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To explore changes of protein-coding genes and non-coding transcripts, as well as their interactions upon MDV infection in chickens, transcriptomic sequencing was conducted in eight RNA samples extracted from chicken CD4+ T cells isolated from individuals of F₀ generation before and after Marek’s disease viral infection. F0 generation chickens include two highly inbred parental lines 6₃ and 7₂. All chicken lines were maintained at the Avian Disease and Oncology Laboratory (ADOL) (Bacon et al., 2000).

A total of 274 candidate lincRNAs were identified from chickens of the two parental lines 6₃ and 7₂, using the data analysis pipeline as previously described (He et al., 2015). Differentially expressed lincRNAs were captured between infected and non-infected chickens by a p-value less than 0.2 ( Table 1 and Supplementary Sheet 1 ). The 16 differentially expressed lincRNAs were selected to confirm their expression in CD4+ T cells by qPCR, and results for 94% of lincRNAs analyzed were consistent with RNA-seq results ( Figure S1 ).

Linc-GALMD1 was found to be significantly differentially expressed between infected and non-infected chickens for MD resistant line 6₃, but no expression difference was found for susceptible line 7₂ ( Figure 1 ). To explore possible roles for linc-GALMD1 in chicken Marek’s disease, loss-of-function experiments of linc-GALMD1 were performed in the MSB1 cell line that is a commercial MDV-transformed CD4+ T-cell line derived from lymphomas of chickens with MD. Primarily, the structure of linc-GALMD1 was detected in CD4+ T cells and MSB1 cells by ordinary PCR with the primer pairs designed spanning over two exons of linc-GALMD1. PCR cloning and sequencing were conducted and further indicated that linc-GALMD1 exists in both CD4+ T cells and MSB1 cells ( Figure 2A ).

To perform loss-of-function experiments of linc-GALMD1, we generated five lentiviral-based short hairpin RNAs (shRNAs) targeting linc-GALMD1, including one negative control shRNA in MSB1 cells. Figure 2B shows that shRNA2 and shRNA4 successfully targeted linc-GALMD1 and significantly reduced its expression compared to negative control in MSB1 cells (p-value < 0.01, t-test). The best shRNA, shRNA2, was selected for final knockdown experiments and further RNA sequencing in MSB1 cells.

Following the linc-GALMD1 knockdown in MSB1 cells, RNA sequencing results demonstrated that linc-GALMD1 significantly affected expression of 290 genes (|log2fold change| ≥ 1 and FDR ≤ 0.01) ( Figure 2C ), which are distributed on all chromosomes with only 3% of differentially expressed genes located on chromosome 11 that contains linc-GALMD1 ( Figure 3A ). This suggests that linc-GALMD1 is more likely to affect gene expression in trans. To examine whether linc-GALMD1 could affect gene expression in cis, 10 neighboring genes upstream and another 10 downstream of linc-GALMD1 were analyzed to determine their expression change upon the linc-GALMD1 knockdown and MDV infection, respectively ( Figure 3B ). The results indicated that very few genes were up-regulated after the linc-GALMD1 knockdown. However, their expression was significantly changed after MDV infection in line 6₃ and 7₂. Also, some genes had distinct reactions to MDV infection in resistant line 6₃ and susceptible line 7₂. Consequently, we hold the view that the effects of MDV infection on these 20 neighboring genes are far greater than the effects of linc-GALMD1.

In our previous study, we identified enhancers in MSB1 cells by deoxyribonuclease sequencing (He et al., 2014). An enhancer at 243 kb upstream of linc-GALMD1 and another one at 2 Mb downstream were captured ( Figure S2 ). Furthermore, the conservation of linc-GALMD1 and neighboring enhancers was investigated. Linc-GALMD1 and the upstream enhancer were not found in the human and mouse, but the downstream enhancer, next to AKTIP gene, was found across the chicken, human, and mouse, which provided some clues for decoding the chicken genome to explore regulatory elements involved in molecular mechanisms of MD.

To further characterize differentially expressed genes (DEGs) induced by MDV and linc-GALMD1, respectively, differential expression of genes was analyzed in CD4+ T cells between MDV-infected and non-infected chickens in line 6₃ and 7₂, and before and after the linc-GALMD1 knockdown in MSB1 cells as well. A total of 116 DEGs were found between infected- and non-infected chickens for line 6₃, in which 71 genes were expressed higher in non-infected chickens than infected, namely, up-regulated genes; and the remaining 45 genes were down-regulated after MDV infection. For line 7₂, 105 DEGs were found, and 68 genes were up-regulated and 37 were down-regulated after MDV infection (|log2fold change| ≥ 1 and FDR ≤ 0.1, Figure 4A and Figure S3 ). Gene Ontology (GO) analysis of these genes demonstrated that most genes are involved in nucleic acid binding and nucleotide-binding processes. In addition, a portion of genes participates in immune activities. Interestingly, most down-regulated genes that were highly expressed in infected chickens actively participate in viral reproduction, immune response, cell proliferation, cell killing, and cell death compared to up-regulated genes for both of line 6₃ and 7₂, which indicates that the immune systems of infected chickens were activated to defeat MDV infection ( Figure S4 ). Ten genes were differentially expressed in both lines 6₃ and 7₂ ( Figure 4B ), in which two genes had distinct expression directions in the two chicken lines, seven genes were down-regulated, and one gene was up-regulated for both lines. These two line-specific genes are IGLL1 (ENSGALG00000021139) and 5.8S rRNA (ENSGALG00000025656). IGLL1 gene was lowly expressed in infected chickens for resistant line 6₃, but it was highly expressed in infected chickens for susceptible line 7₂, which implies that IGLL1 could be a susceptible gene or a marker for clinical diagnosis of MD.

As mentioned above, the linc-GALMD1 knockdown significantly affected the expression of 290 genes ( Figure 2C ), in which 64 genes were up-regulated and 226 genes were down-regulated after the linc-GALMD1 knockdown. By GO analysis of these DEGs, we found that these genes were very active in extracellular and intracellular regions and they participate in macromolecular complex binding and transmembrane transporter activities, which hinted that this lincRNA could play roles in the cell nucleus, cell cytoplasm, and intercellular regions to support transporter activities. It is worth noting that down-regulated genes are preferentially involved more in immune response, cell motion, cell death, leukocyte activation and migration, as well as lymphocyte costimulation than up-regulated genes ( Figure 5 ), which suggested that the immune system became less active after the linc-GALMD1 knockdown in MSB1 cells, which is different from responses of the immune system to MDV infection that is the immune system may be activated when a chicken is infected by MDV.

Based on integrated analysis of RNA sequencing upon MDV infection and upon the linc-GALMD1 knockdown, only three genes were found to be differentially expressed after linc-GALMD1 knockdown in MSB1 cells, and after MDV infection in CD4+T cells for line 6₃ ( Figure 4C ). The results indicated that their expression was significantly decreased after the linc-GALMD1 knockdown. In addition, their expression was lower in infected chickens compared to non-infected chickens for resistant line 6₃, but no significant expression difference was found for line 7₂, which further illustrated that linc-GALMD1 could play roles during MDV infection for chickens.

To determine whether MDV could interact with linc-GALMD1, MDV infection was performed in CEF cells with and without linc-GALMD1 knockdown by shRNA2 and shRNA4, respectively. It was known that MDV induces rapid-onset T-cell lymphomas in chickens and that T-cell transformation requires the expression of a viral protein called Meq, whose changes in the coding sequence of Meq correlated with increased virulence (Kumar et al., 2012). Figure 6 demonstrates that linc-GALMD1 was significantly knocked down by shRNA2 and shRNA4 compared to a negative control shRNA upon MDV infection ( Figure 6A ). Meanwhile, Meq copy numbers increased after the linc-GALMD1 knockdown compared to the condition with MDV infection and a negative control shRNA. Meq copy numbers increased more when linc-GALMD1 was knocked down more by shRNA4 than by shRNA2 (p-value ≤ 0.01, Figure 6B ). These results indicate that linc-GALMD1 could repress MD virus progression during MDV infection in chickens.

Line 6₃ and line 7₂, which are known as MD-resistant and MD-susceptible lines, respectively (USDA-ARS, Avian Disease and Oncology Laboratory, East Lansing, Michigan, USA), were used in this study. The animal challenge trials were carried out in accordance with guidelines established and approved by the USDA, ADOL Animal Care and Use Committee (ACUC) (April, 2005), and the Guide for the Care and Use of Laboratory Animals by Institute for Laboratory Animal Research (2011). Chickens from each of the lines/crosses were divided into two groups, one was challenged with a very virulent plus (vv+) strain of MDV (648A passage 40) with a dosage of 500 plaque-forming units (PFU) per bird intra-abdominally in the fifth day post-hatch, the other was maintained as uninfected control. Two chickens were sampled from the MDV challenged groups and two from the control group for each line and each cross. Peripheral blood of the chickens was collected at 21-days post-infection, and peripheral blood mononuclear cells (PBMC) were separated from anti-coagulated peripheral blood by standard preparation method. After removing clumps (if any) and dead cells, PBMC were incubated with Mouse Anti-Chicken CD4-PE (Cat. No. 8210-09, Southern Biotech) at room temperature for 15 min, and then the CD4+ T cells were finally isolated by EasySep^® Positive PE Selection Kit (Cat. No. 18557, STEMCELL Technologies) following the manufacturer’s instructions. The cell number and purity were quantified by flow cytometric analysis. Only those cell samples with purity greater than 90% were used for subsequent analyses. Highly pure CD4+ T cell samples were flash-frozen in liquid nitrogen immediately and then stored at −80°C until RNA extraction.

A total of eight RNA samples were extracted from chicken CD4+ T cells using Direct-zol™ RNA Miniprep Kit (Cat. No. R2050, Zymo Research), and mRNA isolation was performed by Oligotex mRNA Mini Kit (Cat. No. 70022, QIAGEN). Then mRNA was used to synthesize the first and the second strand cDNA using SuperScript™ III Reverse Transcriptase (Cat. No. 18080-093, Invitrogen) and NEBNext^® mRNA Second Strand Synthesis Module (Cat. No. E6111S, NEB). After purification, the double-stranded cDNA (dscDNA) was fragmented into ∼300 bp. Then the library for sequencing on the Illumina HiSeq 2000 Analyzer was constructed as previously described (He et al., 2015). Finally, cluster generation and sequencing analysis were performed on the Illumina Hiseq 2000 following the manufacturer’s protocol.

After sequencing quality test by FastQC, the first 6 bp and the last 4 bp for all reads were trimmed off, and then all trimmed reads with 40 bp of length for each sample were mapped to the chicken genome (galGal3) individually by Bowtie 1.2.0. The numbers of reads that fell in each gene were counted by htseq-count, and DEGs between infected and non-infected chickens were analyzed by edgeR with the criteria of |log2fold change| ≥ 1 and FDR ≤ 0.01.

Transcriptomic sequencing reads for each sample were mapped to the chicken genome (galGal3) individually using TopHat 2.0.6 and assembled individually with cufflinks. Putative lincRNAs were identified with our previous pipeline in chickens (He et al., 2015). The expression levels of all protein-coding genes and lincRNAs were estimated using cufflinks and they were represented with FPKM (Fragments Per Kilobase of transcript per Million mapped reads). To obtain the expression patterns of lincRNAs and protein-coding genes, log2 fold changes between infected and non-infected chickens were calculated for each chicken line. Those lincRNAs and protein-coding genes with |log2fold change| ≥ 1 and FDR ≤ 0.1 were selected for substantial analysis. The conservation of non-coding RNAs and enhancers was investigated with the VISTA Enhancer Browser (https://enhancer.lbl.gov).

The protocols of mRNA extraction and dsDNA synthesis were the same as those mentioned above. Real-time PCR using SYBR Green PCR Kit was utilized to validate differentially expressed lincRNAs and genes in CD4+ T cells between infected and non-infected chickens with iCycler iQ PCR System (Bio-Rad). The annealing temperature was set at 60°C. The primer pairs were designed within exons and the length of the amplicons was between 50 and 200 bp. All primers were designed using Primer3 (http://fokker.wi.mit.edu/primer3/input.htm), and the detailed information is listed in Table S1 . qPCR reaction was run with triplicate using the program as follows: pre-incubation (95°C for 10 min), 40 cycles of amplification (95°C for 10 s, 60°C for 10 s, and 72°C for 10 s), melting curves using a heat ramp and cool down. Cycle threshold values (Ct values) were obtained from iCycler iQ PCR software. The expression levels of lincRNAs and genes were normalized against GAPDH complementary DNA in the corresponding samples. The relative fold enrichment of each treatment group was calculated by comparing the enrichment value for the given primer pair to GAPDH. The copy numbers of the Meq gene were calculated against PCCA as the reference gene (Wang et al., 2010). Data were analyzed using a two-tailed Student’s t-test. The differences were considered to be statistically significant at p-value < 0.05.

To confirm the structure of linc-GALMD1 in CD4+ T cells and MSB1 cells, the primer pairs were designed to span over two exons of linc-GALMD1 and the length of the amplicons was between 80 and 500 bp ( Table S2 ). Touchdown-PCR was performed to amplify linc-GALMD1 with conventional PCR, and chicken genomic DNA was used as control using the following reaction: 5 µl of GoTaq^® Hot Start Green Master Mix (Promega, USA), 1 µl of primer mix (10 µM), 1 µl of cDNA, and 3 µl of UltraPure^® Distilled Water (Invitrogen, USA) to a total volume of 10 µl. The optimal PCR program was 94°C for 5 min, 3 cycles of amplification (94°C for 1 min, 68°C for 1 min, and 72°C for 2 min), 3 cycles of amplification [94°C for 1 min, (68-3i)°C (i = 1 to 5) for 1 min, and 72°C for 2 min], 30 cycles of amplification (94°C for 1 min, 50°C for 1 min, and 72°C for 2 min), and a final extension step 72°C for 10 min. PCR products were run on 1.5% TBE-buffered agarose gel at 90 V for 1 h, and 230 bp fragments were excised from the gel to purify by QIAquick Gel Extraction Kit (QIAGEN, USA).

To confirm that sequences of linc-GALMD1 existed in CD4+ T cells and MSB1 cells, cloning sequencing of PCR products was conducted. The details were as follows. The purified PCR products were ligated into pGEM-T Vector (pGEM-T Vector System I, Promega, USA), transformed into DH5α competent cells (Z-Competent E. Coli Cells—Strain Zymo 5α, ZYMO Research, USA), and screened for successful insertions (blue-white selection) after incubation at 37°C overnight. In the next step, five white colonies from each sample were cultured overnight in a 37°C shaker. Plasmid DNA was isolated using Zyppy Plasmid Miniprep Kit (ZYMO Research, USA). M13 reverse primer and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) was employed for sequencing in the ABI 3730 machine as described by the manufacturer.

The MDV-transformed lymphoblastoid MSB-1 cells were obtained from Dr. C. Itakura’s lab, Department of Veterinary Pathology, Tottori University, Tottora, Japan and grown at 37°C in 5% CO₂ in RPMI 1640 medium containing 10% fetal bovine serum. The chicken embryo fibroblast (CEF) cells were isolated from fertile eggs at day 10 and grown at 37°C in 5% CO₂ in M199 tissue culture medium supplemented with 10% FBS. All reagents for cell culture were purchased from Life Technologies (CA, USA).

The shRNAs were designed and hypothesized by GenePharma Biotech (Shanghai, China). Four shRNAs for interfering linc-GALMD1 and a negative control (NC) were cloned into a shuttle vector (LV3-pGLV-h1-GFP-puro) labeled by GFP (green fluorescent protein) and incorporated into a lentiviral vector, respectively. The sequences of shRNAs were listed in Table S3 .

MDCC-MSB1 cells were plated at a density of 5×10⁵ cells per well in 24-well plates. Cells were infected with 40 µl of a lentiviral shRNA stock (2×10⁸ TU/ml) and incubated at 37°C for 72 h until green fluorescence was observed by fluorescence microscope. The total RNA extraction and qRT-PCR were performed as described above.

CEF cells were plated at a density of 4×10⁵ cells per well in 24-well plates. They were co-infected with 2000 PFU MDV (CVI988, Beijing Lingyu Biological Technology Co., Ltd.) and 40 µl of lentiviral shRNA2 or shRNA4 plus NC (2×10⁸ TU/ml). The cell lesion and green fluorescence were observed at 96 h after infection, and then cells were harvested for isolating the total RNAs and DNA.

MDCC-MSB1 cells were plated in 24-well plates, and cells were infected with a lentiviral shRNA2 and an empty lentiviral vector as well as a NC in triplicates. After cells were harvested, the total RNAs were extracted and dscDNA was produced as described above. Then the library for sequencing on the Illumina HiSeq 2500 Platform was generated using the NEBNext^® Ultra^™ Directional RNA Library Prep Kit for Illumina^® (Cat. No. E7420L, NEB) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Products were purified by QIAquick PCR Purification Kit (Cat No. 28106, QIAGEN), and library quality was assessed on Qubit^® Fluorometer by Qubit^® dsDNA HS Assay Kit (Cat. No. Q32851, Thermo Fisher). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Cat. No. PE-401-3001, Illumina) following the manufacturer’s protocol. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2000 V4 PE125 platform and paired-end reads were generated.

After filtering out paired reads with adaptors and dirty reads, all clean reads for each sample were mapped to the chicken genome (galGal4) individually by Tophat v2.0.9, and the average concordant pair alignment rate was 70%. Mapped transcripts were assembled individually with cufflinks and then transcripts from all samples were merged together with cuffmerge to build a consensus set of transcripts across samples. DEGs between samples with linc-GALMD1 knockdown and with negative lentiviral vector were screened by cuffdiff based on cuffmerge results. Gene expression levels were represented with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and log2 transformation was used for normalization. To obtain gene expression patterns, log2 fold change before and after the linc-GALMD1 knockdown in MSB1 cells was calculated. Those genes with |log2fold change| ≥ 1 and FDR ≤ 0.01 were selected for clustering of expression profiles based on hierarchal clustering with complete linkage and visualized using heatmaps.