also known to regulate various biological pathways through gene regulation.MethodsThis study analysed the lung transcriptome data and identified the role of genes and long non-coding RNAs in differential immune resistance. The computational pipeline, FHSpipe, as used in our previous studies on analysis of harderian gland and trachea transcriptome was used to identify genes and lncRNAs. This was followed by differential expression analysis, functional annotation of genes and lncRNAs, identification of transcription factors, microRNAs and finally validation using qRT-PCR.Results and discussionA total of 8219 novel lncRNAs were identified. Of them, 1263 lncRNAs and 281 genes were differentially expressed. About 66 genes were annotated with either an immune-related GO term or pathway, and 12 were annotated with both. In challenge and breed-based analysis, most of these genes were upregulated in Fayoumi compared to Leghorn, and in timepoint-based analysis, Leghorn challenge chicken showed downregulation between time points. A similar trend was observed in the expression of lncRNAs. Co-expression analysis has revealed several lncRNAs co-expressing with immune genes with a positive correlation. Several genes annotated with non-immune pathways, including metabolism, signal transduction, transport of small molecules, extracellular matrix organization, developmental biology and cellular processes, were also impacted. With this, we can understand that Fayoumi chicken showed upregulated immune genes and positive cis-lncRNAs during both the non-challenged and NDV-challenge conditions, even without viral transcripts in the tissue. This finding shows that these immune-annotated genes and coexpressing cis-lncRNAs play a significant role in Fayoumi being comparatively resistant to NDV compared to Leghorn. Our study affirms and expands upon the outcomes of previous studies and highlights the crucial role of lncRNAs during the immune response to NDV.ConclusionThis analysis clearly shows the differences in the gene expression patterns and lncRNA co-expression with the genes between Leghorn and Fayoumi, indicating that the lncRNAs and co-expressing genes might potentially have a role in differentiating these breeds. We hypothesise that these genes and lncRNAs play a vital role in the higher resistance of Fayoumi to NDV than Leghorn. This study can pave the way for future studies to unravel the biological mechanism behind the regulation of immune-related genes.

引言：新喀里多尼亚病是家禽业中一个重要的难题，其全球性的影响灾难性。肺脏是重要的器官之一，其中包含支气管相关淋巴组织，并在免疫反应中发挥着至关重要的作用。莱格亨和法尤米品种在抵抗新喀里多尼亚病方面存在差异。基因和长非编码RNA（lncRNA）已知通过基因调控调节多种生物通路。方法：本研究分析了肺转录组数据，并确定了基因和长非编码RNA在差异免疫抵抗力中的作用。本研究采用了我们之前在分析泪腺和气管转录组研究中使用的计算流程FHSpipe，以识别基因和lncRNA。随后进行了差异表达分析、基因和lncRNA的功能注释、转录因子和微RNA的识别，以及最终通过qRT-PCR进行验证。结果与讨论：共鉴定出8219个新的lncRNA。其中，1263个lncRNA和281个基因表现出差异表达。大约66个基因被注释为与免疫相关的GO术语或通路，其中12个同时被注释。在挑战和品种分析中，与莱格亨相比，法尤米中的大多数这些基因上调，而在时间点分析中，莱格亨挑战鸡在时间点之间表现出下调。lncRNA的表达也呈现出相似的趋势。共表达分析揭示了与免疫基因共表达并呈正相关的一些lncRNA。一些注释为非免疫通路（包括代谢、信号转导、小分子运输、细胞外基质组织、发育生物学和细胞过程）的基因也受到影响。因此，我们可以理解，法尤米鸡在非挑战和NDV挑战条件下均表现出上调的免疫基因和正向顺式lncRNA，即使在组织中没有病毒转录物。这一发现表明，这些免疫注释基因和共表达的顺式lncRNA在法尤米相对于莱格亨对NDV具有较高抵抗力方面起着重要作用。本研究证实并扩展了先前研究的成果，并突出了lncRNA在NDV免疫反应中的关键作用。结论：这一分析明确显示了莱格亨和法尤米在基因表达模式和基因与lncRNA共表达方面的差异，表明lncRNA和共表达基因可能在这些品种的区分中发挥作用。我们假设这些基因和lncRNA在家禽对NDV的抵抗力方面比莱格亨更高起着至关重要的作用。本研究为未来研究揭开免疫相关基因调控背后的生物学机制铺平了道路。