穿山甲新冠相关病毒高度多样性警示潜在的生态风险

Min-Sheng Peng; Jian-Bo Li; Zheng-Fei Cai; Hang Liu; Xiaolu Tang; Ruochen Ying; Jia-Nan Zhang; Jia-Jun Tao; Ting-Ting Yin; Tao Zhang; Jing-Yang Hu; Ru-Nian Wu; Zhong-Yin Zhou; Zhi-Gang Zhang; Li Yu; Yong-Gang Yao; Zheng-Li Shi; Xue-Mei Lu; Jian Lu; Ya-Ping Zhang

doi:10.24272/j.issn.2095-8137.2021.334

穿山甲新冠相关病毒高度多样性警示潜在的生态风险

doi: 10.24272/j.issn.2095-8137.2021.334

彭旻晟^{1, 2, 3, #, ,},
李建波^{1, 2, #},
蔡正飞^{4, #},
刘行^{1, 2, #},
唐小鹿^{5, #},
应若晨⁵,
张嘉楠⁶,
陶家军⁶,
尹婷婷¹,
张涛⁴,
胡靖杨⁴,
吴汝念¹,
周中银¹,
张志刚⁴,
于黎⁴,
姚永刚^{2, 3, 7},
石正丽⁸,
吕雪梅^{1, 2, 9},
陆剑^5, ,,
张亚平^{1, 2, 3, 4, 9, ,}

1.
中国科学院昆明动物研究所遗传资源与进化国家重点实验室, 云南省畜禽分子生物学重点实验室, 云南昆明 650201, 中国
2.
中国科学院大学昆明生命科学学院, 云南昆明 650204, 中国
3.
中国科学院昆明动物研究所中国科学院明动物研究所-香港中文大学生物资源与疾病分子机理联合实验室, 云南昆明 650201, 中国
4.
云南大学云南生物资源保护与利用国家重点实验室, 云南昆明 650091, 中国
5.
北京大学生命科学学院蛋白质与植物基因研究国家重点实验室, 生物信息中心, 北京 100871, 中国
6.
博瑞迪生物技术有限公司, 河北石家庄 050035, 中国
7.
中国科学院昆明动物研究所中国科学院-云南省动物模型与人类疾病机理重点实验室, 云南昆明 650201, 中国
8.
中国科学院武汉病毒研究所中国科学院高致病性病原生物学与生物安全重点实验室, 湖北武汉 430071, 中国
9.
中国科学院动物进化与遗传前沿交叉卓越创新中心, 云南昆明 650201, 中国

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出版历程
- 收稿日期: 2021-09-22
- 录用日期: 2021-11-09
- 网络出版日期: 2021-11-10
- 刊出日期: 2021-11-18

The high diversity of SARS-CoV-2-related coronaviruses in pangolins alerts potential ecological risks

Min-Sheng Peng^{1, 2, 3, #
, ,},
Jian-Bo Li^{1, 2, #},
Zheng-Fei Cai^{4, #},
Hang Liu^{1, 2, #},
Xiaolu Tang^{5, #},
Ruochen Ying⁵,
Jia-Nan Zhang⁶,
Jia-Jun Tao⁶,
Ting-Ting Yin¹,
Tao Zhang⁴,
Jing-Yang Hu⁴,
Ru-Nian Wu¹,
Zhong-Yin Zhou¹,
Zhi-Gang Zhang⁴,
Li Yu⁴,
Yong-Gang Yao^{2, 3, 7},
Zheng-Li Shi⁸,
Xue-Mei Lu^{1, 2, 9},
Jian Lu^{5
, ,},
Ya-Ping Zhang^{1, 2, 3, 4, 9
, ,}

1.
State Key Laboratory of Genetic Resources and Evolution, Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
2.
Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
3.
KIZ/CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
4.
State Key Laboratory for Conservation and Utilization of Bio-resources in Yunnan, Yunnan University, Kunming, Yunnan 650091, China
5.
State Key Laboratory of Protein and Plant Gene Research, Center for Bioinformatics, School of Life Sciences, Peking University, Beijing 100871, China
6.
Molbreeding Biotechnology Co., Ltd., Shijiazhuang, Hebei 050035, China
7.
Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan 650201, China
8.
CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
9.
Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, Yunnan 650201, China

Funds: This work was supported by the National Key Research and Development Projects of the Ministry of Science and Technology of China, National Key Research and Development Program of China (2021YFC0863300), Ministry of Agriculture of China (2016ZX08009003-006), Key Program of Chinese Academy of Sciences (KJZD-SW-L11), and Animal Branch of the Germplasm Bank of Wild Species, Chinese Academy of Sciences (the Large Research Infrastructure Funding)

More Information

Corresponding author: E-mail: pengminsheng@mail.kiz.ac.cn; luj@pku.edu.cn; zhangyp@mail.kiz.ac.cn

#Authors contributed equally to this work

摘要

摘要: 了解新冠病毒（SARS-CoV-2）的起源和进化历史将为COVID-19疫情的防控以及潜在的人兽共患病的预防提供重要见解。穿山甲携带新冠相关病毒，但其在病毒起源和进化中扮演的角色尚不清楚。我们对采集自4个穿山甲物种163只个体的172份组织样本进行新冠相关病毒筛选，在4只爪哇穿山甲（Manis javanica）和1只中华穿山甲（M. pentadactyla）的肌肉样本中检测到了病毒核酸阳性信号。对穿山甲线粒体DNA的系统地理分析表明这5份阳性穿山甲样本可能起源于东南亚。针对病毒核酸含量低且降解严重的特点，我们采用古代DNA研究中广泛使用的目标捕获测序技术，从中华穿山甲样本中获得了长为22895 nt的新冠相关病毒部分基因组序列（MP20）。系统发育分析显示MP20与广西查获的马来亚穿山甲携带的新冠相关病毒亲缘关系密切，聚类为一个支系。蝙蝠冠状病毒通过重组对这一支系的穿山甲新冠相关病毒具有遗传贡献。对原始测序数据的进一步分析显示穿山甲中新冠相关病毒的遗传多样性大大高于此前的预期。穿山甲中新冠相关病毒的潜在感染性和高度的遗传多样性提示了人兽共患的致病性冠状病毒进化和传播的生态风险。
- 新型冠状病毒 /
- 穿山甲 /
- 重组 /
- 多样性 /
- 测序 /
- 线粒体DNA
Abstract: Understanding the zoonotic origin and evolution history of SARS-CoV-2 will provide critical insights for alerting and preventing future outbreaks. A significant gap remains for the possible role of pangolins as a reservoir of SARS-CoV-2 related coronaviruses (SC2r-CoVs). Here, we screened SC2r-CoVs in 172 samples from 163 pangolin individuals of four species, and detected positive signals in muscles of four Manis javanica and, for the first time, one M. pentadactyla. Phylogeographic analysis of pangolin mitochondrial DNA traced their origins from Southeast Asia. Using in-solution hybridization capture sequencing, we assembled a partial pangolin SC2r-CoV (pangolin-CoV) genome sequence of 22895 bp (MP20) from the M. pentadactyla sample. Phylogenetic analyses revealed MP20 was very closely related to pangolin-CoVs that were identified in M. javanica seized by Guangxi Customs. A genetic contribution of bat coronavirus to pangolin-CoVs via recombination was indicated. Our analysis revealed that the genetic diversity of pangolin-CoVs is substantially higher than previously anticipated. Given the potential infectivity of pangolin-CoVs, the high genetic diversity of pangolin-CoVs alerts the ecological risk of zoonotic evolution and transmission of pathogenic SC2r-CoVs.
- SARS-CoV-2 /
- Pangolin /
- Recombination /
- Diversity /
- Sequencing /
- mtDNA
#Authors contributed equally to this work
注释:

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Figure 1. Genetic characterization of pangolin-CoVs in pangolin muscles

A: The five samples that yielded positive RT-PCR signals of SC2r-CoVs. The nomenclature of mitochondrial DNA (mtDNA) clusters used for inferring the possible geographic origins of the samples is given in reference (Hu et al., 2020). B: Sequencing depth of clean reads remapped to MP20 using nucleotide extraction and enrichment technologies. The genome organization of the SARS-CoV-2 reference strain MN908947 is shown. C: Sliding window analysis of nucleotide sequence similarity between SARS-CoV-2 and SC2r-CoVs/SARSr-CoVs from bats and pangolins. D: Maximum likelihood tree based on the alignment of the 22 genomes with the substitution model GTR+I+G4. Bootstrap values calculated from 1000 replicates are shown.

下载: 全尺寸图片幻灯片

Figure 2. The numbers of overlapping iHVs between pangolin viral sequencing libraries at the genome-wide level (A) and at the level of the S gene (B)

下载: 全尺寸图片幻灯片

Figure 3. Recombination analysis of the pangolin-CoV-GXC sublineage

A: Similarity plot comparing 21 viruses related to MP20 from successfully captured coding sequences (CDSs). ^*: Region for which a recombination signal was detected. SC2r-CoVs are displayed in warm colors, and SARSr-CoVs are displayed in cool colors. B: Similarity plot comparing 20 viruses related to the pangolin-CoV GXP5L. ^*: Region for which a recombination signal was detected. C, D: Neighboring-joining phylogenetic trees for regions with a recombination signal based on all positions (C) and based on only the third positions (D) in the codons. The bootstrap values calculated from 1000 replicates and branch scale bars are shown.

下载: 全尺寸图片幻灯片

Table 1. The number of putative iHVs identified in the pangolin-CoV-GXC sublineage

Library	GXP1E	GXP5L	GXP5E	GXP4L	GXP2V	Total iHVs (union)
Median coverage (25%, 75%)	420 (56, 2558)	341 (37, 1547)	829 (130, 3662)	247 (126, 443)	133 (88, 254)	Total iHVs (union)
Genome-wide
Synonymous	93	61	139	15	1	217
Nonsynonymous	162	107	181	31	8	336
Frameshift	105	85	108	22	114	264
Stop-gain	3	1	0	0	0	3
Intergenic	7	9	2	3	20	29
Other	3	2	2	0	0	3
Total	373	265	432	71	143	852
S gene
Synonymous	31	8	13	1	0	36
Nonsynonymous	63	20	28	6	0	73
Frameshift	23	20	16	6	18	46
Stop-gain	1	0	0	0	0	1
Other	2	1	1	0	0	2
Total	120	49	58	13	18	158
The SRA accession Nos. are as follows: SRR11093266 (GXP1E), SRR11093267 (GXP5L), SRR11093268 (GXP5E), SRR11093269 (GXP4L), and SRR11093271 (GXP2V). Sites with coverage of ≥150X and ≥10 reads covering the alternative alleles in each library.

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