An EAV-HP insertion in the promoter region of SLCO1B3 has pleiotropic effects on chicken liver metabolism based on the transcriptome and proteome analysis

Solute carrier organic anion transporter 1B3 (SLCO1B3) is an important liver primarily highly expressed gene, its encoded protein (OATP1B3) involved in the transport of multi-specific endogenous and exogenous substances. We previously reported that an EAV-HP inserted mutation (IM+) in the 5ʹ flanking region of SLCO1B3 was the causative mutation of chicken blue eggs, and a further research showed that IM+ significantly reduced the expression of SLCO1B3 in liver. Herein, we confirmed a cholate response element (IR-1) played an important role in activating SLCO1B3 and in vitro experiments showed that the activation of IR-1 can be significantly reduced by the EAV-HP IM+ . We performed transcriptome and proteomic analysis using the same set of IM+ and IM− liver tissues from Yimeng hens (a Chinese indigenous breed) to study the effect of SLCO1B3 and OATP1B3 expression reduction on chicken liver function. The results showed that common differential expression pathways were screened out from both transcriptome and proteome, in which fatty acid metabolism and drug metabolism—cytochrome P450 were significantly enriched in the KEGG analysis. The lipid-related metabolism was weakened in IM+ group, which was validated by serum biochemical assay. We unexpectedly found that EAV-HP fragment was highly expressed in the liver of the IM+ chickens. We cloned the EAV-HP full-length transcript and obtained the complete open reading frame. It is worth noting that there was some immune related differential expressed genes, such as NFKBIZ, NFKBIA, and IL1RL1, which were higher expressed in the IM+ group, which may due to the high expression of EAV-HP. Our study showed that EAV-HP IM+ reduced the expression of SLCO1B3 in liver, resulting in the decrease of fatty metabolism and exogenous substance transport capacity. The mutation itself also expressed in the liver and may be involved in the immune process. The mechanism needs further study.

related to Rotor syndrome and hyperbilirubinemia, which blocks bile salts from the blood from being transported into the liver [11][12][13][14] . Furthermore, OATP1B3 also plays an important role in xenobiotic metabolism, which includes drugs and toxins 15,16 . The substrates transported by OATP1B3 are mostly ligands for nuclear receptors, such as the farnesoid X receptor (FXR), pregnenolone X receptor (PXR), and aryl hydrocarbon receptor (AHR) [17][18][19][20] , suggesting that OATP1B3 in the liver could regulate gene expression by xenobiotic-activated transcription factors. As an important endogenous and exogenous transporter protein, chicken OATP1B3 in the liver is also considered to play an important role during metabolism.
In a previous study, we have cloned a partial endogenous retroviral (EAV-HP) insertion in the 5ʹ flanking region of the SLCO1B3, this insertion promoted the ectopic high expression of SLCO1B3 in shell-gland, and it was found to cause chickens to have blue eggshells 21 . A post-gene mapping study conducted genetic investigation of the SLCO1B3 [22][23][24] ; however, its function in chickens has not been widely investigated. Recently, an expression profile of SLCO1B3 was outlined in different tissues between the EAV-HP inserted mutation (IM+) and wild type (IM−) hens, and it indicated that chicken SLCO1B3 also mainly specific expressed in the liver and its expression was significantly decreased in the livers of IM+ chickens 25 . This means that IM+ chickens could be used as an animal model to investigate the function of SLCO1B3 in liver.
Transcriptome and proteome analysis techniques in recent years have advanced to enable differentially expressed genes and proteins in the same tissues to be profiled. Here, to explore the possible effects of the decreased SLCO1B3 expression on the expression of other genes and associated biological pathways in the IM+ Yimeng chickens, we have collected chicken livers and performed RNA-seq and utilized tandem mass tag (TMT) techniques, to analyze their transcriptome and proteome profiles. We are trying to reveal that the inserted mutation of SLCO1B3 leads to the different expression regulation patterns in shell-gland and liver, and also affects its function in these two tissues.

Results
Identification of the IR-1 like element in the upstream of SLCO1B3 in vitro. In order to identify the upstream regulatory element that activate chicken SLCO1B3 expression, we constructed luciferase vectors with different length of the 5′ region of SLCO1B3 to do dual luciferase assays in chicken liver hepatocellular carcinoma (LMH) cells (Fig. 1A). LMH cells have been extensively used as model for ligand-dependent activation of endogenously expressed nuclear receptors 17,26 . We found that in the absence of activator, the region covering ~ 2 Kb upstream of the SLCO1B3 showed no obvious activation compared with pGL3-Basic vector (Fig. 1B). It has been reported that SLCO1B3 can be regulated by bile acids in human 17 . We speculate that the regulation of SLCO1B3 expression also depends on bile acids stimulation. Then, we used different concentrations of chenodeoxycholic acid (CDCA, one of the bile acids) to culture LMH cells. After incubating 36 h with CDCA, SLCO1B3 expression was detected using quantitative real-time PCR (qPCR). The expression of SLCO1B3 increased with the increase of CDCA content, and it did not increase after 50uM concentration (Fig. 1C).
The luciferase assays showed that − 202 ~ + 7 bp fragment vector had the highest activities with 50 μM CDCA stimulation (Fig. 1D). In this region, we predicted an inverted hexanucleotide repeat motif (IR-1) like element 25 , which can be stimulated by bile acids. When we mutant the IR-1 like element (Fig. 1E), the expression of dual luciferase report gene decreased (Fig. 1F), the same thing happens when we add partial EAV-HP insertion upstream to the IR-1 like element. Though the dual luciferase experiment, we verified the existence of IR-1 like element in the upstream of SLCO1B3 as activator, and the EAV-HP insertion reduced the transcriptional activation of IR-1 like (Fig. 1F).
Differential expression of the genes screened from RNA-seq and the proteome. Six liver tissue cDNA libraries were established (n = 3 IM+ and n = 3 IM− hens), which represented the case control samples with or without the EAV-HP insertion in the promoter of SLCO1B3. The RNA-seq generated from 4.25 Gb to 5.78 Gb of clean reads for each library, with an average of 5.24 and 4.70 Gb of paired-end reads for the IM+ and IM− groups, respectively. The clean reads were used for all further analyses. Of the reads in each library, 87.3% to 92.2% were uniquely mapped to the chicken reference genome, and the average mapping rates were 89.45% and 89.33%, for the IM+ and IM− groups, respectively (Table 1).
After the assembly, 12,802 genes were identified using the RNA-seq analysis, of which 989 were not annotated. We used the annotated genes to select the differentially expressed genes (DEGs). A total of 142 DEGs were selected, using the criteria of a fold change ≥ 2 and a false discovery rate (P adjust) ≤ 0.05, and of these, 67 were upregulated and 75 were downregulated. The details of the DEGs are listed in Table S1.
Proteome analysis was performed using the same samples as for the transcriptome. By comparing the reference genome, we identified a total of 3481 proteins. The criteria for selecting differentially expressed proteins (DEPs) were a fold change ≥ 1.2 and a P value ≤ 0.05. There were 75 DEPs identified, of which 31 were upregulated and 44 were downregulated. Detailed information of the DEPs is listed in Table S2. SLCO1B3 and OATP1B3 expression levels in the RNA-seq and proteome. The chickens used in this study were randomly selected from the same population of Yimeng chickens according to the eggshell color. We previously reported different levels of SLCO1B3 expression in the liver, which were associated with the IM insert mutation in Yimeng chickens 25 . As predicted, RNA-seq analysis revealed that the expression levels of SLCO1B3 were lower in IM+ chickens than in IM− chickens ( Fig. 2A), as were the expression levels of the OATP1B3 protein (Fig. 2B). The difference in the expression of SLCO1B3 between IM+ and IM− chickens was also validated by the qPCR analysis (Fig. S1), the primers for which have been described in a previous study 25  www.nature.com/scientificreports/  www.nature.com/scientificreports/ pared with those in IM− chickens. Herein, we used IM+ Yimeng chickens as the SLCO1B3 knockdown model to perform functional analysis in the liver using a gene enrichment method.
Functional enrichment analysis of the DEGs. The regulation network differences for the SLCO1B3 between the IM+ and IM− hens were investigated. We performed a functional enrichment analysis of the DEGs using DAVAID 6.8 online software. The DEGs were significantly enriched in the pathways, including the tryptophan metabolism, metabolic pathways, drug metabolism-cytochrome P450, Retinol metabolism, Tyrosine metabolisminsulin resistance, and fatty acid metabolism (Fig. 3A). The main GO terms included iron ion binding, cell, response to antibiotic, NAD binding, and oxidative deethylation (Fig. S2A). There were also some DEGs that could be classified according to their gene symbol and function. The liver microsome CYP450 category for the DEGs included CYP2C18, CYP1A1, CYP1A2, and CYP3A4, which play an important role in xenobiotics and drug metabolism. The lipid biosynthesis DEG category included SCD, FASN, ELOVL6, THRSP, and ME1. In addition, some immune related genes, such as NFKBIA, NFKBIZ highly expressed in the IM+ chickens, IL1R1L highly expressed in the IM− chickens. We will try to discuss this phenomenon later.
Comparative analysis of the transcriptome and proteome. In the functional analysis, fatty acid metabolism and drug metabolism-cytochrome P450 were all significantly enriched in the two omics studies. Furthermore, some key genes involved in fatty acid metabolism, such as SCD and FASN in the proteome, had lower expression levels in IM+ chickens than in IM− chickens. The proteome analysis results validated the transcriptome analysis. We noted that some transcript factor genes in the RNA-seq were significantly different between the IM+ and IM− chickens, for example, THRSP, AHR, and IGFBP1, whereas they were not identified in the proteome analysis. This may be because the transcript factor protein content was exceptionally low in the tissues. These transcription factors could be the direct reason for the gene expression regulation of the fatty acids and exogenous substance metabolism.

Differences in serum biochemical indexes between IM + and IM− hens.
To reflect the difference of lipid metabolism between IM+ and IM− chickens, serum biochemical indexes of the two groups were detected. The IM+ and IM− Yimeng hens came from the same herd, the same period, the same feeding conditions. Six serum biochemical indexes including APOA1, APOB, TBA, HDL-C, LDL-C and VLDL-C, were measured to show the difference of lipid metabolism between IM+ and IM− chickens. Five of them showed significant difference between groups. Serum concentrations of HDL C, LDL C, and TBA in the IM+ birds were significantly lower than those in the IM− birds (P ≤ 0.05), and serum APOA1 and APOB concentrations in the IM+ birds were extreme significantly lower than those in the IM− birds (P ≤ 0.01), while no significant difference was detected in VLDL-C (Table S3). www.nature.com/scientificreports/ EAV-HP transcript identified and full-length open reading frame cloned. We noticed that there is one transcript (LOC107051636) significantly differentially expressed between IM+ and IM− chickens in the RNA-seq, the fold change was up to 52 times. After sequence alignment, we found that the transcript had 84.77% similarity to the EAV-HP fragment (GeneBank: JF837512). We then compared the clean reads number of transcriptome sequencing with the EAV-HP sequence, we found that the comparable reads of IM+ group were all more than 10,000 reads, while the reads number in IM-group were very low (Fig. 5A). We speculated that the differential expression of EAV-HP transcript has relationship to the EAV-HP IM+ in the upstream of SLCO1B3.
After comparison in the database, we found that there were 15 specific SNPs between the EAV-HP IM+ and other EAV-HP sequence in the genome (Table S4). Therefore, we calculated the number of reads with the above specific SNPs, the results showed that more than 95% of the mapped reads in each SNP locus in the IM+ individuals contained special SNPs, while no specific SNP was contained in reads of IM− individuals (Table S5). This indicated that the EAV-HP transcript highly expressed in the liver of IM+ chickens come from the EAV-HP insertion in the 5ʹ region of SLCO1B3.
To ensure the EAV-HP transcript transcriptional start sites and orientation, two orientation (left and right, shown in Fig. 5B) 5ʹ and 3ʹ RACE experiments were performed, the results showed that the left RACE did not clone any fragment, while the right direction RACE successfully amplified specific fragments (Fig. 5C), suggesting that the EAV-HP transcription starts in the reverse orientation. The full-length EAV-HP transcript sequence was also cloned, which is a new EAV-HP transcript starting from the R region of the long terminal region (LTR) and ended in another R region of the LTR (Fig. 5D), having a complete open reading frame (Fig. S3).
qPCR verification of DEGs. To confirm the repeatability and accuracy of the RNA-seq gene expression data obtained from the chicken liver libraries, qPCR was carried out on 9 selected DEGs: transporter genes SLC16A1, transcription factor genes (THRSP, IGFBP1, AHR), antibacterial peptide gene (LEAP2), fatty acid synthesis gene (ELOVL6), inflammatory factor gene (NFBKBZ), microsome CYP450 representative gene (CYP2C18), and the EAV-HP transcript were also selected for gene expression validation. The qPCR analysis results matched well with the RNA-seq results ( Table 2), indicating that the RNA-seq analysis results are reliable. The correlation coefficient between the qPCR and RNA-seq was 0.997 (P < 0.001), indicating good reproducibility of the differential expression results obtained by the RNA-seq.

Discussion
In humans, SLCO1B3 is mainly expressed in the liver and its encoded protein OATP1B3, plays an important role in endogenous and exogenous transport processes, which has a closely relationship with bile acids and drug metabolism in liver 3,20 . Our previous research also showed that chicken SLCO1B3 was mainly expressed in liver, and the SLCO1B3 homolog gene SLCO1B1 had exceptionally low expression levels in the liver 25 . This may indicate that in chickens, SLCO1B3 plays more important role in the endogenous and exogenous transport processes in the liver. On one hand, the EAV-HP insertion upstream of SLCO1B3 gene resulted in the chicken blue eggshell phenotype, on the other hand, it also reduced the expression level in liver. In this study, we compared the liver transcriptome and proteome of IM+ and IM− groups. These two groups come from the same population, under the same feeding environment, and have high similarity in genetic and environmental background, which means that the differentially expressed genes and pathways found in the transcriptome and proteome are more related to the variation and down-regulation of SLCO1B3 gene. So here we used the IM+ hens as SLCO1B3 gene knockdown animal models to investigate the function of this gene.
Both transcriptome and proteome analysis confirmed the enrichment of fatty acid metabolism pathway. Comparison with IM− group, the expression level of genes in this pathway was mainly significantly decreased in IM+ group. THRSP can regulate FASN, ACL, and ME1 expression after activation by the thyroid hormone 27 . Estradiol-17β glucuronide and estrone-3-sulfate are the most important estrogens in vertebrate animals, and could be transported by OATP1B3, which may control fatty acid metabolism in livers of laying hens [28][29][30][31] . In conclusion, we speculated that SLCO1B3 could regulate liver fatty acid metabolism by transporting thyroid hormones, estrogens, and bile acids indirectly, which are all ligands of OATP1B3 3,11,32 . Unlike mammals, more than 70% of bird fatty acid synthesis occurs in the liver 33 . The liver fatty acid metabolism was lower in IM+ hens and was also reflected in the serum biochemical parameters. The apolipoprotein cholesterol in the serum could affect the accumulation of nutrients in the egg yolk, especially the cholesterol 34 . The nutrient content of the IM+ Yimeng chicken eggs should be investigated further in future investigations.
It was also noted that the metabolism of xenobiotics and drugs by the cytochrome P450 pathway was enriched in the RAN-Seq and proteome analyses. Transcriptome analysis revealed several DEGs categories, including the liver microsome CYP450s. CYP450s play a very important role in the metabolism of arachidonic acid, prostaglandins, retinol, retinoic acid, and cholecalciferol 35 . Some studies have summarized the function of liver microsome CYP450s in the metabolism of drugs in humans 36 . The nuclear receptors AHR can regulate the expression of the liver microsome CYP450s either continuously or when induced 37,38 , and some ligands, including various endogenous and xenobiotic substrates, can be transported by OATP1B3. OATP1B3 may thus regulate the metabolism of xenobiotics by controlling the activity of CYP450s 18,19,39,40 The results of the present study are consistent with previous reports about the involvement of human SLCO1B3 in the metabolism of xenobiotics in the liver 15,16,41 . From this perspective, the chickens with blue eggshells could function as a liver SLCO1B3 gene knockdown model for drug and toxic metabolism experiments in the future.
Herein, we propose a schematic diagram of the function of the chicken SLCO1B3 gene in the liver tissue (Fig. 6), by combining the enrichment analysis of the RNA-seq and proteome with the gene function of human SLCO1B3. Chicken OATP1B3 protein may regulate the activity of ligands-dependent on transcription factors, to regulate the expression of genes related to fatty acid metabolism and exogenous substance metabolism, by www.nature.com/scientificreports/ controlling the amount that endogenous and exogenous substances are transferred (including bile acids, thyroid hormone, estrogens, etc.) into the liver cells. Although some transcription factors (e. g., ESR and PPAR) were not significantly expressed or detected in the RNA-seq and proteome. www.nature.com/scientificreports/ It was reported that IR-1 is an important element which can regulated the human SLCO1B3 gene expression in the liver 17 . Our LMH cells experiment analysis revealed that there is a predicted IR-1 like element in the 5ʹflanking region of SLCO1B3, which also can be activated by the bile acids. It helped us to understand why EAV-HP insertion decreased the expression of SLCO1B3 in the liver. Furthermore, a chicken specific bile acid activated element IR-1 like has been cloned and validated in this study, it will be helpful to study the expression of cholate responsive genes in the future.
Endogenous retroviruses widely exist in vertebrate genomes. EAV-HP is a subtype of chicken endogenous virus and has been investigated for almost 30 years and is commonly found in avian species 42 . It is widely accepted that Avian Leukosis Virus-J (ALV-J) originated from the recombination of EAV-HP and an ancient ALV 43 . EAV-HP expression occurs during the embryonic period 44 , and with an immune tolerance when hatched meat-type chickens are infected with ALV-J 43 . Since its emergence, ALV-J has caused high levels of economic loss. However, whether the expression of EAV-HP can induce chicken immunity has not been reported. In the study of human endogenous retrovirus, it was found that endogenous retrovirus was related to human innate immune response [45][46][47] , which should arouse our attention to the relationship between endogenous retrovirus and immunity.
It is widely accepted that most endogenous retroviral elements are silenced in the genome, however, there are still some reports showed that some subtypes of retroviruses are expressed in embryonic stage in humans 47 .
Here the EAV-HP transcript was identified in the IM+ group, while it was barely detected in the IM− group with RNA-seq and qPCR analyses. There are at least seven almost complete EAV-HP sequences identified in the most recent chicken reference database (Table S4), but only the EAV-HP insertion in the promoter region of SLCO1B3 was highly expressed. Promoters of the chicken SLCO1B3 have been discussed in a previous investigation 25 . However, the EAV-HP transcript RACE analysis in this study showed that its high expression levels may not be due to the promoter of the chicken SLCO1B3 gene, but a detailed investigation is required to better understand this. EAV HP transcripts have a complete open reading frame (Fig. S3), which means that some viral protein elements (env, gag) can be translated. These protein fragments may play the role of antigen and cause immune  www.nature.com/scientificreports/ response. The biosynthesis of antibiotics pathways and the regulation of inflammatory response terms were identified in our omic analysis, as some immune genes, such as NFKBIZ, NFKBIA, and IL1RL1, and some proteins such as PPIA, that function to inhibit viruses 48,49 was differentially expressed between two groups. However, the comprehensive effects of the EAV-HP transcripts on the chicken immune system, its health and some potential roles in the new virus formation require further investigation.
In conclusion, we cloned a chicken cholate responsive element in the 5ʹ flanking region of SLCO1B3, which is helpful to understand the expression of SLCO1B3 in IM+ individuals, and is conducive to the future research of chicken bile salt response genes. We provided new insights into the functions of SLCO1B3 in the metabolism of lipids and xenobiotics in chickens by comparing the Transcription and proteomic expression between IM+ and IM− individuals. EAV-HP insertion greatly decreased the expression of SLCO1B3 and activated itself expression at the same time. The high expression of EAV-HP is firstly reported here, which may induce liver immune response and need further study. Our study provided another example of pleiotropic variation in domestic animals, it might be useful in the current breeding of the blue eggshell chickens.

Methods
Animals and sample preparation. Yimeng chickens (Fig. 7A) was separated into IM+ or IM− groups, which can be selected based on the eggshell color (Fig. 7B C), were obtained from Shandong Longsheng Agriculture and Animal Husbandry Group Co., Ltd., Linyi City, Shandong Province, China, for use in this study. In this case, the IM+ chickens were all heterozygotes, with one copy of the EAV-HP insertion, while the IM− chickens were all recessive homozygotes. Six hens (n = 3 IM+ and n = 3 IM−) were slaughtered at the same age (approximately 300 days). Furthermore, 2 mL whole blood samples were collected from the wing veins of 42 chickens (21 IM+ chickens and 21 IM− chickens) without providing an anticoagulant. Serum was separated from the whole blood samples by centrifugation at 3000×g for 10 min at room temperature, and then stored in 1.5 mL centrifuge tubes at − 20 °C, for the biochemical analysis. The liver tissues were divided into two and immediately snapfrozen in liquid nitrogen and stored at − 80 °C, for later analysis. The molecular identification method of the IM insert mutation in Yimeng chickens was performed according to a previously described multiple PCR method 21 .
All procedures and experiments performed in this study were approved by the Animal Care and Use Committee of China Agricultural University (Approval no. XK257), all methods were performed in accordance with ARRIVE guidelines (https:// arriv eguid elines. org), and all methods were carried out in accordance with relevant guidelines and regulations.
Vector construction, cell culture, transient transfection and dual luciferase assay. About 2000 bp (IM−) and ~ 4.8 Kb (IM+ , including 4.2 Kb EAV-HP insertion) upstream region from the transcription start site of the SLCO1B3 was cloned by Taq DNA polymerase and ligated into the pMD19-T vector (Takara Biomedical Technology (Beijing) Co., Ltd, Beijing, China) as per the manufacturer's guidelines. After sequencing by a Sanger method (Sino Geno Max, Beijing, China), seven different length fragments of the upstream of SLCO1B3 was cloned using PCR with primers contained specific restriction site listed in Table S6, then they were inserted into the multiple copy region of pGL3-Basic vector (Promega, Beijing, China). A chemical synthesis method was used to the mutated vector construction (Beijing Genomics Institute, Beijing, China). Constructed plasmids were extracted using the plasmid midi kit (Tiangen Biotech Co., Ltd, Beijing, China) as per the manufacturer's guidelines. Chenodeoxycholic acid (CDCA) (Sigma-Aldrich, MO, USA) dissolved in DMSO into a storage solution of 100 mM. LMH cells were cultured in Waymouth's medium plus 10% fetal bovine serum, 1% penicillin/ streptomycin and different concentration CDCA. All reagents for cell culture were purchased from Invitrogen/ Gibco. Cells were seeded into 24-cell plates (Corning, NY, USA) and were co-transfected with 180 ng pGL3-construct and 20 ng pRL-TK using Lipofectamine 2000 (Invitrogen, CA, USA) as per the manufacturer's guidelines. www.nature.com/scientificreports/ Each construct has three replicates, including pGL3-Basci vector. After 36 h, firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega) as per the manufacturer's guidelines.

RNA-seq library preparation and sequencing.
Total RNA was extracted from the liver using an RNA prep Pure Kit (Tiangen) as per the manufacturer's guidelines. Six mRNA libraries were constructed and RNA degradation and contamination were visualized on 1% agarose gels. RNA purity was checked using a NanoPhotometer spectrophotometer (Implen, CA, USA), and concentrations were determined using a Nanodrop 2000 (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) as per the manufacturer's guidelines. A total of 3 μg RNA from each sample was used as the input material for RNA sample preparations. The mRNA sequence libraries were constructed using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Inc., MA, USA), according to the manufacturer's recommendations. Subsequently, the mRNA libraries were sequenced on an Illumina Hiseq 2500 platform, and 100-bp paired-end reads were generated.
Read mapping. The clean reads were obtained from the raw reads by removing the reads containing adapters, poly-N regions, and low-quality reads. The Q20 scores and Q30 scores of the clean data were calculated. The clean reads were mapped to the chicken genome assembly (galGal6), which was downloaded from the Ensembl database. The reference genome index was constructed using Bowtie v2.0.6, and the clean paired-end reads were aligned to the reference genome using TopHat v2.0.9 50,51 . The mapped reads of each sample were assembled using Cufflinks (v2. . The parameters were set as follows: Search parameters were trypsin specificity, carbamidomethyl as a fixed modification, oxidation and phosphorylation as variable modifications, with two allowed missed cleavages per peptide; three maximum allowed variable PTM per peptide Precursor mass tolerance was set at 15 ppm, and fragment ion tolerance at 0.02 Da. Protein identifications were only considered confident if at least two unique peptides with at least two spectra were identified. The protein expression profile was done by hierarchical clustering to create an expressional profile of differentially expressed protein groups between IM+ and IM− chickens.

Measurement of serum biochemical parameters.
Six serum biochemical parameters were assessed using kits, including a high-density lipoprotein cholesterol (HDL-C) kit, low-density lipoprotein cholesterol (LDL-C) kit, apolipoprotein A-I (APOAI) kit, and apolipoprotein B (APOB) kit were purchased from Biosino Bio-Technology and Science Inc. (Beijing, China). Furthermore. a total bile acid kit was purchased from Zhejiang Weiyi Bio-tech. Co., Ltd. (Zhejiang, China) and a very low-density lipoprotein cholesterol (VLDL) kit was purchased from the Beijing Sino-UK Institute of Biology Technology (Beijing, China). These parameters were measured by following the manufacturer's instructions for all kits using a Mindray IM-420 fully automated clinical chemistry analyzer (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) at the Beijing Sino-UK Institute of Biology Technology (Beijing, China).
Cloning the full-length EAV-HP transcript. The IM+ Yimeng chicken liver tissue RNA for RNA-seq was used for the 5ʹRACE and 3ʹRACE experiments. The RACE experiments followed the protocol of SMARTer RACE 5ʹ/3ʹ Kit (Clontech, Mountain View, CA, USA). Gene RACE primers (including nested primers) were designed for the two orientations as the transcriptional start site for the EAV-HP could not be confirmed. In addition, another primer (AP) set was designed for the entire cDNA sequence of the EAV-HP, as the products of the corresponding RACE primer sets could not complete the entire sequence. The primer sequences are presented in Table S7, and the PCR products were sequenced using the Sanger method (Sino Geno Max).