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 22 895 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.
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。.
Keywords: Diversity; Pangolin; Recombination; SARS-CoV-2; Sequencing; mtDNA.