Table6_Breed and timepoint-based analysis of chicken harderian gland transcriptome during Newcastle disease virus challenge.XLSX

Introduction: Newcastle disease is a highly infectious disease caused by the Newcastle Disease Virus (NDV) and has a devastating financial impact on the global chicken industry. It was previously established that Leghorn and Fayoumi breeds of chicken exhibit variable resistance against NDV infection. The harderian gland is the less studied tissue of the chicken, known to play an essential role in the immune response.
Methods: Our previous study, we reported differential gene expression and long noncoding RNAs (lncRNAs) between challenged and non-challenged chickens in the Harderian gland transcriptomic data. Now, we report the analysis of the same data studying the differential expression patterns between Leghorn and Fayoumi and between different timepoints during disease. First, the pipeline FHSpipe was used for identification of lncRNAs, followed by differential expression analysis by edgeR (GLM), functional annotation by OmicsBox, co-expression analysis using WGCNA and finally validation of selected lncRNAs and co-expressing genes using qRT-PCR.
Results: Here, we observed that Leghorn showed a higher number of upregulated immune-related genes than Fayoumi in timepoint-based analysis, especially during the initial stages. Surprisingly, Fayoumi, being comparatively resistant, showed little difference between challenged and non-challenged conditions and different time points of the challenge. The breed-based analysis, which compared Leghorn with Fayoumi in both challenged and non-challenged conditions separately, identified several immune-related genes and positive co-expressing cis lncRNAs to be upregulated in Fayoumi when compared to Leghorn in both challenged and non-challenged conditions.
Discussion: The current study shows that Leghorn, being comparatively more susceptible to NDV than Fayoumi, showed several immune-related genes and positive co-expressing cis lncRNAs upregulated in challenged Leghorn when compared to non-challenged Leghorn and also in different timepoints during challenge. While, breed-based analysis showed that there were more upregulated immune genes and positive cis-lncRNAs in Fayoumi than Leghorn. This result clearly shows that the differences in the expression of genes annotated with immune-related GO terms and pathways, i.e., immune-related genes and the co-expressing cis-lncRNAs between Leghorn and Fayoumi, and their role in the presence of differences in the resistance of Leghorn and Fayoumi chicken against NDV.
Conclusion: These immune-genes and cis-lncRNAs could play a role in Fayoumi being comparatively more resistant to NDV than Leghorn. Our study elucidated the importance of lncRNAs during the host defense against NDV infection, paving the way for future research on the mechanisms governing the genetic improvement of chicken breeds.

引言：新城疫（Newcastle disease）是由新城疫病毒（Newcastle Disease Virus, NDV）引发的高度传染性疾病，对全球肉鸡产业造成极具破坏性的经济损失。既往研究已证实，来航鸡（Leghorn）与法尤姆鸡（Fayoumi）对NDV感染表现出不同程度的抗性。哈德氏腺（Harderian gland）是鸡体内研究较少的组织，已知其在免疫应答中发挥关键作用。
方法：在本团队前期研究中，我们已基于哈德氏腺（Harderian gland）转录组数据，报道了攻毒与未攻毒鸡只间的差异基因表达及长链非编码RNA（long noncoding RNAs, lncRNAs）谱。本次研究则针对同一数据集，分析来航鸡与法尤姆鸡之间，以及疾病进程中不同时间点的差异表达模式。分析流程如下：首先通过分析管线FHSpipe鉴定lncRNAs，随后采用edgeR（广义线性模型，GLM）进行差异表达分析，借助OmicsBox完成功能注释，利用WGCNA进行共表达分析，最终通过qRT-PCR验证筛选出的lncRNAs及其共表达基因。
结果：本研究基于时间点的分析显示，来航鸡的上调免疫相关基因数量多于法尤姆鸡，尤其在感染初期。值得注意的是，抗性相对更强的法尤姆鸡在攻毒与未攻毒状态，以及不同攻毒时间点之间的基因表达差异极小。在品种水平的分析中，我们分别在攻毒与未攻毒状态下比较来航鸡与法尤姆鸡的转录组，发现在两种状态下，法尤姆鸡体内均有多个免疫相关基因及正向共表达顺式作用lncRNAs（cis lncRNAs）呈现上调表达。
讨论：本研究证实，相较于法尤姆鸡，对NDV更为易感的来航鸡，在攻毒后以及感染进程的不同时间点，体内均存在多个免疫相关基因及正向共表达顺式作用lncRNAs的上调现象。而品种水平的分析则显示，法尤姆鸡体内的上调免疫基因及正向顺式作用lncRNAs数量多于来航鸡。该结果清晰表明，来航鸡与法尤姆鸡之间，免疫相关基因本体（GO）条目及通路注释的基因（即免疫相关基因）与共表达顺式作用lncRNAs的表达差异，与其对NDV的抗性差异密切相关。
结论：上述免疫基因及顺式作用lncRNAs可能是法尤姆鸡相较于来航鸡对NDV抗性更强的关键因素。本研究阐明了lncRNAs在宿主抵御NDV感染过程中的重要作用，为后续解析鸡品种遗传改良的调控机制奠定了研究基础。