内源性逆转录病毒序列在猪基因组中的搜寻及其在中西方猪品种中的拷贝数变异和序列多样性调查

Jia-Qi Chen; Ming-Peng Zhang; Xin-Kai Tong; Jing-Quan Li; Zhou Zhang; Fei Huang; Hui-Peng Du; Meng Zhou; Hua-Shui Ai; Lu-Sheng Huang

doi:10.24272/j.issn.2095-8137.2021.379

内源性逆转录病毒序列在猪基因组中的搜寻及其在中西方猪品种中的拷贝数变异和序列多样性调查

doi: 10.24272/j.issn.2095-8137.2021.379

1.
江西农业大学猪遗传改良与养殖技术国家重点实验室, 江西南昌330045, 中国

计量
- 文章访问数: 674
- HTML全文浏览量: 311
- PDF下载量: 124
- 被引次数: 0
出版历程
- 收稿日期: 2022-03-05
- 录用日期: 2022-04-12
- 网络出版日期: 2022-04-15
- 刊出日期: 2022-05-18

Scan of the endogenous retrovirus sequences across the swine genome and survey of their copy number variation and sequence diversity among various Chinese and Western pig breeds

1.
State Key Laboratory for Swine Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China

Funds: This study was supported by the National Swine Industry and Technology System of China (nycytx-009), and National Natural Science Foundation of China (31672383)

More Information

Corresponding author: E-mail: aihsh@hotmail.com; lushenghuang@hotmail.com

摘要

摘要: 在猪-人异种移植中，猪内源性逆转录病毒（PERV）的传播风险备受关注。然而，PERV在猪基因组中的分布、它们在欧亚猪中的遗传变异以及它们的进化史尚不清楚。我们根据序列相似性在当前猪参考基因组（11.1版）中搜寻了PERV，检测到36个长的完整或接近完整的PERV（lcPERV）和23个短的不完整PERV（siPERV）。除了三种已知PERV（PERV-A、-B和-C），该研究还检测到四种新的PERV类型（PERV-JX1、-JX2、-JX3和-JX4）。进化分析表明，新发现的PERV更古老，PERV-B可能在50万年前经历了多样性骤降。通过分析63个高质量猪全基因组测序数据，我们发现中国猪的PERV拷贝数(32.0 ± 4.0)低于西方猪的（49.1 ± 6.5）。此外，中国猪的PERV序列多样性也低于西方猪。在这些lcPERV中，西方猪的PERV-A和-JX2显著高于中国猪。值得注意的是，巴马香猪的PERV拷贝数最低（27.8±5.1），其中一头巴马香猪没有PERV-C，且PERV拷贝数是最低的（23）。这表明巴马香猪更适合作为异种移植供体进行筛选或被修饰。此外，我们还鉴定了451个PERV插入多态性位点，其中86个是10个中国和西方猪品种所共有的。我们的研究发现为PERV的基因组分布、变异、进化和可能的生物学功能提供了系统的见解。
- 猪内源性逆转录病毒 /
- 中西方猪 /
- 拷贝数变化 /
- 进化 /
- 生物功能预测
Abstract: In pig-to-human xenotransplantation, the transmission risk of porcine endogenous retroviruses (PERVs) is of great concern. However, the distribution of PERVs in pig genomes, their genetic variation among Eurasian pigs, and their evolutionary history remain unclear. We scanned PERVs in the current pig reference genome (assembly Build 11.1), and identified 36 long complete or near-complete PERVs (lcPERVs) and 23 short incomplete PERVs (siPERVs). Besides three known PERVs (PERV-A, -B, and -C), four novel types (PERV-JX1, -JX2, -JX3, and -JX4) were detected in this study. According to evolutionary analyses, the newly discovered PERVs were more ancient, and PERV-Bs probably experienced a bottleneck ~0.5 million years ago (Ma). By analyzing 63 high-quality porcine whole-genome resequencing data, we found that the PERV copy numbers in Chinese pigs were lower (32.0±4.0) than in Western pigs (49.1±6.5). Additionally, the PERV sequence diversity was lower in Chinese pigs than in Western pigs. Regarding the lcPERV copy numbers, PERV-A and -JX2 in Western pigs were higher than in Chinese pigs. Notably, Bama Xiang (BMX) pigs had the lowest PERV copy number (27.8±5.1), and a BMX individual had no PERV-C and the lowest PERV copy number (23), suggesting that BMX pigs were more suitable for screening and/or modification as xenograft donors. Furthermore, we identified 451 PERV transposon insertion polymorphisms (TIPs), of which 86 were shared by all 10 Chinese and Western pig breeds. Our findings provide systematic insights into the genomic distribution, variation, evolution, and possible biological function of PERVs.
- PERVs /
- Chinese and Western pigs /
- Copy number variation /
- Evolutionary history /
- Biological function prediction

HTML全文

Figure 1. Prediction pipeline of PERV types in an individual using the resequencing data based on the unique k-mers

An example was given to determine whether the individual CNWB01 contained the PERV (PERV-JX1-17-41.4M). The result indicated CNWB01 had PERV-JX1-17-41.4M in its genome.

下载: 全尺寸图片幻灯片

Figure 2. 59 PERVs in the S. Scrofa reference genome

A: The positions of PERVs in the reference genome. NW965: NW_018084965; NW136: NW_018085136; NW086: NW_018085086; NW255: NW_018085255; NW331: NW_018085331; NW293: NW_018085293; NW154: NW_018085154; NW193: NW_018085193; NW355: NW_018085355. siPERV: short incomplete PERV. B: Maximum-likelihood (ML) phylogenetic tree of 36 long complete or near-complete PERVs (lcPERVs) without LTR. In the tree, 3 complete PERVs (PERV-A: AY099323.1, PERV-B: AY099324.1, and PERV-C: KY352351.1) were included, and an MLV (MLV-J01998.1) was set as the outgroup. The color on the outer circle represents the detection rate of PERVs on the genome by 10 breeds (n=63). The bluer the color, the more pigs have this PERV copy, and the redder the color, the fewer pigs. C: Maximum-likelihood (ML) phylogenetic tree of 36 lcPERVs with LTRs. The color on the outer circle represents LTR types. D: ORF prediction of 62 PERVs and their LTRs. 59 PERVs were identified from the swine reference genome Build 11.1, and 3 complete PERVs (PERV-A: AY099323.1, PERV-B: AY099324.1, and PERV-C: KY352351.1) were downloaded from the NCBI Genbank database. ORFs of gag, pol, env were predicted in three forward reading frames. For example, gag1 means gag ORF predicted in forward frame 1; gag2 means gag ORF predicted in forward frame 2; gag3 means gag ORF predicted in forward frame 3. The sequences of pol-probe and env-probe were downloaded from the reference (Yang et al., 2015).

下载: 全尺寸图片幻灯片

Figure 3. Evolutionary histories of 39 lcPERVs and their 5' LTRs

The evolutionary tree of lcPERVs is on the left. Yellow triangle: PERV contains full gag, pol and env ORFs; Yellow circular: the pol ORF is complete but the other ORFs are incomplete. The phylogenetic tree was estimated using 40 ERV internal sequences (including 3 PERV reference sequences and one outgroup MLV) by BEAST. We set the divergence time between MLV and PERV-A-AY099323.1 to be 96 Ma with confidence intervals from 91 to 101 Ma same as the divergence time of pig and mice in TimeTree database. The LTR tree is on the right. 5’ LTRs of 39 lcPERVs were used, and 5' LTR of MLV was used as an outgroup. The divergence time was the same as above. PERV and its corresponding LTR are connected with a solid line.

下载: 全尺寸图片幻灯片

Figure 4. Correlation between PERV copy number and LTR copy number measured by method 1 (mapping-to-genome method)

The correlation between the copy number of PERV and LTR in ten pig breeds was shown in the scatter plot. The box plot at the top shows the difference of PERV copy number between Chinese and Western pig breeds, and the box plot at the right shows the difference of LTR copy number between Chinese and Western pig breeds. ^***: P<0.0001.

下载: 全尺寸图片幻灯片

Figure 5. PERV copy number and PERV types in 63 Chinese and Western pigs

A: The percentage of different PERV types in 63 Chinese and Western pigs. B: Copy number difference of each PERV type between Chinese and Western pig breeds. ^***: P<0.0001. C: Heatmap of PERVs with a significant difference in copy number between Chinese and Western pig breeds.

下载: 全尺寸图片幻灯片

Figure 6. Differences in PERV sequence diversity, copy number of PERVs with transposable potentiality and TIPs between Chinese and Western pig breeds

A: Sequence diversity of PERVs without LTRs in Eurasian pig breeds. The number of k-mer types in the reads aligned to PERVs without LTRs can reflect the sequence diversity of PERVs. The left venn diagram shows the numbers of specific k-mer types of PERVs not shared and common k-mer types of PERVs shared between all Chinese and Western pigs; the middle one shows the relation between Chinese and Western domestic pigs; the right one shows the relation between Chinese and Western wild boars. B: The percentage of PERVs with transposable potentiality in 63 Chinese and Western pigs. The above bar plot with pink background shows the percentage of seven PERVs with complete gag, pol, and env ORF in all Chinese (32) and Western (31) pigs. The bar plot below with green background shows the percentage of four PERVs with incomplete gene structure but with complete pol ORF in all Chinese and Western pigs. C: Total copy number of eleven PERVs with transposition potentialities in different pig breeds. D: PERV TIPs in ten Chinese and Western pig breeds. The left horizontal bar plot shows the total number of PERV TIPs in each pig breed. The top bar plot shows the number of PERV TIPs for each intersection. Only 86 all breed-common, 6 Chinese pig breed-common, 6 Western pig breed-common, and 150 breed-specific TIP loci are showed.

下载: 全尺寸图片幻灯片

Table 1. Classification and descriptive statistics of all 59 PERVs in the Sus scrofa reference genome (assembly Build 11.1).

No.	Category¹	Subclass	PERV name	Chromosome / unplaced contig	Strand	Position	Length²	Unique k-mer³	Detection rate⁴
1	lcPERV	PERV-A	PERV-A-1-132.0M	1	+	132020281–132028361	8 081	253	0.57
2	lcPERV	PERV-A	PERV-A-1-262.2M	1	+	262166347–262175262	8 916	35	0.38
3	lcPERV	PERV-A	PERV-A-3-10.6M	3	−	10660619–10669533	8 915	647	0.24
4	lcPERV	PERV-A	PERV-A-5-92.1M	5	−	92185133–92194050	8 918	226	0.56
5	lcPERV	PERV-A	PERV-A-7-105.7M	7	+	105710884–105719193	8 310	119	0.17
6	lcPERV	PERV-A	PERV-A-8-51.6M	8	+	51570546–51579460	8 915	466	0.94
7	lcPERV	PERV-A	PERV-A-12-28.2M	12	+	28221374–28230287	8 914	95	0.43
8	lcPERV	PERV-A	PERV-A-13-142.0M	13	−	142030847–142039759	8 913	451	1
9	lcPERV	PERV-A	PERV-A-13-146.7M	13	−	146750532–146759521	8 990	520	1
10	lcPERV	PERV-A	PERV-A-15-66.8M	15	−	66864153–66873044	8 892	500	0.44
11	lcPERV	PERV-A	PERV-A-17-3.7M	17	−	3794010–3802709	8 700	411	1
12	lcPERV	PERV-A	PERV-A-17-33.1M	17	+	33062883–33071800	8 918	930	0.41
13	lcPERV	PERV-Alike	PERV-Alike-U965-14.7kb	NW_018084965	+	14674–22827	8 154	1 933	0
14	lcPERV	PERV-A	PERV-A-U136-0.5M	NW_018085136	−	563793–572725	8 933	1 205	0.86
15	lcPERV	PERV-A	PERV-A-X-73.7M	X	−	73752986–73761903	8 918	577	0.51
16	lcPERV	PERV-A	PERV-A-Y-20.1M	Y	−	20194782–20203572	8 791	983	0.46
17	lcPERV	PERV-A	PERV-A-Y-21.8M	Y	−	21867301–21876035	8 735	920	0.38
18	lcPERV	PERV-A	PERV-A-Y-25.2M	Y	+	25245606–25253905	8 300	11	0.63
19	lcPERV	PERV-B	PERV-B-3-51.1M	3	−	51108601–51117360	8 760	197	0.86
20	lcPERV	PERV-B	PERV-B-4-45.5M	4	−	45599494–45608252	8 759	132	0.62
21	lcPERV	PERV-B	PERV-B-8-15.3M	8	+	15319428–15328187	8 760	177	0.76
22	lcPERV	PERV-B	PERV-B-9-138.8M	9	−	138895584–138904340	8 757	51	0.46
23	lcPERV	PERV-B	PERV-B-11-38.2M	11	+	38201361–38210116	8 756	386	0.86
24	lcPERV	PERV-B	PERV-B-14-119.7M	14	+	119667621–119676299	8 679	486	0.46
25	lcPERV	PERV-B	PERV-B-15-110.9M	15	−	110958945–110967782	8 838	374	0.44
26	lcPERV	PERV-B	PERV-B-16-59.6M	16	+	59571885–59580646	8 762	141	0.44
27	lcPERV	PERV-B	PERV-B-U086-42.1kb	NW_018085086	−	42158–50876	8 719	564	0.81
28	lcPERV	PERV-B	PERV-B-U255-1.9kb	NW_018085255	+	1949–10743	8 795	242	0.54
29	lcPERV	PERV-B	PERV-B-U331-13.8kb	NW_018085331	+	13836–22597	8 762	85	0.87
30	lcPERV	PERV-C	PERV-C-14-62.5M	14	−	62550397–62557645	7 249	652	0.78
31	lcPERV	PERV-JX1	PERV-JX1-7-21.2M	7	+	21236160–21244976	8 817	1 387	1
32	lcPERV	PERV-JX1	PERV-JX1-17-41.4M	17	−	41467519–41476101	8 583	1 343	1
33	lcPERV	PERV-JX1	PERV-JX1-U293-0.8M	NW_018085293	+	820148–829192	9 045	2 015	1
34	lcPERV	PERV-JX2	PERV-JX2-X-71.4M	X	+	71402222–71409912	7 691	1 472	0.49
35	lcPERV	PERV-JX3	PERV-JX3-3-17.8M	3	+	17778858–17788086	9 229	1 460	1
36	lcPERV	PERV-JX4	PERV-JX4-2-76.6M	2	−	76648002–76655788	7 787	859	1
37	siPERV	PERV-A	sPERV-A-6-10.4M	6	+	10402614–10407657	5 044	20	0.57
38	siPERV	PERV-A	sPERV-A-X-55.1M	X	+	55079139–55083211	4 073	168	0.86
39	siPERV	PERV-B	sPERV-B-1-38.5M	1	+	38459013–38465006	5 994	61	0.48
40	siPERV	PERV-B	sPERV-B-X-112.8M	X	−	112842765–112847657	4 893	108	0.76
41	siPERV	PERV-C	sPERV-C-5-29.4M	5	+	29356367–29361638	5 272	639	0.78
42	siPERV	PERV-C	sPERV-C-11-29.1M	11	−	29084884–29090328	5 445	611	0.57
43	siPERV	PERV-C	sPERV-C-13-146.75M	13	−	146759522–146764990	5 469	19	0.56
44	siPERV	PERV-C	sPERV-C-13-146.76M	13	−	146764392–146769858	5 467	34	0
45	siPERV	PERV-C	sPERV-C-X-81.5M	X	−	81543240–81545899	2 660	178	0.56
46	siPERV	PERV-Alike	sPERV-Alike-U293-0.9M	NW_018085293	+	909902–914028	4 127	729	0
47	siPERV	PERV-Clike	sPERV-Clike-U154-3.2kb	NW_018085154	+	3178–6483	3 306	505	0
48	siPERV	PERV-Clike	sPERV-Clike-U193-4.0kb	NW_018085193	+	3949–7250	3 302	232	0.57
49	siPERV	PERV-Clike	sPERV-Clike-U355-19.3kb	NW_018085355	+	19304–23938	4 635	604	0.81
50	siPERV	PERV-Clike	sPERV-Clike-Y-25.269M	Y	−	25269797–25273827	4 031	16	0.24
51	siPERV	PERV-Clike	sPERV-Clike-Y-25.29M	Y	−	25299007–25302057	3 051	0	/
52	siPERV	PERV-JX2like	sPERV-JX2like-12-23.0M	12	−	22986893–22991220	4 328	704	1
53	siPERV	PERV-JX3like	sPERV-JX3like-15-116.3M	15	+	116321677–116334781	13 105	1 206	1
54	siPERV	PERV-JX3like	sPERV-JX3like-17-9.5M	17	−	9536784–9541504	4 721	854	1
55	siPERV	PERV-JX3like	sPERV-JX3like-X-72.2M	X	+	72244018–72249170	5 153	907	0.49
56	siPERV	PERV-UC	sPERV-UC1-9-63.2M	9	−	63235598–63238136	2 539	405	0.63
57	siPERV	PERV-UC	sPERV-UC2-13-21.3M	13	+	21308535–21310732	2 198	422	0.78
58	siPERV	PERV-UC	sPERV-UC3-Y-24.4M	Y	+	24407502–24411155	3 654	644	0.24
59	siPERV	PERV-UC	sPERV-UC4-Y-25.261M	Y	+	25261084–25264796	3 713	728	0.16
¹: LcPERV indicates long and complete or near-complete PERV; siPERV indicates short incomplete PERV. ²: The pol gene of sPERV-JX3like-15-116.3M was inserted by a 6 302 bp long sequence containing LINE1 transposon and (T)n simple repeat, which resulted in the length of the PERV increasing to 13 105 bp. ³: The number of unique k-mer (k=31) of PERV on pig reference genome. ⁴: The detection proportion of the PERV in the 63 Chinese and Western pigs; since the PERV sPERV-Clike-Y-25.29M has no unique k-mers, it cannot be detected in the tested pigs.

下载: 导出CSV

Table 2. Average PERV and LTR copy numbers in ten pig breeds

Pig breed	Average PERV copy No. (n, Method 1)	Average PERV copy No. (n, Method 2)	Average LTR copy No. (n, Method 1)	Average LTR copy No. (n, Method 2)
Western pigs	49.1±6.5	48.2±6.3	254.8±30.0	204.7±26.7
Duroc	45.3±6.3	44.0±6.2	224.5±18.2	172.6±16.8
European wild boar	43.3±4.5	42.5±4.5	234.0±10.6	192.0±8.8
Landrace	55.6±4.0	54.6±4.0	280.9±18.4	230.4±16.1
Large White	46.1±5.0	45.7±4.2	248.8±21.7	197.0±15.0
White Duroc	51.9±4.1	50.7±4.0	271.3±29.1	219.1±23.0
Chinese pigs	32.0±4.0	32.0±4.2	197.8±10.9	160.4±13.6
Bama Xiang	27.8±5.1	28.5±5.4	198.5±12.7	164.0±13.6
Chinese wild boar	29.6±3.1	29.6±3.5	189.9±12.4	150.7±18.2
Erhualian	33.9±2.5	33.8±2.5	196.5±12.6	159.9±11.8
Laiwu	34.0±2.6	33.9±3.4	202.0±7.3	163.6±11.3
Tibetian	34.0±2.1	34.1±3.4	202.4±4.6	163.9±12.3
P-value¹	6.63×10^-12	7.90×10^-11	1.01×10^-07	3.64×10^-06
¹: P-value indicates the comparison result of PERV/LTR copy numbers between Chinese and Western pig breeds by student’s t-test. Number in bold indicates the minimum copy number detected by this method.

下载: 导出CSV

参考文献(77)

[1]	Ai HS, Fang XD, Yang B, Huang ZY, Chen H, Mao LK, et al. 2015. Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing. Nature Genetics, 47(3): 217−225. doi: 10.1038/ng.3199
[2]	Ai HS, Zhang MP, Yang B, Goldberg A, Li WB, Ma JW, et al. 2021. Human-mediated admixture and selection shape the diversity on the modern swine (Sus scrofa) Y chromosomes. Molecular Biology and Evolution, 38(11): 5051−5065. doi: 10.1093/molbev/msab230
[3]	Bartosch B, Stefanidis D, Myers R, Weiss R, Patience C, Takeuchi Y. 2004. Evidence and consequence of porcine endogenous retrovirus recombination. Journal of Virology, 78(24): 13880−13890. doi: 10.1128/JVI.78.24.13880-13890.2004
[4]	Bartosch B, Weiss RA, Takeuchi Y. 2002. PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B. Journal of General Virology, 83(9): 2231−2240. doi: 10.1099/0022-1317-83-9-2231
[5]	Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, et al. 2009. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics, 25(8): 1091−1093. doi: 10.1093/bioinformatics/btp101
[6]	Carpentier MC, Manfroi E, Wei FJ, Wu HP, Lasserre E, Llauro C, et al. 2019. Retrotranspositional landscape of Asian rice revealed by 3000 genomes. Nature Communication, 10(1): 24. doi: 10.1038/s41467-018-07974-5
[7]	Chen H, Huang M, Yang B, Wu ZP, Deng Z, Hou Y, et al. 2020a. Introgression of Eastern Chinese and Southern Chinese haplotypes contributes to the improvement of fertility and immunity in European modern pigs. Gigascience, 9(3): giaa014. doi: 10.1093/gigascience/giaa014
[8]	Chen N. 2004. Using RepeatMasker to identify repetitive elements in genomic sequences. Current Protocols in Bioinformatics, 5(1): 4.10.1−4.10.14.
[9]	Chen YC, Chen MY, Duan XY, Cui J. 2020b. Ancient origin and complex evolution of porcine endogenous retroviruses. Biosafety and Health, 2(3): 142−151. doi: 10.1016/j.bsheal.2020.03.003
[10]	Cohen CJ, Lock WM, Mager DL. 2009. Endogenous retroviral LTRs as promoters for human genes: a critical assessment. Gene, 448(2): 105−114. doi: 10.1016/j.gene.2009.06.020
[11]	Cooper DKC. 2003. Clinical xenotransplantion—how close are we?. The Lancet, 362(9383): 557−559. doi: 10.1016/S0140-6736(03)14118-9
[12]	Denner J. 2008. Recombinant porcine endogenous retroviruses (PERV-A/C): a new risk for xenotransplantation?. Archives of Virology, 153(8): 1421−1426. doi: 10.1007/s00705-008-0141-7
[13]	Denner J. 2016a. Expression and function of endogenous retroviruses in the placenta. APMIS, 124(1-2): 31−43. doi: 10.1111/apm.12474
[14]	Denner J. 2016b. How active are porcine endogenous retroviruses (PERVs)?. Viruses, 8(8): 215. doi: 10.3390/v8080215
[15]	Denner J. 2018. Why was PERV not transmitted during preclinical and clinical xenotransplantation trials and after inoculation of animals?. Retrovirology, 15(1): 28. doi: 10.1186/s12977-018-0411-8
[16]	Denner J, Tönjes RR. 2012. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clinical Microbiology Reviews, 25(2): 318−343. doi: 10.1128/CMR.05011-11
[17]	Deorowicz S, Kokot M, Grabowski S, Debudaj-Grabysz A. 2015. KMC 2: fast and resource-frugal k-mer counting. Bioinformatics, 31(10): 1569−1576. doi: 10.1093/bioinformatics/btv022
[18]	Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7(1): 214. doi: 10.1186/1471-2148-7-214
[19]	Fan X, Abbott TE, Larson D, Chen K. 2014. BreakDancer: identification of genomic structural variation from paired-end read mapping. Current Protocols in Bioinformatics, 45: 15.6.1−11.
[20]	Fiebig U, Fischer K, Bähr A, Runge C, Schnieke A, Wolf E, et al. 2018. Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs. Xenotransplantation, 25(4): e12445. doi: 10.1111/xen.12445
[21]	Frantz LAF, Schraiber JG, Madsen O, Megens HJ, Bosse M, Paudel Y, et al. 2013. Genome sequencing reveals fine scale diversification and reticulation history during speciation in Sus. Genome Biology, 14(9): R107.
[22]	Frantz LAF, Schraiber JG, Madsen O, Megens HJ, Cagan A, Bosse M, et al. 2015. Evidence of long-term gene flow and selection during domestication from analyses of Eurasian wild and domestic pig genomes. Nature Genetics, 47(10): 1141−1148. doi: 10.1038/ng.3394
[23]	Garkavenko O, Wynyard S, Nathu D, Muzina M, Muzina Z, Scobie L, et al. 2008. Porcine endogenous retrovirus transmission characteristics from a designated pathogen-free herd. Transplantation Proceedings, 40(2): 590−593. doi: 10.1016/j.transproceed.2008.01.051
[24]	Gel B, Serra E. 2017. karyoploteR: an R/Bioconductor package to plot customizable genomes displaying arbitrary data. Bioinformatics, 33(19): 3088−3090. doi: 10.1093/bioinformatics/btx346
[25]	Grandbastien MA. 1998. Activation of plant retrotransposons under stress conditions. Trends in Plant Science, 3(5): 181−187. doi: 10.1016/S1360-1385(98)01232-1
[26]	Groenen MAM. 2016. A decade of pig genome sequencing: a window on pig domestication and evolution. Genetics Selection Evolution, 48(1): 23. doi: 10.1186/s12711-016-0204-2
[27]	Groenen MAM, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, et al. 2012. Analyses of pig genomes provide insight into porcine demography and evolution. Nature, 491(7424): 393−398. doi: 10.1038/nature11622
[28]	Harrison I, Takeuchi Y, Bartosch B, Stoye JP. 2004. Determinants of high titer in recombinant porcine endogenous retroviruses. Journal of Virology, 78(24): 13871−13879. doi: 10.1128/JVI.78.24.13871-13879.2004
[29]	Hénaff E, Zapata L, Casacuberta JM, Ossowski S. 2015. Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution. BMC Genomics, 16(1): 768. doi: 10.1186/s12864-015-1975-5
[30]	Kabát P, Tristem M, Opavský R, Pastorek J. 1996. Human endogenous retrovirus HC2 is a new member of the S71 retroviral subgroup with a full-length pol gene. Virology, 226(1): 83−94. doi: 10.1006/viro.1996.0630
[31]	Karlas A, Irgang M, Votteler J, Specke V, Özel M, Kurth R, et al. 2010. Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C. Annals of Transplantation, 15(2): 45−54.
[32]	Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30(4): 772−780. doi: 10.1093/molbev/mst010
[33]	Kent WJ. 2002. BLAT-the BLAST-like alignment tool. Genome Research, 12(4): 656−664.
[34]	Kozak CA. 1984. Differential expression of murine leukemia virus loci in chemically induced hybrid cells. Journal of Virology, 51(3): 876−879. doi: 10.1128/jvi.51.3.876-879.1984
[35]	Krüger L, Kristiansen Y, Reuber E, Möller L, Laue M, Reimer C, et al. 2019. A comprehensive strategy for screening for xenotransplantation-relevant viruses in a second isolated population of Göttingen minipigs. Viruses, 12(1): 38. doi: 10.3390/v12010038
[36]	Krüger L, Stillfried M, Prinz C, Schroder V, Neubert LK, Denner J. 2020. Copy number and prevalence of porcine endogenous retroviruses (PERVs) in German Wild Boars. Viruses, 12(4): 419. doi: 10.3390/v12040419
[37]	Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6): 1547−1549. doi: 10.1093/molbev/msy096
[38]	Kumar S, Stecher G, Suleski M, Hedges SB. 2017. TimeTree: a resource for timelines, timetrees, and divergence times. Molecular Biology and Evolution, 34(7): 1812−1819. doi: 10.1093/molbev/msx116
[39]	Kunarso G, Chia NY, Jeyakani J, Hwang C, Lu XY, Chan YS, et al. 2010. Transposable elements have rewired the core regulatory network of human embryonic stem cells. Nature Genetics, 42(7): 631−634. doi: 10.1038/ng.600
[40]	Lanciano S, Cristofari G. 2020. Measuring and interpreting transposable element expression. Nature Reviews Genetics, 21(12): 721−736. doi: 10.1038/s41576-020-0251-y
[41]	Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9(4): 357−359. doi: 10.1038/nmeth.1923
[42]	Larson G, Dobney K, Albarella U, Fang MY, Matisoo-Smith E, Robins J, et al. 2005. Worldwide phylogeography of wild boar reveals multiple centers of pig domestication. Science, 307(5715): 1618−1621. doi: 10.1126/science.1106927
[43]	Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. 1997. Two sets of human-tropic pig retrovirus. Nature, 389(6652): 681−682. doi: 10.1038/39489
[44]	Lee D, Lee J, Yoon JK, Kim NY, Kim GW, Park C, et al. 2011. Rapid determination of perv copy number from porcine genomic DNA by real-time polymerase chain reaction. Animal Biotechnology, 22(4): 175−180. doi: 10.1080/10495398.2011.595294
[45]	Lee JH, Webb GC, Allen RDM, Moran C. 2002. Characterizing and mapping porcine endogenous retroviruses in Westran pigs. Journal of Virology, 76(11): 5548−5556. doi: 10.1128/JVI.76.11.5548-5556.2002
[46]	Letunic I, Bork P. 2019. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Research, 47(W1): W256−W259. doi: 10.1093/nar/gkz239
[47]	Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25(14): 1754−1760. doi: 10.1093/bioinformatics/btp324
[48]	Lian ZX, Rogel-Gaillard C, Li N, Chardon P, Wu CX. 2002. Copy number polymorphism of endogenous retrovirus sequence in Chinese local pig breeds by Semi-PCR. Acta Veterinaria et Zootechnica Sinica, 33(6): 521−524.
[49]	Liu G, Li Z, Pan M, Ge M, Wang Y, Gao Y. 2011. Genetic prevalence of porcine endogenous retrovirus in Chinese experimental miniature pigs. Transplantation Proceedings, 43(7): 2762−2769. doi: 10.1016/j.transproceed.2011.06.061
[50]	Mang R, Maas J, Chen XH, Goudsmit J, Van Der Kuyl AC. 2001. Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding. Journal of General Virology, 82(8): 1829−1834. doi: 10.1099/0022-1317-82-8-1829
[51]	Matos MC, Pinheiro A, Melo-Ferreira J, Davis RS, Esteves PJ. 2021. Evolution of Fc receptor-like scavenger in mammals. Frontiers in Immunology, 11: 590280. doi: 10.3389/fimmu.2020.590280
[52]	Morales JF, Snow ET, Murnane JP. 2003. Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors. Mutagenesis, 18(2): 151−158. doi: 10.1093/mutage/18.2.151
[53]	Morgulis A, Coulouris G, Raytselis Y, Madden TL, Agarwala R, Schäffer AA. 2008. Database indexing for production MegaBLAST searches. Bioinformatics, 24(16): 1757−1764. doi: 10.1093/bioinformatics/btn322
[54]	Niebert M, Tönjes RR. 2005. Evolutionary spread and recombination of porcine endogenous retroviruses in the Suiformes. Journal of Virology, 79(1): 649–654.
[55]	Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, et al. 2017. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science, 357(6357): 1303−1307. doi: 10.1126/science.aan4187
[56]	Patience C, Switzer WM, Takeuchi Y, Griffiths DJ, Goward ME, Heneine W, et al. 2001. Multiple groups of novel retroviral genomes in pigs and related species. Journal of Virology, 75(6): 2771−2775. doi: 10.1128/JVI.75.6.2771-2775.2001
[57]	Patience C, Takeuchi Y, Weiss RA. 1997. Infection of human cells by an endogenous retrovirus of pigs. Nature Medicine, 3(3): 282−286. doi: 10.1038/nm0397-282
[58]	Premachandra HKA, La Cruz FLD, Takeuchi Y, Miller A, Fielder S, O’Connor W, et al. 2017. Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence. Scientific Reports, 7(1): 9386. doi: 10.1038/s41598-017-07419-x
[59]	Quereda JJ, Herrero-Medrano JM, Abellaneda JM, García-Nicolás O, Martínez-Alarcón L, Pallarés FJ, et al. 2012. Porcine endogenous retrovirus copy number in different pig breeds is not related to genetic diversity. Zoonoses and Public Health, 59(6): 401−407. doi: 10.1111/j.1863-2378.2012.01467.x
[60]	Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6): 841−842. doi: 10.1093/bioinformatics/btq033
[61]	Rombel IT, Sykes KF, Rayner S, Johnston SA. 2002. ORF-FINDER: a vector for high-throughput gene identification. Gene, 282(1-2): 33−41. doi: 10.1016/S0378-1119(01)00819-8
[62]	Scheef G, Fischer N, Krach U, Tönjes RR. 2001. The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells. Journal of Virology, 75(15): 6933−6940. doi: 10.1128/JVI.75.15.6933-6940.2001
[63]	Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. 2003. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 13(11): 2498−2504. doi: 10.1101/gr.1239303
[64]	Sykes M, Sachs DH. 2019. Transplanting organs from pigs to humans. Science Immunology, 4(41): eaau6298. doi: 10.1126/sciimmunol.aau6298
[65]	Sypniewski D, Machnik G, Mazurek U, Wilczok T, Smorag Z, Jura J, et al. 2005. Distribution of porcine endogenous retroviruses (PERVs) DNA in organs of a domestic pig. Annals of Transplantation, 10(2): 46−51.
[66]	Tönjes RR, Niebert M. 2003. Relative age of proviral porcine endogenous retrovirus sequences in Sus scrofa based on the molecular clock hypothesis. Journal of Virology, 77(22): 12363−12368. doi: 10.1128/JVI.77.22.12363-12368.2003
[67]	Wang T, Zeng J, Lowe CB, Sellers RG, Salama SR, Yang M, et al. 2007. Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53. Proceedings of the National Academy of Sciences of the United States of America, 104(47): 18613−18618. doi: 10.1073/pnas.0703637104
[68]	Warr A, Affara N, Aken B, Beiki H, Bickhart DM, Billis K, et al. 2020. An improved pig reference genome sequence to enable pig genetics and genomics research. Gigascience, 9(6): giaa051. doi: 10.1093/gigascience/giaa051
[69]	Wilson CA, Wong S, VanBrocklin M, Federspiel MJ. 2000. Extended analysis of the in vitro tropism of porcine endogenous retrovirus. Journal of Virology, 74(1): 49−56. doi: 10.1128/JVI.74.1.49-56.2000
[70]	Yan GR, Guo TF, Xiao SJ, Zhang F, Xin WS, Huang T, et al. 2018. Imputation-based whole-genome sequence association study reveals constant and novel loci for hematological traits in a large-scale swine F₂ resource population. Frontiers in Genetics, 9: 401. doi: 10.3389/fgene.2018.00401
[71]	Yang B, Cui LL, Perez-Enciso M, Traspov A, Crooijmans RPMA, Zinovieva N, Schook LB, et al. 2017. Genome-wide SNP data unveils the globalization of domesticated pigs. Genetics Selection Evolution, 49(1): 71. doi: 10.1186/s12711-017-0345-y
[72]	Yang LH, Güell M, Niu D, George H, Lesha E, Grishin D, et al. 2015. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 350(6264): 1101−1104. doi: 10.1126/science.aad1191
[73]	Yang YG, Sykes M. 2007. Xenotransplantation: current status and a perspective on the future. Nature Reviews Immunology, 7(7): 519−531. doi: 10.1038/nri2099
[74]	Yoon JK, Choi J, Lee HJ, Cho Y, Gwon YD, Jang Y, et al. 2015. Distribution of porcine endogenous retrovirus in different organs of the hybrid of a Landrace and a Jeju domestic pig in Korea. Transplantation Proceedings, 47(6): 2067−2071. doi: 10.1016/j.transproceed.2015.05.023
[75]	Yue YN, Xu WD, Kan YN, Zhao HY, Zhou YX, Song XB, et al. 2021. Extensive germline genome engineering in pigs. Nature Biomedical Engineering, 5(2): 134−143. doi: 10.1038/s41551-020-00613-9
[76]	Zhang L, Huang YM, Si JL, Wu YJ, Wang M, Jiang QY, et al. 2018. Comprehensive inbred variation discovery in Bama pigs using de novo assemblies. Gene, 679: 81−89. doi: 10.1016/j.gene.2018.08.051
[77]	Zhang P, Yu P, Wang W, Zhang L, Li SF, Bu H. 2010. An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues. In Vitro Cellular & Developmental Biology - Animal volume, 46(5): 408−410.