背景与目的：蝙蝠是最重要的动物源性病原体的自然宿主之一，一些全球性重大传染病，如严重急性呼吸综合征冠状病毒（Severe acute respiratory syndrome-coronavirus, SARS-CoV）、中东呼吸综合征病毒（Middle east respiratory syndrome coronavirus, MERS-CoV）、以及近几年引起全球大流行的新型冠状病毒肺炎（Corona Virus Disease 2019, COVID-19）的病原体2019新型冠状病毒（SARS-CoV-2），都被发现直接或间接与蝙蝠有关。研究表明，蝙蝠能够携带多种高致病性病毒，却很少表现出明显的疾病症状，其独特的免疫系统可以允许其在抵抗病毒感染的同时，又不引起过激的炎症反应。然而，当前对于蝙蝠免疫系统的工作机制、基因组适应特性以及其与病毒共存甚至“共进化”的具体分子机制了解有限。此外，蝙蝠基因组中可能存在与免疫相关的重要基因模块及特异性进化特征，这对于理解蝙蝠的抗病毒能力至关重要，但具体机制尚未完全揭示。因此，还需要结合高质量的基因组数据和实验验证，系统地解析蝙蝠独特的基因组特征及抗病毒机制。

方法：本研究通过对菲菊头蝠（Rhinolophus pusillus）进行全基因组测序和分析，结合NCBI上已发表的19个其他蝙蝠基因组和另外55个哺乳动物基因组，共75个哺乳动物物种进行了系统且全面的比较基因组学分析，包括：全基因组共线性分析，基因组中整合的内源性逆转录病毒识别，基因家族扩张与收缩分析，正选择基因识别等等。随后，对筛选到的正选择基因进行了实验验证。

结果：通过三代PacBio HiFi测序及三维Hi-C测序和组装，本研究获得了一个高质量染色体级的菲菊头蝠基因组数据（测序深度达26 ×），组装的基因组大小为2.19 Gb。组装质量评估显示，菲菊头蝠组装的基因组完整性较高，93.57 %的基因组碱基序列成功对齐到31条染色体级别的scaffolds，约96.93 %的组装包含跨越兆碱基对的高质量序列。通过将二代Illumina测序数据mapping到组装的基因组，发现菲菊头蝠的基因组覆盖率很高，高达99.77 %。而且，BUSCO分析显示9,226个哺乳动物BUSCO基因中的96.0 %被组装的菲菊头蝠基因组覆盖。菲菊头蝠蛋白编码基因注释完整度也较高，约97.2 %BUSCO蛋白被完整注释。将菲菊头蝠组装和注释完成的基因组进行全基因组共线性分析，发现菲菊头蝠与马铁菊头蝠（菊头蝠科）之间存在高度的基因组共线性，而与埃及果蝠（狐蝠科）的共线性较低。此外，菲菊头蝠的7号染色体与其他哺乳动物的X染色体之间高度同源。本研究使用OrthoFinder软件，共鉴定出6,360个1:1同源基因集，并构建了涵盖75个物种的系统发育进化树，结果高度支持猛兽有蹄类假说，并估算阴翼手目与阳翼手目的分歧时间约为6.8千万年前。对20个蝙蝠基因组和7个外群哺乳动物基因组中整合的内源性逆转录病毒（endogenous retroviruse, ERVs）进行了分析。结果表明，与其他哺乳动物相比，蝙蝠基因组中Gammaretroviruses、Spumaretroviruses和Deltaretroviruses的整合情况显著更多，提示相较于其他哺乳动物，蝙蝠基因组中整合了多样化的ERVs。识别翼手目、菊头蝠超科及菊头蝠科的基因家族扩张与收缩情况，发现翼手目中与免疫相关的基因家族，例如MHC、TUBB和TRIM家族基因，发生了收缩，这其可能与蝙蝠的免疫适应性有关。随后，本研究通过分支位点模型，筛选出正选择基因，发现菊头蝠超科中与“病毒基因组复制”相关的基因受到正选择。在75个哺乳动物物种中，每个目至少有577个基因经历了正选择，其中翼手目的正选择基因对“免疫反应”过程的富集结果最显著，菊头蝠超科的正选择基因对“病毒基因组复制”过程的富集最为显著，尤其是菊头蝠超科的TRIM38基因受到的正选择作用最强。而且ACE2基因在菊头蝠科中也受到明显的正选择作用。本研究的实验结果显示，菲菊头蝠（北京）的TRIM38蛋白对EV71病毒复制表现出更强的抑制能力。这一过程可能依赖于其B-Box和Coiled-coil结构域的功能。不同地域来源的菲菊头蝠TRIM38的功能也存在变异，例如来自北京的菲菊头蝠TRIM38抗病毒功能最强，来自广西菲菊头蝠TRIM38的RING结构域缺失突变可能降低了其抗病毒能力。此外，研究成功构建了菲菊头蝠的永生化细胞系，为后续研究提供了重要的细胞模型。

结论：总体而言，本研究提供了高质量的菲菊头蝠基因组数据，揭示了其独特的基因组特征及抗病毒机制，为未来在蝙蝠进化、免疫及病毒适应性研究方面打下了坚实的基础。

Background and objectives: Bats are one of the most important natural reservoirs for zoonotic pathogens. Several major global infectious diseases, such as severe acute respiratory syndrome-coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and the pathogen of the recent global pandemic Corona Virus Disease 2019 (COVID-19), SARS-CoV-2, have been found to be directly or indirectly associated with bats. Studies have shown that bats can carry numerous highly pathogenic viruses yet rarely exhibit obvious clinical symptoms. Their unique immune system allows them to resist viral infections without triggering excessive inflammatory responses. However, the current understanding of the working mechanisms is limited in the genomic adaptive traits of bat immune systems, and the specific molecular mechanisms of its coexistence or even "co-evolution" with viruses. Furthermore, the bat genome may harbor important immune-related gene modules and specific evolutionary features that are crucial for understanding their antiviral capabilities, but the specific mechanisms have not been fully elucidated. Therefore, it is necessary to systematically analyze the unique genomic features and antiviral mechanisms of bats by combining high-quality genomic data with experimental validation.

Methods: This study conducted a systematic and comprehensive comparative genomics analysis of 75 mammalian species by whole genome sequencing and analysis of the lesser horseshoe bat (Rhinolophus pusillus), combined with 19 other bat genomes and 55 mammalian genomes published on NCBI. The analysis included whole genome collinearity analysis, identification of endogenous retroviruses (ERVs) integrated into the genome, analysis of gene family expansion and contraction, and identification of positive selection genes. Subsequently, experimental verification was conducted on the selected positive selection genes.

Results: Through third-generation PacBio HiFi sequencing and three-dimensional Hi-C sequencing and assembly, this study obtained a high-quality, chromosome-level genome assembly for Rhinolophus pusillus (sequencing depth of 26×), with an assembled genome size of 2.19 Gb. Assembly quality assessment revealed high completeness, with 93.57% of the genomic base sequences successfully assigned to 31 chromosome-level scaffolds, and approximately 96.93% of the assembly consisting of high-quality sequences spanning megabase pairs. Mapping of second-generation Illumina sequencing data to the assembled genome showed a high coverage of 99.77%. Furthermore, BUSCO analysis indicated that 96.0% of the 9,226 mammalian BUSCO genes were covered by the assembled Rhinolophus pusillus genome. The completeness of protein-coding gene annotation was also high, with approximately 97.2% of BUSCO proteins fully annotated. Whole-genome synteny analysis of the assembled and annotated Rhinolophus pusillus genome revealed a high degree of genomic synteny with the greater horseshoe bat (Rhinolophus ferrumequinum, family Rhinolophidae) but lower synteny with the Egyptian fruit bat (Rousettus aegyptiacus, family Pteropodidae). Additionally, chromosome 7 of Rhinolophus pusillus showed high homology with the X chromosome of other mammals. Using the OrthoFinder software, 6,360 1:1 single-copy orthologous gene sets were identified and were used to constructe a phylogenetic tree for the 75 species. The results strongly support the Fereuungulata hypothesis and estimated the divergence time between Yinpterochiroptera and Yangochiroptera to be approximately 68 million years ago. Analysis of endogenous retroviruses (ERVs) integrated into the genomes of 20 bat species and 7 outgroup mammalian species showed significantly more integration of Gammaretroviruses, Spumaretroviruses, and Deltaretroviruses in bats compared to other mammals, indicating that bat genomes have a more diverse range of ERV integrations. Identification of gene family expansion and contraction in Chiroptera, Rhinolophoidea, and Rhinolophidae revealed that immune-related gene families, such as MHC, TUBB, and TRIM, contracted in bats, which may be related to their immune adaptability. Subsequently, through a branch-site model, positively selected genes were identified. It was found that genes related to "viral genome replication" were under positive selection in Rhinolophoidea. Among the 75 mammalian species, at least 577 genes underwent positive selection in each order, with positively selected genes in Chiroptera being most significantly enriched in immune response processes, and positively selected genes in Rhinolophoidea being most significantly enriched in viral genome replication processes. In particular, the TRIM38 gene in Rhinolophoidea was found to be strongly under positive selection. Additionally, the ACE2 gene also showed notable positive selection in Rhinolophidae. The experimental results of this study demonstrated that the TRIM38 protein from Rhinolophus pusillus (Beijing) exhibited stronger inhibitory effects on enterovirus 71 (EV71) replication. This process is likely dependent on the functionality of its B-Box and Coiled-coil domains. Variants in the TRIM38 sequences from different geographical sources were also observed, with the TRIM38 from Beijing showing the strongest antiviral activity while the RING domain deletion mutation in the TRIM38 from Guangxi might reduce its antiviral capability. Furthermore, an immortalized cell line of Rhinolophus pusillus was successfully established, providing an important cellular model for further research. 

Conclusions: Overall, this study provides high-quality genomic data for Rhinolophus pusillus, revealing its unique genomic characteristics and antiviral mechanisms, thus establishing a solid foundation for future research on bat evolution, immunity, and viral adaptation.