De novo detection of ChERVs in the chicken genome
Two different pipelines were employed for mining ChERVs in the chicken genome 5.0 (Fig. 1). Firstly, ChERVs were detected by LTRharvest and annotated with LTRdigest. A total of 17,353 pairs of putative LTRs in the chicken genome were identified with LTRharvest. These putative LTR pairs were filtered down to 337 ERV candidates by LTRdigest. Additionally, 99 unique ERV candidates were revealed by MGEScan. After merging the ERV candidates identified by the two pipelines, we identified a total of 436 ChERV candidates in the chicken genome (Fig. 1A, Additional file 2: Table S1). The full lengths of these 436 ChERV candidates range from 2,555 to 21,180 bp, and their LTR lengths vary from 104 to 1781 bp.
To classify the detected ChERVs, the RT domain sequences were used to build a multiple alignment and compute phylogenetic trees using the neighbor-joining method implemented in MEGA 6. Among the 436 ChERVs identified in this study, 384 ChERVs have a RT domain conserved enough to be aligned confidently for phylogenetic analysis. According to the phylogenetic tree analysis, ChERVs were classed into three groups and 15 families, denoted group I to III and families GGERV1 to 15 (Fig. 1B and Additional file 3: Table S2). Most of the ChERVs (247) included in the phylogenetic analysis belong to the group III and were classified into eight families including GGERV11 to GGERV15 (Fig. 1B). Group II ChERV contains the most ERV families ranging from GGERV 3 to GGERV10 comprised of 126 elements (Fig. 1B). Only 11 ChERVs in the GGERV1 and GGERV2 belong to the group I (Fig. 1B). Additionally, 52 ChERVs were marked as unclassified members (Additional file 3: Table S2).
Identification and analysis of (potentially cis-Target) genes that occur in the neighborhood of ChERVs
Mapping 436 ChERV candidates to chicken chromosomes, we found that a lot of ChERVs are inserted on chromosome 1, 2, 3, 4 and sex chromosome Z, W (Fig. 1C). Especially, ChERVs insertion on the W chromosome is very high (Fig. 1C). Furthermore, to identify host genes that may be regulated by ChERVs, we analyzed the positional relationship between ChERVs and their neighboring genes. A total of 414 encoding genes, 223 long non-coding RNA (lncRNA) and 60 non-coding RNA (ncRNA) such as miRNA and snRNA were identified within 100 kb of upstream and downstream of these 436 ChERVs (Additional file 4: Table S3).
The 414 encoding genes were identified as potential cis-target genes of 202 ChERVs. GO and KEGG were employed to further explore the potential functions of these genes. GO biological process analysis showed that most of the candidate cis-target genes were enriched in “cellular process”, “developmental process”, “single-organism process”, “immune system process”, “response to stimulus”, “metabolic process”, and etc (Fig. 1D, Additional file 5: Table S4). KEGG analysis illustrated mainly enrichment in “Ubiquinone and other terpenoid-quinone biosynthesis”, “Focal adhesion”, “Ubiquitin mediated proteolysis”, “FoxO signaling pathway”, “VEGF signaling pathway”, and “RIG-I-like receptor signaling pathway” (Fig. 1E, Additional file 5: Table S4). Based on the analysis of GO and KEGG, 19 immune-related genes such as TRAF6, IL7R, TRAF3IP1, MAPK12 were identified within 100 kb of upstream and downstream of 17 ChERVs (Additional file 6: Fig. S1, Additional file 5: Table S4).
Transcriptome Analysis of ChERVs in ALV-J, AIV, MDV and APEC infection
To obtain global view of ChERVs expression after pathogenic microbial infection in chicken, four published transcriptome raw data about ALV-J, AIV, MDV and APEC infection were used to match Gallus_gallus-5.0 genome for deep analysis [23–26]. The analysis resulted in the identification of 258 (59.4%) ChERVs that were transcribed in ALV-J-infected spleens or their controls (Fig. 2A, Additional file 7: Table S5). Compared to uninfected sample, the expression levels of 15 ChERVs were significantly up-regulated and 34 ChERVs were significantly down-regulated in ALV-J-infected spleen (Fig. 2B, Additional file 8: Table S6). 299 (68.6%) ChERVs were detected to be transcribed in H5N1-infected chicken ileum samples and PBS-infected ileum samples at 1 dpi (Fig. 2C, Additional file 7: Table S5). Scatter plot showed that only 3 ChERVs were significantly up-regulated and 15 ChERVs were significantly down-regulated in H5N1-infected chicken ileum samples (Fig. 2D, Additional file 8: Table S6). The transcriptional profiling of ChERVs in MDV-infected spleen and uninfected spleen at 14 dpi showed that 240 (55.0%) ChERVs were transcribed (Fig. 2E, Additional file 7: S5). Of these 240 ChERVs, 23 ChERVs were significantly up-regulated and 43 ChERVs were significantly down-regulated in MDV-infected spleen samples (Fig. 2F, Additional file 8: Table S6). 219 (50.2%) ChERVs were detected to be transcribed in APEC-infected bursa samples and uninfected samples at 5 dpi (Fig. 2G, Additional file 7: Table S5). Further analysis showed that the expression of 15 ChERVs were significantly up-regulated and 2 ChERVs were significantly down-regulated in APEC-infected bursa (Fig. 2H, Additional file 8: Table S6). From the results of ChERV transcriptomes analysis in the present study, the expression levels of most of the ChERVs are very low (Fig. 2).
For the differentially expressed ChERVs, the venn diagram showed that only one common ChERV was in the four ERV transcriptomes analysis after ALV-J, AIV, MDV and APEC infection (Fig. 3A). This common ERV was numbered ChERV-3 in this study. Coincidentally, heat map showed that ChERV3 was significantly down-regulated after ALV-J, AIV, MDV and APEC infection (Fig. 3B). Furthermore, the predicted ChERV-3 sequence was used to perform phylogenetic analysis with the verified chicken endogenous retroviruses including ev1, ev3, SD0501, EAV-HP, ALVE-B9/10/11, MLV-related ChERV and Ovex1. Phylogenetic tree showed that ChERV-3 was closely related to ev1, ev3, SD0501and ALVE-B9/10/11 (Fig. 3C). By comparing with the sequences of these known chicken endogenous retroviruses, we further determined the exact information of ChERV-3 (Fig. 3D). ChERV-3 has a complete retroviral structure with a length of 7,524 bp and is located on chromosome 1 (Fig. 3D).
The expression of ChERV-3 envelope protein was inhibited after ALV-J infection
To verify whether ChERV-3 expression was inhibited after infection with pathogenic microorganisms, ALV-J was selected for further in vitro experiments. Figure 4A showed that the endogenous expression of ChERV-3 env gene was significantly inhibited after ALV-J infection at 24 and 48 hpi. Furthermore, ChERV-3 env gene was synthesized and transfected into DF1 cells. After 24 h post transfection (hpt), DF1 cells were infected by ALV-J. Compared to the uninfected cells, the qPCR result showed that the exogenous ChERV-3 env expression was significantly inhibited by ALV-J infection at 24 hpi (48 hpt) and 48 hpi (72 hpt) (Fig. 4B). Furthermore, at the protein level, the exogenous expression of ChERV-3 envelope protein was also suppressed after ALV-J infection at the 24 hpi (48 hpt) and 48 hpi (72 hpt) (Fig. 4C).
ChERV-3 Envelope Protein Has No Effect On ALV-J Production
Given that the expression of ChERV-3 envelope protein is inhibited by ALV-J infection, we speculate whether the envelope protein of ChERV-3 affects ALV-J replication. Compared to the empty vector control, transfection of ChERV-3 env did not affect ALV-J replication in DF1 cells at 24 and 48 hpi (Fig. 5A, B). However, the expression levels of all the ISGs detected in our study were significantly up-regulated in the ChERV-3 env transfected DF1 cells compared with the empty vector transfected group at 24 hpt (Fig. 5C).