JIA-2019-11

2590 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 would cause the atrophy of immune organs (thymus and bursa of fabricius) leading to immuno-suppression (Witter et al . 1981). REV damages T lymphocytes in the thymus and spleen, causing reduced secretion of interleukin (IL) 2 (Hrdlicková et al . 1994). This decrease leads to a reduction in the number of T cells, and causes serious immune dysfunction when secreting the pro-inflammatory cytokines influence the differentiation of T cells into helper T cell and cytotoxic T cells, which have different functions (Kim et al . 2004). Nucleotide-binding oligomerization domain 1 (NOD1), a newly discovered protein receptor, plays an important role in the innate immunity of a host against pathogen infection by promoting the expression of genes related to cytokines, and by influencing the release of inflammatory factors (Kim et al . 2004). In the NOD1 signaling pathway, receptor-interacting protein 2 (RIP2) is an important mediator of the secretion of inflammatory cytokines (Yan and Liu 2017). Tumor necrosis factor receptor-associated factor 5 (TRAF5) interacts with RIP2 (McCarthy et al . 1998), implicating the involvement of TRAF5 in the regulation of the NOD1 signaling pathway in the immune response, and reducing the secretion levels of pro-inflammatory cytokines (such as IL8 and IL18) (Bi et al . 2018). On the other hand, long non-coding RNA (lncRNA), defined non-coding RNA with a length of over 200 nt, is essential to the growth and differentiation of cells (Dey et al . 2014), the development of tumors (Hu et al . 2017), and other functions. LncRNA inhibits or promotes the expression of target genes (Feng et al . 2006; Ponting et al . 2009), specifically at the transcriptional level, post-transcriptional level, and epigenetic level (Chen et al . 2010; Bai et al . 2015). LncRNA is involved in regulating various immune diseases (Li et al . 2014; Wang et al . 2015; Zhang et al . 2016), and previous studies reported the pathogenesis of REV from genome, transcriptome, and proteome (Miao et al . 2015; Li et al . 2016; Xue et al . 2017). However, there is scant knowledge of the regulatory mechanism of lncRNA in the immune response to REV in chickens. In this study, the lymphocytes from chicken blood cultured in vitro were used, RNA sequencing was performed, novel lncRNAs related to the NOD1 pathway were identified, and the regulatory mechanism of lncRNA to the NOD1 pathway in the immune response of chicken lymphocytes after REV infection was clarified. The data will provide a new view for the understanding of the resistance mechanism to REV in chicken blood lymphocytes. 2. Materials and methods 2.1. Cells and REV infection Using citric acid dextrose as an anticoagulant, the blood was collected from the wing vein of 21-day-old, specific pathogen-free, Rugao chickens purchased from the Poultry Institute, ChineseAcademy of Agricultural Sciences (Yangzhou, Jiangsu, China). According to themanufacturer’s instructions, the lymphocytes were derived using a Chicken Peripheral Blood Lymphocyte Separator Kit (Code number: LTS1090C, Haoyang Co., Ltd., Tianjin, China). The cells (1×10 5 mL –1 ) were divided into two groups, and incubated with RPMI-1640 medium containing 10% fetal bovine serum for 24 h. One group was infected (5×10 5 TCID 50 /0.1 mL, multiplicity of infection of 10) with 200 μLREV strain HA1101 (GenBank accession number: KF305089.1, obtained from the Key Laboratory of Jiangsu Preventive Veterinary Medicine, China). The other group was the uninfected control. Cells were harvested after 36 h for subsequent analysis. 2.2. Preparation of chicken model of REV infection Considering the death from virus injections, 200 SPF Rugao pure line chickens were purchased from the Poultry Institute, Chinese Academy of Agricultural Sciences, and randomly distributed at birth into the infected and control groups. For the REV infection model, the aforementioned REV strain was used to infect chickens (5×10 5 TCID 50 /0.1 mL) by intramuscular injection. Uninfected control birds received the same volume of RPMI-1640 medium. Birds were raised in an environmentally controlled room. Feed and water were provided ad libitum during the experiment. Diets were formulated as per existing recommendations (Nutrient Requirements of Yellow-feathered Broiler, NY/T 33-2004, China). Two weeks later, 10 chickens with similar weights were chosen from the surviving chickens in each group, and the blood was collected from the wing vein to derive the blood lymphocytes for subsequent experiments. All animal experiments were approved and guided by the Animal Care and Use Committee of Yangzhou University. All experimental procedures were performed in accordance with the Administration Act of Experimental Animals Use and Care in Jiangsu Province (No. 115th Jiangsu Province Government Notice in 2008). 2.3. Extraction of genomicDNAandPCRamplification Genomic DNA was isolated using the Blood Genome DNA Extraction Kit (BioTeke Co., Ltd., Beijing, China). A targeted fragment of the long terminal repeat (LTR) of REV (GenBank accession no. S70398) was amplified by PCR. Primers are listed in Table 1. For each PCR procedure, 100 ng of genomic DNA was used as template in the presence of 1.5 mmol L –1 MgCl 2 , 200 mmol L –1 dNTP, 2.5 U Taq polymerase, and 50 pmol of each forward and reverse