Riding apoptotic bodies for cell-cell transmission by African swine fever virus
非洲猪瘟病毒通过凋亡小体进行细胞间传递

Jun Han ${}^{\mathrm{a},2}$${}^{\mathrm{a},2}$^(a,2){ }^{\mathrm{a}, 2}, and Hanchun Yang ${}^{\mathrm{a},2}$${}^{\mathrm{a},2}$^(a,2){ }^{\mathrm{a}, 2} (ID)
韩俊 ${}^{\mathrm{a},2}$${}^{\mathrm{a},2}$^(a,2){ }^{\mathrm{a}, 2} 和杨汉春 ${}^{\mathrm{a},2}$${}^{\mathrm{a},2}$^(a,2){ }^{\mathrm{a}, 2} (ID)

Edited by Bernard Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD; received June 7, 2023; accepted October 25, 2023
编辑：伯纳德·莫斯，美国国立过敏和传染病研究所，马里兰州贝塞斯达；收到日期：2023 年 6 月 7 日；接受日期：2023 年 10 月 25 日

ASFV | cell to cell transmission | apoptotic bodies | immune evasion
ASFV | 细胞间传播 | 凋亡小体 | 免疫逃逸
African swine fever virus (ASFV) is an economically devastating pathogen to the world swine industry and represents a major threat to the world swine-producing regions.
非洲猪瘟病毒（ASFV）对全球猪肉产业造成了经济上的严重损失，并对世界猪肉生产地区构成了重大威胁。
It was first reported in Africa in 1921 and then spread to Europe on three occasions during the past 100 y (1).
它于 1921 年首次在非洲被报道，随后在过去 100 年中三次传播到欧洲（1）。
In 2018, ASFV spread to China, the first country in Asia for ASF outbreaks and has since devastated the Chinese swine production, leading to colossal economic losses (2-4). Up to now, many countries in Africa, Europe, and Asia remain infected with ASFV (5-7).
在 2018 年，非洲猪瘟病毒（ASFV）传播到中国，这是亚洲首个发生非洲猪瘟疫情的国家，至今已对中国的猪肉生产造成了严重破坏，导致巨大的经济损失（2-4）。到目前为止，非洲、欧洲和亚洲的许多国家仍然受到 ASFV 的感染（5-7）。
ASFV is a nucleocytoplasmic large DNA virus with a diameter around 200 nm that is wrapped by two layers of membranes separated by capsid and is the only member of the Asfarviridae family (8-10).
ASFV 是一种直径约 200 纳米的核细胞质大型 DNA 病毒，外面包裹着两层膜，膜之间由衣壳分隔，是 Asfarviridae 家族的唯一成员（8-10）。
Clinically, ASFV can cause high fever and systematic hemorrhage of wild and domestic pigs with a mortality rate up to $100\mathrm{\%}(11,12)$$100\mathrm{\%}(11,12)$100%(11,12)100 \%(11,12). Currently, there are no effective vaccines or treatments available.
临床上，非洲猪瘟病毒（ASFV）可以导致野生和家猪高烧和系统性出血，死亡率高达 $100\mathrm{\%}(11,12)$$100\mathrm{\%}(11,12)$100%(11,12)100 \%(11,12) 。目前，没有有效的疫苗或治疗方法可用。

ASFV mainly infects cells of macrophage-monocyte lineage (13).
ASFV 主要感染巨噬细胞-单核细胞谱系的细胞（13）。
The past studies have shown that this virus can use endocytosis and macropinocytosis for host entry (13-16), with much focusing on post-receptor binding stages, such as uncoating and membrane fusion, that involve a series of viral factors (e.g., E248R, E199L, B117L, etc.) (17-20), but the early entry events such as the receptor usage and binding have remained poorly understood.
过去的研究表明，这种病毒可以通过内吞作用和大吞噬作用进入宿主（13-16），主要集中在受体结合后的阶段，如去壳和膜融合，这些过程涉及一系列病毒因子（例如，E248R、E199L、B117L 等）（17-20），但早期进入事件，如受体的使用和结合，仍然了解得很少。
Antibodies are thought to play a role in control of ASFV infection, as passive transfer of colostrum or serum antibodies from convalescent pigs can result in reduced viremia and delay the onset of clinical signs and mortality post ASFV challenge (21-26).
抗体被认为在控制非洲猪瘟病毒（ASFV）感染中发挥作用，因为从康复猪体内被动转移初乳或血清抗体可以减少病毒血症，并延迟 ASFV 挑战后临床症状和死亡的出现（21-26）。
Despite that ASFV-specific antibodies can mediate virus neutralization in plaque reduction assays, a fraction of the infective virus populations remains nonneutralizable even under the circumstance of high antibody titers $(21,26)$$(21,26)$(21,26)(21,26). Moreover, sera from pigs immunized with live-attenuated ASFV and fully protected against parental virus displayed incomplete neutralization with dose-dependent neutralizing efficacies (21, 22, 26).
尽管 ASFV 特异性抗体可以在斑块减少实验中介导病毒中和，但即使在高抗体滴度的情况下，仍有一部分感染性病毒群体保持非中和状态。此外，接种了活减毒 ASFV 并对亲本病毒完全保护的猪的血清显示出不完全中和，且中和效能呈剂量依赖性（21, 22, 26）。
Paradoxes as such suggest the existence of thus far unidentified mechanisms for ASFV dissemination and evasion of humoral immunity.
悖论本身暗示了迄今为止尚未识别的非洲猪瘟病毒传播和逃避体液免疫的机制的存在。

Extracellular vesicles (EVs) are lipid bilayer-enveloped nanoparticles that are derived from cells and found in plasma, serum, urine, and many other biophysiological fluids (27, 28).
细胞外囊泡（EVs）是由细胞衍生的脂质双层包裹的纳米颗粒，存在于血浆、血清、尿液以及许多其他生物生理液体中（27, 28）。
EVs are divided into three major subgroups by size, biogenesis, content, and function, including exosomes ( 40 to 100 nm ), microvesicles ( 100 to 500 nm ), and apoptotic bodies ( 500 to $5,000\mathrm{nm}$$5,000\mathrm{nm}$5,000nm5,000 \mathrm{~nm} ) (27-29). EV lipid membrane offers protection for their cargo transferring
EVs 根据大小、生物发生、内容和功能分为三个主要亚组，包括外泌体（40 到 100 纳米）、微囊泡（100 到 500 纳米）和凋亡小体（500 到 $5,000\mathrm{nm}$$5,000\mathrm{nm}$5,000nm5,000 \mathrm{~nm} ）（27-29）。EV 脂质膜为其转运的货物提供保护。

ASFV is a leading threat to the world pork production and has evolved a mechanism to avoid antibody neutralization, contributing to viral persistence within infected pigs.
ASFV 是全球猪肉生产的主要威胁，已经演化出一种机制以避免抗体中和，从而导致病毒在感染猪体内的持续存在。
We report here that ASFV highjacks the cellular efferocytosis pathway to ride the apoptotic bodies for cell-cell transmission of viral outer-membrane-free particles.
我们在此报告，非洲猪瘟病毒劫持细胞吞噬凋亡体的途径，以便通过凋亡体进行病毒外膜无颗粒的细胞间传播。
The finding sheds light on the mechanisms of ASFV entry and transmission and also provides a means of viral immune evasion of antibodies.
该发现揭示了非洲猪瘟病毒（ASFV）进入和传播的机制，并提供了一种病毒逃避抗体免疫的手段。

Author affiliations: ${}^a$${}^a$^(a){ }^{a} National Key Laboratory of Veterinary Public Health Security, Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People’s Republic of China; and ${}^{\mathrm{b}}$${}^{\mathrm{b}}$^(b){ }^{\mathrm{b}} China Animal Disease Control Center, Beijing 100125, People’s Republic of China
作者单位： ${}^a$${}^a$^(a){ }^{a} 中国农业大学兽医学院，国家兽医公共卫生安全重点实验室，农业农村部动物流行病学重点实验室，北京 100193，中华人民共和国；以及 ${}^{\mathrm{b}}$${}^{\mathrm{b}}$^(b){ }^{\mathrm{b}} 中国动物疾病控制中心，北京 100125，中华人民共和国

Author contributions: L.Z., J.H., and H.Y. designed research; P.G., J.W., W.W., H.W., M.Y., and Y.Q. performed research; P.G., L.Z., J.W., H.W., Y.H., Y.Z., X. Ge, and X. Guo contributed new reagents/analytic tools; P.G., L.Z., X.
作者贡献：L.Z.、J.H. 和 H.Y. 设计了研究；P.G.、J.W.、W.W.、H.W.、M.Y. 和 Y.Q. 进行了研究；P.G.、L.Z.、J.W.、H.W.、Y.H.、Y.Z.、X. Ge 和 X. Guo 贡献了新的试剂/分析工具；P.G.、L.Z.、X.
Ge, J.H., and H.Y. analyzed data; and P.G., J.H., and H.Y. wrote the paper.
葛, J.H. 和 H.Y. 分析了数据；P.G., J.H. 和 H.Y. 撰写了论文。
The authors declare no competing interest.
作者声明没有竞争利益。
This article is a PNAS Direct Submission.
这篇文章是 PNAS 直接提交的。
Copyright © 2023 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
版权 © 2023 作者。由 PNAS 出版。本文根据创意共享署名-非商业性使用-禁止演绎 4.0 许可证 (CC BY-NC-ND) 分发。
${}^1$${}^1$^(1){ }^{1} P.G., L.Z., and J.W. contributed equally to this work.
${}^1$${}^1$^(1){ }^{1} P.G.、L.Z. 和 J.W. 对本工作贡献相同。
${}^2$${}^2$^(2){ }^{2} To whom correspondence may be addressed. Email: hanx0158@cau.edu.cn or yanghanchun1@cau.edu.cn.
${}^2$${}^2$^(2){ }^{2} 通信可寄至。电子邮件：hanx0158@cau.edu.cn 或 yanghanchun1@cau.edu.cn。
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas. 2309506120/-/DCSupplemental.
本文包含在线支持信息，网址为 https://www.pnas.org/lookup/suppl/doi:10.1073/pnas. 2309506120/-/DCSupplemental。
Published November 20, 2023.
发布于 2023 年 11 月 20 日。
to recipient cells from degradation catalysts and other destructive substances that may exist in extracellular spaces. They carry cellular or viral components as cargo such as nucleic acids, proteins, and virions for intercellular communication (28, 30).
从降解催化剂和可能存在于细胞外空间的其他破坏性物质中，传递到接受细胞。它们携带细胞或病毒成分作为货物，如核酸、蛋白质和病毒颗粒，用于细胞间通信（28, 30）。
Especially, viruses could utilize EVs for cell-to-cell transmission (27, 31-36). This strategy can give many viruses the ability to evade host immune surveillance and antibody neutralization, which is conducive to the efficient viral spread and pathogenesis (31).
特别是，病毒可以利用外泌体进行细胞间传播（27，31-36）。这一策略使许多病毒能够逃避宿主的免疫监视和抗体中和，有利于病毒的高效传播和致病机制（31）。
Since ASFV has been reported to induce cellular apoptosis at late stage of infection $(37,38)$$(37,38)$(37,38)(37,38), we hypothesize that ASFV might utilize EVs for cell-to-cell transmission.
由于有报道称 ASFV 在感染后期会诱导细胞凋亡 $(37,38)$$(37,38)$(37,38)(37,38) ，我们假设 ASFV 可能利用外泌体进行细胞间传播。
We show that the apoptotic bodies (ApoBDs) derived from ASFV-infected macrophages contain viral particles, and ASFV could selectively exploit ApoBD efferocytosis as an alternative pathway for virus entry and cell-cell transmission. The detailed results are described below.
我们展示了来自非洲猪瘟病毒（ASFV）感染的巨噬细胞的凋亡小体（ApoBDs）含有病毒颗粒，ASFV 可以选择性地利用 ApoBD 的吞噬作用作为病毒进入和细胞间传播的替代途径。详细结果如下所述。

ASFV Induces Apoptosis of Primary Macrophages to Shed ApoBDs. The diameter of ASFV particles is approximately 200 nm (9).
ASFV 诱导初级巨噬细胞凋亡以释放 ApoBDs。ASFV 颗粒的直径约为 200 纳米（9）。
If the intercellular transmission of this virus happens, the apoptotic bodies (ApoBDs) will be the most ideal EVs for delivering ASFV particles considering their large sizes.
如果这种病毒的细胞间传播发生，凋亡小体（ApoBDs）将是传递非洲猪瘟病毒颗粒的最理想的外泌体，因为它们的尺寸较大。
In line with this hypothesis, ASFV encodes a number of proteins for regulating cellular apoptosis, including anti-apoptotic proteins (e.g., A179L, A224L, DP71L, EP153R, etc.) and pro-apoptotic proteins (e.g., p54, E199L, etc.) (38-41).
根据这一假设，非洲猪瘟病毒编码多种调节细胞凋亡的蛋白质，包括抗凋亡蛋白（如 A179L、A224L、DP71L、EP153R 等）和促凋亡蛋白（如 p54、E199L 等）（38-41）。
To understand the temporal dynamics of apoptotic induction, we infected primary porcine alveolar macrophages (PAMs) with ASFV strain HN09 at a multiplicity of infection (MOI) of 0.1 and monitored the activation of caspase-3 in a time course manner (Fig. 1).
为了理解凋亡诱导的时间动态，我们以感染率（MOI）为 0.1 感染了初级猪肺泡巨噬细胞（PAMs）与 ASFV HN09 株，并以时间进程的方式监测了 caspase-3 的激活（图 1）。
The cleaved form of caspase-3 was detectable around 12 hours post infection (hpi), became visibly increased at 24 hpi , and then reached the plateau around 48 to 60 hpi (Fig. 1A), a time point when the virus replication hit the peak (Fig. 1B).
在感染后约 12 小时（hpi），可检测到切割形式的 caspase-3，24 hpi 时明显增加，然后在 48 到 60 hpi 时达到平台期（图 1A），此时病毒复制达到峰值（图 1B）。
The same was observed for the infection by a green fluorescent protein (GFP)-tagged recombinant virus (ASFV-GFP), in which the MGF360-18R loci was replaced by GFP under p72 promoter while the substitution did not affect virus growth or interferon induction (SI Appendix, Fig. S1 $A-D$$A-D$A-DA-D ). The differential interference contrast (DIC) microscopy revealed in both cases the membrane-blebbing morphology of infected PAMs that gave rise to large EVs, which were similar to the size of ApoBDs (Fig. $1C$$1C$1C1 C and $E$$E$EE; SI Appendix, Fig. S1 $E$$E$EE and $G$$G$GG ). The results were further confirmed by transmission electron microscopy (TEM) showing that the large EVs were around the ASFV-positive PAMs and contained a variety of cellular components (Fig. $1D$$1D$1D1 D and SI Appendix, Fig. S1F). DNA staining confirmed that ASFV-infected PAMs underwent nuclear fragmentation and chromatin condensation (SI Appendix, Fig. S1H).
在感染绿色荧光蛋白（GFP）标记的重组病毒（ASFV-GFP）时也观察到了相同的现象，其中 MGF360-18R 位点在 p72 启动子下被 GFP 替代，而这种替代并未影响病毒的生长或干扰素的诱导（SI 附录，图 S1 $A-D$$A-D$A-DA-D ）。差分干涉对比（DIC）显微镜显示，在这两种情况下，感染的 PAMs 表现出膜突起形态，产生了与 ApoBDs 大小相似的大 EVs（图 $1C$$1C$1C1 C 和 $E$$E$EE ；SI 附录，图 S1 $E$$E$EE 和 $G$$G$GG ）。通过透射电子显微镜（TEM）进一步确认了结果，显示大 EVs 围绕 ASFV 阳性的 PAMs，并包含多种细胞成分（图 $1D$$1D$1D1 D 和 SI 附录，图 S1F）。DNA 染色确认 ASFV 感染的 PAMs 经历了核碎裂和染色质凝聚（SI 附录，图 S1H）。

The identity of the large EVs was further validated by staining of ASFV-GFP-infected PAMs with Alexa-568-conjugated Annexin V (A5-Alexa568), a cell nonpermeable fluorescent reporter for phosphatidylserine (PS) lipids being on the outer leaflet of plasma membrane as a hallmark of apoptotic cells or ApoBDs.
大型电动汽车的身份通过用 Alexa-568 标记的 Annexin V（A5-Alexa568）对 ASFV-GFP 感染的 PAM 进行染色进一步验证，Alexa-568 是一种细胞不可渗透的荧光报告分子，用于标记磷脂酰丝氨酸（PS）在细胞膜外层的存在，作为凋亡细胞或 ApoBDs 的标志。
Meanwhile, the cells were treated with staurosporine (STS) as a positive control. The entire plasma membrane of ASFV-GFP-infected or STS-treated PAMs as well as the EVs were positive for Annexin V (Fig. $1F$$1F$1F1 F and SI Appendix, Fig. S2 $A$$A$AA and B). Further three-dimensional (3D) reconstruction revealed that the GFP sphere was entirely enclosed by Annexin V-positive membranes (Fig. $1F$$1F$1F1 F and SIAppendix, Fig. S2C). Moreover, ASFV-GFP-infected PAMs continuously shed GFP-positive vesicles with increasing amount as the infection progressed as revealed by live-cell imaging (SI Appendix, Fig. S3 $A$$A$AA and $B$$B$BB and Movie S1). Thus, ASFV infection can actively lead to cell apoptosis and formation of ApoBDs.
与此同时，细胞用斯托罗霉素（STS）处理作为阳性对照。ASFV-GFP 感染的 PAMs 以及 EVs 的整个质膜对 Annexin V 呈阳性（图 $1F$$1F$1F1 F 和 SI 附录，图 S2 $A$$A$AA 和 B）。进一步的三维（3D）重建显示，GFP 球体完全被 Annexin V 阳性膜包围（图 $1F$$1F$1F1 F 和 SI 附录，图 S2C）。此外，ASFV-GFP 感染的 PAMs 随着感染的进展持续释放 GFP 阳性囊泡，数量逐渐增加，这通过活细胞成像得以揭示（SI 附录，图 S3 $A$$A$AA 和 $B$$B$BB 以及电影 S1）。因此，ASFV 感染可以主动导致细胞凋亡和 ApoBDs 的形成。

ASFV-Containing ApoBDs Can Be Swallowed by Neighboring Macrophages to Establish a Secondary Infection. We performed a more detailed time-course assay to understand the tempodynamics of ApoBD formation by using WT ASFV.
含 ASFV 的 ApoBD 可以被邻近的巨噬细胞吞噬，从而建立二次感染。我们进行了更详细的时间过程实验，以了解使用 WT ASFV 形成 ApoBD 的温动力学。
The timelapse confocal/DIC imaging showed that only a few ApoBDs were observed at early stages ( 6 to 12 hpi ) in a high-dose infection $(\mathrm{MOI}=10)$$(\mathrm{MOI}=10)$(MOI=10)(\mathrm{MOI}=10), whereas large numbers of ApoBDs were formed around 18 hpi (SI Appendix, Fig. S4 $A$$A$AA and $B$$B$BB ), fitting well with the documented viral replication cycle (42). At later time point, many cells detached from plates. In a low-dose infection (MOI $=0.1$$=0.1$=0.1=0.1 ), the ASFV-associated ApoBDs became detectable around 24 hpi and were produced in large amount around 48 to 60 hpi (SI Appendix, Fig. S5A), coinciding with detectable caspase-3 activation (Fig. $1A$$1A$1A1 A and SI Appendix, Fig. S1C). Interestingly, this phenomenon was not observed in PAMs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (SI Appendix, Fig. S5B), an arterivirus of swine that also induces cellular apoptosis (43).
时间推移共聚焦/DIC 成像显示，在高剂量感染的早期阶段（6 到 12 小时后感染），仅观察到少量 ApoBD，而在 18 小时后感染时形成了大量 ApoBD（见附录 SI，图 S4），与已记录的病毒复制周期相符（42）。在后期，许多细胞从培养板上脱落。在低剂量感染（MOI）中，ASFV 相关的 ApoBD 在 24 小时后感染时开始可检测，并在 48 到 60 小时后感染时大量产生（见附录 SI，图 S5A），与可检测的 caspase-3 活化相吻合（图和附录 SI，图 S1C）。有趣的是，这一现象在感染猪繁殖与呼吸综合症病毒（PRRSV）的 PAM 中并未观察到（见附录 SI，图 S5B），PRRSV 是一种也会诱导细胞凋亡的猪类动脉病毒（43）。
Further analyses by IFA and DIC showed that ASFV infection induced beaded-apoptopodia containing ASFV structural protein p30 and other viral proteins, and these filopodia radiated outward from the center of apoptotic cells to neighboring ASFV-negative macrophages (Fig. $2A$$2A$2A2 A and SI Appendix, Fig. S6 A-C). Further TEM analysis demonstrated that the beaded-apoptopodia contained lots of virion particles (Fig. 2 B, Top) and so were the shedded ApoBDs (Fig. 2 B, Bottom).
进一步的 IFA 和 DIC 分析显示，ASFV 感染诱导了含有 ASFV 结构蛋白 p30 和其他病毒蛋白的珠状凋亡伪足，这些伪足从凋亡细胞的中心向邻近的 ASFV 阴性巨噬细胞辐射（图 $2A$$2A$2A2 A 和 SI 附录，图 S6 A-C）。进一步的透射电子显微镜分析表明，珠状凋亡伪足中含有大量病毒颗粒（图 2 B，顶部），脱落的 ApoBDs 也是如此（图 2 B，底部）。

We used ASFV-GFP to infect PAMs at a low MOI to track the release and movement of ApoBDs and to determine whether those GFP-positive ApoBDs could be phagocytosed via neighboring cells to induce a secondary infection.
我们使用 ASFV-GFP 以低感染剂量感染 PAMs，以追踪 ApoBDs 的释放和移动，并确定这些 GFP 阳性的 ApoBDs 是否可以通过邻近细胞被吞噬，从而引发二次感染。
The time-lapse live-cell imaging showed that the infected PAMs undergo membrane-blebbing, induction of ApoBDs, and subsequent transmission of GFPpositive ApoBDs to a recipient cell to establish a productive infection (Fig. 2C and Movie S2).
时间推移活细胞成像显示，感染的 PAMs 经历了膜突起、ApoBDs 的诱导，以及随后将 GFP 阳性 ApoBDs 传递给受体细胞以建立有效感染（图 2C 和电影 S2）。
Interestingly, once the ApoBD contacted a recipient cell, it could be quickly phagocytosed; this was accompanied by quick disappearance of GFP fluorescence (likely being quenched in an acidic environment and then degraded) and then a slow reappearance of GFP fluorescence around 6 h post phagocytosis (Movie S2), suggesting the start of a new infection.
有趣的是，一旦 ApoBD 接触到受体细胞，它就会被迅速吞噬；这伴随着 GFP 荧光的快速消失（可能是在酸性环境中被淬灭然后降解），然后在吞噬后约 6 小时左右 GFP 荧光缓慢重新出现（电影 S2），这表明新感染的开始。
Representative images along the time course in the video are shown in Fig. 2C. Thus, ApoBDs can serve as a vehicle for ASFV spread in macrophages.
视频中时间过程的代表性图像如图 2C 所示。因此，ApoBDs 可以作为 ASFV 在巨噬细胞中传播的载体。

The Virions Loaded within ApoBDs Are Exclusive Particles Devoid of the Viral Outer Membrane. The TEM showed that there were three forms of ASFV particles in infected cell culture, and these include intracellular virions, extracellular virions, and ApoBD-associated virions (Fig.
在 ApoBD 中加载的病毒颗粒是缺乏病毒外膜的独特颗粒。透射电子显微镜显示，在感染的细胞培养中存在三种形式的非洲猪瘟病毒颗粒，包括细胞内病毒颗粒、细胞外病毒颗粒和与 ApoBD 相关的病毒颗粒（图）。
3A). Both intracellular and ApoBDassociated virions lacked the outer layer membrane and were hence termed as single-membrane viruses. In contrast, the extracellular virions possess the outer layer membrane and were morphologically mature and hence termed double-membrane viruses.
3A)。细胞内和与 ApoBD 相关的病毒颗粒缺乏外层膜，因此被称为单膜病毒。相比之下，细胞外病毒颗粒具有外层膜，形态上成熟，因此被称为双膜病毒。

To further characterize the biological property, we isolated ApoBDs along with the other two forms of ASFV from ASFVinfected PAMs according to a previously established differential centrifugation procedure $(33,44)$$(33,44)$(33,44)(33,44) as outlined in Fig. 3B. The isolated ApoBDs had a high quality ( $96\mathrm{\%}$$96\mathrm{\%}$96%96 \% ) as monitored by TEM, and it was rare to see the contamination by extracellular virions (Fig. 3C and SI Appendix, Table S1). In addition, both ApoBDs and extracellular virions were strongly positive for PS as analyzed by dot blot assay.
为了进一步表征生物特性，我们根据之前建立的差异离心程序从 ASFV 感染的 PAM 中分离了 ApoBDs 和其他两种形式的 ASFV，如图 3B 所示。分离的 ApoBDs 质量很高（ $96\mathrm{\%}$$96\mathrm{\%}$96%96 \% ），通过透射电子显微镜监测，几乎没有看到细胞外病毒颗粒的污染（图 3C 和 SI 附录，表 S1）。此外，通过点杂交实验分析，ApoBDs 和细胞外病毒颗粒对磷脂酰丝氨酸（PS）均呈强阳性反应。
In contrast, the signal was very weak for intracellular virions, which was suspected to be a background binding (Fig. 3D). The three fractions were all positive for ASFV nucleic acid as determined by qPCR analysis targeting ASFV B646L gene encoding p72 protein (Fig.
相反，细胞内病毒颗粒的信号非常微弱，怀疑是背景结合（图 3D）。通过针对编码 p72 蛋白的 ASFV B646L 基因的 qPCR 分析，三个分馏均对 ASFV 核酸呈阳性（图。
3E), and they were all infectious in PAMs (Fig. 3F and
3E)，它们在 PAMs 中都是传染性的（图 3F 和

Fig. 1. ASFV induces apoptosis of PAMs to shed ApoBDs. (A) Analysis of cellular apoptosis of ASFV-infected cells.
图 1. ASFV 诱导 PAMs 凋亡以释放 ApoBDs。(A) ASFV 感染细胞的细胞凋亡分析。
PAMs were mock-infected or infected with ASFV strain HN09 at an MOI of 0.1 and harvested at indicated times post infection for Western blot with indicated antibodies to caspase-3, $\beta$$\beta$beta\beta-actin, and p30. $\beta$$\beta$beta\beta-actin served as a loading control, and ASFV p30 was used as an indicator of infection. (B) Growth kinetics of ASFV HN09 in PAMs at an MOI of 0.1. © Representative images showing the formation of ApoBDs.
PAMs 被假感染或以 MOI 为 0.1 感染 ASFV HN09 株，并在感染后于指定时间收集样本进行 Western blot，使用针对 caspase-3、 $\beta$$\beta$beta\beta -actin 和 p30 的抗体。 $\beta$$\beta$beta\beta -actin 作为加载对照，ASFV p30 作为感染的指示。 (B) PAMs 中 ASFV HN09 的生长动力学，MOI 为 0.1。© 代表性图像显示 ApoBDs 的形成。
PAMs were mock-infected or infected with ASFV strain HN09 at an MOI of 0.1 and analyzed by DIC microscopy at 48 hpi . (D) Mock- and ASFV-infected PAMs (MOI $=0.1$$=0.1$=0.1=0.1 ) were fixed at 48 hpi and analyzed by TEM. The characteristic ApoBDs (asterisk) and viral factory could be visualized only in ASFV-infected cells. (E) Size distribution of the vesicles around ASFV-infected PAMs $(\mathrm{N}=100)$$(\mathrm{N}=100)$(N=100)(\mathrm{N}=100). The size of vesicles was measured via the scale bar under light microscope and electron microscope. ( $F$$F$FF ) Immunostaining and 3D reconstruction of the ApoBDs. PAMs were infected with ASFV-GFP and stained with A5-Alexa568 at 48 hpi , and the images were reconstructed by Imaris software.
PAMs 在 MOI 为 0.1 的条件下被假感染或感染了 ASFV HN09 株，并在 48 小时后通过 DIC 显微镜进行分析。(D) 在 48 小时后，假感染和 ASFV 感染的 PAMs（MOI $=0.1$$=0.1$=0.1=0.1 ）被固定并通过 TEM 进行分析。只有在 ASFV 感染的细胞中可以观察到特征性的 ApoBDs（星号）和病毒工厂。(E) ASFV 感染的 PAMs 周围囊泡的大小分布 $(\mathrm{N}=100)$$(\mathrm{N}=100)$(N=100)(\mathrm{N}=100) 。囊泡的大小通过光学显微镜和电子显微镜下的比例尺进行测量。( $F$$F$FF ) ApoBDs 的免疫染色和 3D 重建。PAMs 被 ASFV-GFP 感染，并在 48 小时后用 A5-Alexa568 染色，图像通过 Imaris 软件进行重建。
Data information: The images were acquired by Nikon A1 confocal microscope and HITACHI HT7700 electron microscope.
数据资料：图像是通过尼康 A1 共聚焦显微镜和日立 HT7700 电子显微镜获取的。

SI Appendix, Fig. S7A). As a control, the arterivirus PRRSV induced much less shedding of ApoBDs (Fig. 3F and SI Appendix, Fig. S5B).
SI 附录，图 S7A）。作为对照，猪繁殖与呼吸综合症病毒（PRRSV）诱导的 ApoBDs 排泄量要少得多（图 3F 和 SI 附录，图 S5B）。
To further distinguish the nature of ApoBDs from extracellular viruses and to rule out the possibility of contamination by extracellular viruses, we performed a precipitation and dilution assay (Fig. 3 $G$$G$GG and $H$$H$HH ). Series of dilution led to a significant reduction of virus titer in the supernatants (extracellular virions), but did not affect the viruses within the ApoBDs (Fig. 3H).
为了进一步区分 ApoBDs 的性质与细胞外病毒，并排除细胞外病毒污染的可能性，我们进行了沉淀和稀释实验（图 3 $G$$G$GG 和 $H$$H$HH ）。一系列稀释导致上清液中的病毒滴度显著降低（细胞外病毒颗粒），但对 ApoBDs 内的病毒没有影响（图 3H）。

Purified ASFV-Containing ApoBDs Can Be Directly Engulfed by Naive PAMs to Establish a Productive Infection. To further characterize the infection process of ApoBDs, the ApoBDs isolated from ASFV-infected PAMs were incubated with PAMs at $37{}^{\circ }\mathrm{C}$$37{}^{\circ }\mathrm{C}$37^(@)C37{ }^{\circ} \mathrm{C} for 2 h before fixation and staining with monoclonal antibody to p30 or swine serum to ASFV to investigate the vesicle uptake. The
纯化的含 ASFV 的 ApoBDs 可以被天真的 PAMs 直接吞噬，以建立有效感染。为了进一步表征 ApoBDs 的感染过程，从 ASFV 感染的 PAMs 中分离的 ApoBDs 在 $37{}^{\circ }\mathrm{C}$$37{}^{\circ }\mathrm{C}$37^(@)C37{ }^{\circ} \mathrm{C} 时与 PAMs 孵育 2 小时，然后固定并用单克隆抗体对 p30 或猪血清对 ASFV 进行染色，以研究囊泡的摄取。
confocal microscopy showed that the staining-positive ApoBDs could be engulfed by PAMs (Fig. $4A$$4A$4A4 A and $B$$B$BB ). The live-cell imaging showed that GFP-positive ApoBDs were engulfed by PAMs and then gradually lost the fluorescence. At 9 to 12 hpi , the cell began to accumulate visible GFP within gradually increasing fluorescence intensity, suggesting a productive infection (Fig.
共聚焦显微镜显示，染色阳性的 ApoBDs 可以被 PAMs 吞噬（图 $4A$$4A$4A4 A 和 $B$$B$BB ）。活细胞成像显示，GFP 阳性的 ApoBDs 被 PAMs 吞噬后逐渐失去荧光。在 9 到 12 小时感染后，细胞开始积累可见的 GFP，荧光强度逐渐增加，提示发生了有效感染（图。
4C and Movie S3). We also labeled the ApoBDs with A5-Alexa568, showing the signal colocalization of GFP and A5-Alexa568, and the result was the same (SI Appendix, Fig. S6D and Movie S4).
我们还用 A5-Alexa568 标记了 ApoBD，显示了 GFP 和 A5-Alexa568 的信号共定位，结果相同（SI 附录，图 S6D 和电影 S4）。
Thus, these results provide strong evidence that ApoBDs can transmit ASFV particles to naive PAMs to establish an infection.
因此，这些结果提供了有力证据表明 ApoBDs 可以将 ASFV 颗粒传递给天真的 PAMs，从而建立感染。

The production kinetics of three forms of virions was subsequently measured by multistep growth curve analysis at an MOI of 0.1 or 0.01 . The virions in ApoBDs were kept at a very low level at early stage of infection as compared with the amount of
三种病毒颗粒的生产动力学随后通过多步生长曲线分析在 0.1 或 0.01 的感染倍数下进行测量。与感染早期的病毒颗粒数量相比，ApoBDs 中的病毒颗粒保持在非常低的水平。

Fig. 2. ApoBDs derived from ASFV-infected PAMs can be swallowed by neighboring PAMs to establish a secondary infection. (A) Distribution pattern of the ASFV-positive ApoBDs.
图 2. 从 ASFV 感染的 PAM 中衍生的 ApoBD 可以被邻近的 PAM 吞噬，从而建立二次感染。(A) ASFV 阳性 ApoBD 的分布模式。
PAMs on coverslips in six-well plates were mock or infected with ASFV strain HNO9 at an MOI of 0.1. The cells were fixed, permeablized, stained with indicated antibodies, and examined by confocal and DIC microscopy at 48 hpi .
在六孔板的盖玻片上的 PAMs 被假感染或用 ASFV HNO9 株以 0.1 的感染倍数感染。细胞在 48 小时后被固定、透化、用指示抗体染色，并通过共聚焦显微镜和差分干涉对比显微镜进行观察。
The arrows indicated the spreading trend of ASFV-positive ApoBDs. (B) ASFV-infected PAMs were fixed at 48 hpi and analyzed by TEM. © Live-cell imaging of the infection dynamics of ASFV-GFP-infected PAMs and GFP-positive ApoBDs. PAMs were seeded into $35-\mathrm{mm}$$35-\mathrm{mm}$35-mm35-\mathrm{mm} dishes and infected with ASFV-GFP at an MOI of 0.01. At 36 hpi, cells were imaged for time-lapse DIC and confocal microscopy. Representative images were collected from the taken video at the indicated time points.
箭头指示了 ASFV 阳性 ApoBDs 的传播趋势。(B) 在 48 小时后固定的 ASFV 感染 PAMs 并通过透射电子显微镜分析。© ASFV-GFP 感染的 PAMs 和 GFP 阳性 ApoBDs 的感染动态活细胞成像。PAMs 被接种到 $35-\mathrm{mm}$$35-\mathrm{mm}$35-mm35-\mathrm{mm} 培养皿中，并以 0.01 的感染倍数感染 ASFV-GFP。在 36 小时后，细胞进行了时间推移的差示干涉显微镜和共聚焦显微镜成像。从所拍摄的视频中在指定时间点收集了代表性图像。
Data information: The images were acquired by Nikon A1 confocal microscope and HITACHI HT7700 electron microscope.
数据资料：图像是通过尼康 A1 共聚焦显微镜和日立 HT7700 电子显微镜获取的。
extracellular and intracellular virions, but as the infection progressed, the ApoBD-associated viral titer increased rapidly, coinciding with the induction of apoptosis (Fig. $1A$$1A$1A1 A and SI Appendix, Fig. $S1C$$S1C$S1CS 1 C ), and reached to a level comparable to the amount of intracellular virions at 36 to 48 hpi (Fig. 4 D ). At the end of infection, the ApoBD-associated virions became a predominant form (Fig. $4D$$4D$4D4 D ).
细胞外和细胞内病毒颗粒，但随着感染的进展，ApoBD 相关的病毒滴度迅速增加，伴随着凋亡的诱导（图 $1A$$1A$1A1 A 和 SI 附录，图 $S1C$$S1C$S1CS 1 C ），并在感染后 36 到 48 小时达到了与细胞内病毒颗粒相当的水平（图 4 D）。在感染结束时，ApoBD 相关的病毒颗粒成为主要形式（图 $4D$$4D$4D4 D ）。

We also investigated the effect of intervention of cell apoptosis or ApoBD formation on viral propagation.
我们还研究了细胞凋亡或 ApoBD 形成的干预对病毒传播的影响。
We used two drugs, namely Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, and sertraline (Sert), a well-described antidepressant drug that was identified as an inhibitor of ApoBD formation without affecting apoptosis and membrane blebs (45).
我们使用了两种药物，即 Z-VAD-FMK，一种细胞可渗透的全酶抑制剂，以及舍曲林（Sert），一种已知的抗抑郁药，已被确定为 ApoBD 形成的抑制剂，而不影响细胞凋亡和膜泡（45）。
Treatment with either Z-VAD-FMK or Sert barely impaired the ASFV replication but specifically suppressed the ApoBD formation (SI Appendix, Fig. S7 $B$$B$BB and $C$$C$CC ). As a result, this resulted in a significant reduction of ApoBD-associated ASFV, but not the total and intracellular virus yield (SI Appendix, Fig. S7C).
使用 Z-VAD-FMK 或 Sert 的治疗对 ASFV 复制几乎没有影响，但特异性抑制了 ApoBD 的形成（SI 附录，图 S7 $B$$B$BB 和 $C$$C$CC ）。因此，这导致 ApoBD 相关的 ASFV 显著减少，但对总病毒和细胞内病毒产量没有影响（SI 附录，图 S7C）。
Thus, the induction of ApoBDs is not necessary for ASFV replication, but rather serves as a purpose for viral cell-cell transmission.
因此，ApoBDs 的诱导对于 ASFV 复制并不是必需的，而是作为病毒细胞间传播的目的。

ApoBD-Mediated Viral Transmission Is Dependent on Both the PS Lipids and Efferocytosis Receptor TIM4.
ApoBD 介导的病毒传播依赖于 PS 脂质和吞噬凋亡细胞受体 TIM4。
Phosphatidylserine (PS) is a classical “eat-me” signal that normally resides in the inner leaflet of the plasma membrane but flips to the outer membrane of dying cells and ApoBDs (46).
磷脂酰丝氨酸（PS）是一种经典的“吃我”信号，通常位于细胞膜的内层，但在死亡细胞和 ApoBDs 中会翻转到外层（46）。
This signal can be recognized via phagocyte receptors, i.e., Gas6, MFG-E8, TIM4, etc., and masked via PS-binding protein Annexin V $(46,47)$$(46,47)$(46,47)(46,47).
该信号可以通过吞噬细胞受体识别，即 Gas6、MFG-E8、TIM4 等，并通过结合磷脂酰丝氨酸的蛋白质 Annexin V 掩盖。
We prepared intracellular virions, extracellular virions, and ASFV-containing ApoBDs and incubated them, respectively, with Annexin V protein prior to exposure to naive PAMs. Strikingly, treatment with Annexin V significantly inhibited ApoBD-mediated ASFV infection in a dose-dependent manner (Fig.
我们制备了细胞内病毒颗粒、细胞外病毒颗粒和含有非洲猪瘟病毒的 ApoBD，并在暴露于天真 PAM 之前，分别与 Annexin V 蛋白孵育。显著的是，使用 Annexin V 处理显著抑制了 ApoBD 介导的非洲猪瘟病毒感染，且呈剂量依赖性（图）。
$5A-D$$5A-D$5A-D5 A-D ). It also exhibited an inhibitory effect on extracellular virions, which is not surprising as they also were positive for PS in the out leaflet (Fig. 3D). As expected, the treatment did not have effect on the infection of intracellular virions at all (Fig. $5A$$5A$5A5 A and $B$$B$BB ). Thus, the PS is an important factor mediating the infection of both extracellular and ApoBD-associated ASFV infection.
$5A-D$$5A-D$5A-D5 A-D ）。它还对细胞外病毒颗粒表现出抑制作用，这并不令人惊讶，因为它们在外侧叶中也对 PS 呈阳性（图 3D）。如预期的那样，治疗对细胞内病毒颗粒的感染没有任何影响（图 $5A$$5A$5A5 A 和 $B$$B$BB ）。因此，PS 是介导细胞外和 ApoBD 相关 ASFV 感染的重要因素。

We next determined whether the ApoBD-associated ASFV required the efferocytosis receptors on the PAMs for infection.
我们接下来确定了与 ApoBD 相关的 ASFV 是否需要 PAMs 上的吞噬凋亡细胞受体进行感染。

Fig. 3. Purified ApoBDs from ASFV-infected PAMs contain infectious virions. (A) Presence of different forms of virions in ASFV-infected PAMs. (B) Schematic diagram of the procedure for purifying ApoBDs via differential centrifugation.
图 3. 从 ASFV 感染的 PAM 中纯化的 ApoBDs 含有感染性病毒颗粒。(A) ASFV 感染的 PAM 中不同形式病毒颗粒的存在。(B) 通过差速离心纯化 ApoBDs 的程序示意图。
PAMs seeded T-25 flask were infected with ASFV strain HN09 at an MOI of 0.1 . At 60 hpi , the cells and culture were harvested for isolation of ApoBDs.
PAMs 接种的 T-25 培养瓶在 MOI 为 0.1 的条件下感染了 ASFV HN09 株。在 60 小时后，收获细胞和培养液以分离 ApoBDs。
© TEM analysis of the isolated ApoBDs, extracellular virions, and intracellular virions from ASFV-infected PAMs. (D) Dot blot analysis of PS of the three forms of virions.
© 对从 ASFV 感染的 PAMs 中分离的 ApoBDs、细胞外病毒颗粒和细胞内病毒颗粒进行的透射电子显微镜分析。(D) 三种病毒颗粒的磷脂点杂交分析。
The purified virions were resuspended in phosphate buffered saline (PBS) (not to destroy the membrane) or lysed in Radio-Immunoprecipitation Assay (RIPA) buffer, and then spotted onto nitrocellulose membranes, followed by detection with indicated antibodies.
纯化的病毒颗粒被重悬于磷酸盐缓冲盐水（PBS）（以免破坏膜）或在放射免疫沉淀实验（RIPA）缓冲液中裂解，然后点样到硝酸纤维素膜上，随后用指示的抗体进行检测。
PBS or RIPA was spotted as a negative control. (E) qPCR analysis of ASFV nucleic acid in different fractions. ( $F$$F$FF ) Titration of ASFV and PRRSV load of different fractions in PAMs by ${\mathrm{TCID}}_{50}$${\mathrm{TCID}}_{50}$TCID_(50)\mathrm{TCID}_{50}. (G) Schematic presentation of the precipitation and dilution assay. The ApoBD-associated and extracellular virions were isolated same as $(B)$$(B)$(B)(B) and centrifuged at $3,000\mathrm{g}$$3,000\mathrm{g}$3,000g3,000 \mathrm{~g} for 20 min to collect the supernatant, and the pellet was then resuspended by ${\mathrm{RPMI}}^{-1640}$${\mathrm{RPMI}}^{-1640}$RPMI^(-1640)\mathrm{RPMI}^{-1640}. $(H){\mathrm{TCID}}_{50}$$(H){\mathrm{TCID}}_{50}$(H)TCID_(50)(H) \mathrm{TCID}_{50} analysis of virus titer after dilution. Data information: The images were acquired by HITACHI HT7700 electron microscope.
PBS 或 RIPA 被视为阴性对照。(E) 不同分馏中 ASFV 核酸的 qPCR 分析。( $F$$F$FF ) PAMs 中不同分馏的 ASFV 和 PRRSV 载量的滴定由 ${\mathrm{TCID}}_{50}$${\mathrm{TCID}}_{50}$TCID_(50)\mathrm{TCID}_{50} 进行。(G) 沉淀和稀释实验的示意图。ApoBD 相关和细胞外病毒颗粒的分离与 $(B)$$(B)$(B)(B) 相同，并在 $3,000\mathrm{g}$$3,000\mathrm{g}$3,000g3,000 \mathrm{~g} 下离心 20 分钟以收集上清液，然后用 ${\mathrm{RPMI}}^{-1640}$${\mathrm{RPMI}}^{-1640}$RPMI^(-1640)\mathrm{RPMI}^{-1640} 重悬沉淀。 $(H){\mathrm{TCID}}_{50}$$(H){\mathrm{TCID}}_{50}$(H)TCID_(50)(H) \mathrm{TCID}_{50} 稀释后病毒滴度的分析。数据说明：图像由 HITACHI HT7700 电子显微镜获取。

We tested several antibodies against efferocytosis receptors such as Gas6, MFG-E8, and TIM4 but unfortunately only the anti-TIM4 antibody reacted well with PAMs. We found that pretreatment of PAMs with antibodies to TIM4 significantly inhibited ApoBD-mediated ASFV infectivity, and this operated in a dose-dependent manner (Fig.
我们测试了几种针对吞噬细胞受体的抗体，如 Gas6、MFG-E8 和 TIM4，但不幸的是，只有抗 TIM4 抗体与 PAMs 反应良好。我们发现，用抗 TIM4 抗体预处理 PAMs 显著抑制了 ApoBD 介导的 ASFV 感染性，并且这种抑制作用呈剂量依赖性（图）。
$6A-C$$6A-C$6A-C6 A-C ) and that the infectivity of the extracellular virions could also be inhibited to some extent about
$6A-C$$6A-C$6A-C6 A-C ）并且细胞外病毒颗粒的感染性在某种程度上也可以被抑制

40 to $50\mathrm{\%}$$50\mathrm{\%}$50%50 \% (Fig. $6A$$6A$6A6 A and $B$$B$BB ). In contrast, the infectivity of the intracellular ASFV was not affected as expected (Fig. $6A$$6A$6A6 A and $B$$B$BB ). Meanwhile, overexpression of TIM4 or MFG-E8 significantly increased the viral production in extracellular and ApoBD-associated ASFV-infected wild boar lung (WSL) cells by about half a log (Fig. $6D$$6D$6D6 D and $E$$E$EE ), suggesting that TIM4 and MFG-E8 are critical factors for ASFV entry into cells. Together, the above results
40 到 $50\mathrm{\%}$$50\mathrm{\%}$50%50 \% （图 $6A$$6A$6A6 A 和 $B$$B$BB ）。相比之下，细胞内非洲猪瘟病毒（ASFV）的感染性并未如预期那样受到影响（图 $6A$$6A$6A6 A 和 $B$$B$BB ）。与此同时，TIM4 或 MFG-E8 的过表达显著增加了在细胞外和与 ApoBD 相关的 ASFV 感染的野猪肺（WSL）细胞中的病毒产量，约增加了半个对数（图 $6D$$6D$6D6 D 和 $E$$E$EE ），这表明 TIM4 和 MFG-E8 是 ASFV 进入细胞的关键因素。综上所述，以上结果

Fig. 4. The purified ASFV-containing ApoBDs can transmit ASFV and establish a productive infection. PAMs grown on coverslips in six-well plates were incubated with the purified ApoBDs from WT ASFV-infected PAMs at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} for 2 h , followed by cell fixation, permeablization, and staining with the antibodies to p30 (A) or swine serum to ASFV (B).
图 4. 纯化的含 ASFV 的 ApoBDs 可以传播 ASFV 并建立有效感染。在六孔板的盖玻片上培养的 PAMs 与来自 WT ASFV 感染的 PAMs 的纯化 ApoBDs 在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 下孵育 2 小时，随后进行细胞固定、通透化和用抗 p30 抗体(A)或猪血清对 ASFV 进行染色(B)。
© Live-cell imaging of the infection dynamics of the ApoBDs purified from ASFV-GFP-infected PAMs. Representative images were collected from the taken video at indicated time points. Data information: The images were acquired by Nikon A1 confocal microscope. ( $D$$D$DD ) The dynamics of viral load in different fractions. PAMs were infected with WT ASFV or ASFV-GFP at an MOI of 0.1 or 0.01 . At indicated time points, the three forms of virions were isolated and titrated by ${\mathrm{TCID}}_{50}$${\mathrm{TCID}}_{50}$TCID_(50)\mathrm{TCID}_{50} in PAMs.
© 从 ASFV-GFP 感染的 PAM 中纯化的 ApoBDs 的感染动态的活细胞成像。代表性图像是从所拍摄的视频中在指定时间点收集的。数据说明：图像是通过尼康 A1 共聚焦显微镜获取的。( $D$$D$DD ) 不同分馏中病毒载量的动态。PAM 以 0.1 或 0.01 的 MOI 感染 WT ASFV 或 ASFV-GFP。在指定时间点，三种形式的病毒颗粒被分离并通过 ${\mathrm{TCID}}_{50}$${\mathrm{TCID}}_{50}$TCID_(50)\mathrm{TCID}_{50} 在 PAM 中滴定。
suggest that the ASFV-containing ApoBDs can utilize the conventional apoptotic uptake pathway to cell-to-cell transmission.
建议含有 ASFV 的 ApoBDs 可以利用常规的凋亡摄取途径进行细胞间传播。

ApoBD-Mediated Viral Transmission Is Fully Resistant to Swine Sera to ASFV. We investigated ApoBD-mediated ASFV transmission in the presence of swine serum to ASFV. The serum had an IFA titer of more than 50,000 (Fig. 7A) and could inhibit
ApoBD 介导的病毒传播对非洲猪瘟病毒的猪血清完全耐受。我们研究了在非洲猪瘟病毒存在下，ApoBD 介导的非洲猪瘟病毒传播。该血清的 IFA 滴度超过 50,000（图 7A），并能够抑制
the infection efficiency of intracellular virions by about $64\mathrm{\%}$$64\mathrm{\%}$64%64 \% and reduced the virion production by about $0.5\log$$0.5\log$0.5 log0.5 \log but displayed no effect on extracellular and ApoBD-associated virions (Fig. 7 $B-D)$$B-D)$B-D)B-D). However, when the membrane integrity of the ApoBDs was disrupted by three cycles of quick freeze-thaw, the released singlemembrane virions became susceptible to serum neutralization, and the anti-ASFV serum could inhibit the infectivity by about 53%
细胞内病毒颗粒的感染效率降低了约 $64\mathrm{\%}$$64\mathrm{\%}$64%64 \% ，病毒颗粒的产生减少了约 $0.5\log$$0.5\log$0.5 log0.5 \log ，但对细胞外和 ApoBD 相关病毒颗粒没有影响（图 7 $B-D)$$B-D)$B-D)B-D) ）。然而，当 ApoBD 的膜完整性通过三次快速冷冻-解冻被破坏时，释放的单膜病毒颗粒变得易受血清中和，抗 ASFV 血清可以抑制感染性约 53%。

Fig. 5. Blocking PS lipids suppresses ApoBD-mediated ASFV infection. Different forms of ASFV were isolated at 48 hpi from PAMs infected with ASFV strain HN09 at an MOI of 0.1 and then incubated, respectively, with Annexin V protein ( $5\mu \mathrm{g}/\mathrm{mL}$$5\mu \mathrm{g}/\mathrm{mL}$5mug//mL5 \mu \mathrm{~g} / \mathrm{mL} ) prior to exposure to naive PAMs seeded in 24 -well plates. The effect on viral replication was determined by Western blot $(A)$$(A)$(A)(A) and IFA analysis $(B)$$(B)$(B)(B). The infection efficiency was expressed as proportion of p30-positive cells via comparing Annexin V-treated group with control group, respectively. © Dose-dependent effect of Annexin V on ASFV infection by Western blot analysis.
图 5. 阻断 PS 脂质抑制 ApoBD 介导的 ASFV 感染。不同形式的 ASFV 在感染 ASFV HN09 株的 PAMs 中于 48 小时后分离，感染的 MOI 为 0.1，然后分别与 Annexin V 蛋白（ $5\mu \mathrm{g}/\mathrm{mL}$$5\mu \mathrm{g}/\mathrm{mL}$5mug//mL5 \mu \mathrm{~g} / \mathrm{mL} ）孵育，随后暴露于接种在 24 孔板中的天真 PAMs。通过 Western blot $(A)$$(A)$(A)(A) 和 IFA 分析 $(B)$$(B)$(B)(B) 确定对病毒复制的影响。感染效率通过比较 Annexin V 处理组与对照组中 p30 阳性细胞的比例来表示。© Western blot 分析显示 Annexin V 对 ASFV 感染的剂量依赖性效应。
The relative band density of p30 was expressed as percentage compared to the untreated ASFV control (lane 2) after being normalized against $\beta$$\beta$beta\beta-actin in the corresponding lane. (D) Dose-dependent effect of Annexin V on the infection of ApoBD-associated virions by IFA analysis. Data information: Error bars indicate means $\pm$$\pm$+-\pm SDs.
p30 的相对带密度以百分比表示，与未处理的 ASFV 对照（第 2 条）相比，经过与相应条中的 $\beta$$\beta$beta\beta -肌动蛋白标准化后得出。(D) Annexin V 对 ApoBD 相关病毒感染的剂量依赖性影响，通过 IFA 分析得出。数据说明：误差条表示均值 $\pm$$\pm$+-\pm SD。
(SI Appendix, Fig. S8A). As expected, the virions released from ApoBDs were not susceptible to the treatment of either Annexin V or anti-TIM4 antibody (SI Appendix, Fig. S8 B and C).
（SI 附录，图 S8A）。正如预期，从 ApoBDs 释放的病毒颗粒对 Annexin V 或抗 TIM4 抗体的处理均不敏感（SI 附录，图 S8 B 和 C）。
Thus, the ApoBD-mediated antibody evasion depends on its membrane integrity, and the findings highlight efferocytosis as a critical means for ASFV evasion of antibody neutralization.
因此，ApoBD 介导的抗体逃逸依赖于其膜的完整性，研究结果强调了吞噬凋亡细胞作为非洲猪瘟病毒逃避抗体中和的关键手段。

Clinical infections by ASFV often cause hemorrhagic fever of pigs with high fatality and can also lead to persistent infections in the presence of antibodies for years (21, 22, 48). Prominently, immune swine sera are not capable of mediating a complete neutralization
临床感染非洲猪瘟病毒（ASFV）通常会导致猪只出现高致死率的出血热，并且在存在抗体的情况下可能导致持续感染多年（21, 22, 48）。值得注意的是，免疫猪血清无法实现完全中和。
of ASFV in vitro $(21,22)$$(21,22)$(21,22)(21,22), and the mechanisms involved have remained not fully understood. In this study, we report that ASFV takes advantage of the cellular apoptotic pathway and rides the ApoBDs as a form of delivering vehicles for cell-cell transmission.
ASFV 在体外的研究 $(21,22)$$(21,22)$(21,22)(21,22) ，其涉及的机制仍未完全理解。在本研究中，我们报告 ASFV 利用细胞凋亡途径，并借助 ApoBDs 作为细胞间传递的载体。
These vesicles contain infectious but outer layer membrane-free viral particles and are resistant to treatment of neutralizing antibodies. In addition, we identified PS and TIM4 as critical host factors for mediating virus transmission.
这些囊泡含有感染性但没有外层膜的病毒颗粒，并且对中和抗体的治疗具有抵抗力。此外，我们确定了 PS 和 TIM4 作为介导病毒传播的关键宿主因子。
A model for ASFV cell-to-cell transmission is proposed in Fig. 8.
图 8 中提出了一种非洲猪瘟病毒细胞间传播的模型。

Extracellular vesicles play a critical role in intercellular communications by transporting intracellular biomolecules (nucleic acids, proteins, and lipids) $(28,30)$$(28,30)$(28,30)(28,30). Increasing evidence has indicated that many viruses use this mechanism for spread in vivo $(27,31)$$(27,31)$(27,31)(27,31).
细胞外囊泡在细胞间通讯中发挥着关键作用，通过运输细胞内生物分子（核酸、蛋白质和脂质） $(28,30)$$(28,30)$(28,30)(28,30) 。越来越多的证据表明，许多病毒利用这一机制在体内传播 $(27,31)$$(27,31)$(27,31)(27,31) 。

Fig. 6. ApoBD-mediated ASFV infection is dependent on the efferocytosis receptor TIM4. (A) PAMs in 24-well plates were incubated with antibodies to TIM4 $(10\mu \mathrm{g}/\mathrm{mL})$$(10\mu \mathrm{g}/\mathrm{mL})$(10 mug//mL)(10 \mu \mathrm{~g} / \mathrm{mL}) or isotype $\mathrm{lgG}$$\mathrm{lgG}$lgG\operatorname{lgG} for 1 h and then exposed to three forms of virions before performing Western blot analysis at 12 hpi . ( $B$$B$BB ) The same as ( $A$$A$AA ), except by IFA analysis. The infection efficiency was expressed as proportion of p30-positive cells via comparing anti-TIM4 antibody treated group with that of isotype IgG-treated group, respectively. © Dose-dependent analysis.
图 6. ApoBD 介导的 ASFV 感染依赖于吞噬受体 TIM4。(A) 在 24 孔板中的 PAMs 与 TIM4 抗体 $(10\mu \mathrm{g}/\mathrm{mL})$$(10\mu \mathrm{g}/\mathrm{mL})$(10 mug//mL)(10 \mu \mathrm{~g} / \mathrm{mL}) 或同型对照 $\mathrm{lgG}$$\mathrm{lgG}$lgG\operatorname{lgG} 孵育 1 小时，然后暴露于三种病毒颗粒中，随后在 12 小时后进行 Western blot 分析。( $B$$B$BB ) 与( $A$$A$AA )相同，除了采用 IFA 分析。感染效率通过比较抗 TIM4 抗体处理组与同型 IgG 处理组中 p30 阳性细胞的比例来表示。© 剂量依赖性分析。
The same as (A) except that different doses of anti-TIM4 antibody was used. The relative band density of p30 was expressed as percentage compared to the ASFV infection control (lane 1) after being normalized against $\beta$$\beta$beta\beta-actin in the corresponding lane. ( $D$$D$DD and $E$$E$EE ) Effect of overexpression of TIM4 or MFG-E8 on the replication of ASFV in WSL-R4 cells. WSL-R4 cells in 12-well plates were transfected with pCMV-TIM4-HA, pCMV-MFG-E8-HA, or vector control ( $1.5\mu \mathrm{g}$$1.5\mu \mathrm{g}$1.5 mug1.5 \mu \mathrm{~g} ). At 24 h posttransfection, the cells were subjected to Western blot analysis with antibodies to HA tag $(D)$$(D)$(D)(D) or infected with three forms of virions at an MOI of 1.0 for 24 h before being harvested for titration $(E)$$(E)$(E)(E). Data information: Error bars indicate means $\pm$$\pm$+-\pm SDs.
与(A)相同，只是使用了不同剂量的抗 TIM4 抗体。p30 的相对带密度以百分比表示，与 ASFV 感染对照（第 1 条）相比，在相应条带的 $\beta$$\beta$beta\beta -肌动蛋白下进行标准化。( $D$$D$DD 和 $E$$E$EE ) TIM4 或 MFG-E8 的过表达对 WSL-R4 细胞中 ASFV 复制的影响。将 WSL-R4 细胞在 12 孔板中转染 pCMV-TIM4-HA、pCMV-MFG-E8-HA 或载体对照（ $1.5\mu \mathrm{g}$$1.5\mu \mathrm{g}$1.5 mug1.5 \mu \mathrm{~g} ）。在转染后 24 小时，细胞接受 HA 标签抗体 $(D)$$(D)$(D)(D) 的西方印迹分析，或以 MOI 为 1.0 感染三种形式的病毒颗粒 24 小时，然后收集进行滴定 $(E)$$(E)$(E)(E) 。数据说明：误差条表示均值 $\pm$$\pm$+-\pm SD。

Currently, most studies have focused on the utilization of exosomes as an efficient cell-to-cell transmission system, and this is exemplified by many small-to-medium size viruses, even including those nonenveloped viruses, such as hepatitis A virus (HAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HIV, Hepatitis C virus (HCV), herpesviruses, polyomavirus, rotaviruses, noroviruses, and so on (27, 31-33, 35, 36, 49-51).
目前，大多数研究集中在将外泌体作为一种有效的细胞间传递系统的利用上，这在许多小到中等大小的病毒中得到了体现，甚至包括一些非包膜病毒，如甲型肝炎病毒（HAV）、严重急性呼吸综合症冠状病毒 2 型（SARS-CoV-2）、人类免疫缺陷病毒（HIV）、丙型肝炎病毒（HCV）、疱疹病毒、多瘤病毒、轮状病毒、诺如病毒等（27, 31-33, 35, 36, 49-51）。
However, host cells also shed other types of vesicles, including macrovesicles and ApoBDs (28, 31). For the latter, ApoBDs are large vesicles that originate from apoptotic cells and contain residual ingredients of dying cells with a diameter of 500 to $5,000\mathrm{nm}$$5,000\mathrm{nm}$5,000nm5,000 \mathrm{~nm} (46). These vesicles are normally phagocytosed by macrophages to prevent deleterious
然而，宿主细胞还会释放其他类型的囊泡，包括大囊泡和 ApoBDs（28，31）。对于后者，ApoBDs 是来自凋亡细胞的大囊泡，包含死亡细胞的残余成分，直径为 500 到 $5,000\mathrm{nm}$$5,000\mathrm{nm}$5,000nm5,000 \mathrm{~nm} （46）。这些囊泡通常被巨噬细胞吞噬，以防止有害的影响。
impact on the surroundings $(46,47)$$(46,47)$(46,47)(46,47). Although it is common that many viruses use apoptotic mimicry to enhance virus entry and spread $(33,34,52-54)$$(33,34,52-54)$(33,34,52-54)(33,34,52-54), this mechanism in most reported cases is carried out in the form of progeny virions by packing PS into viral envelope when viruses bud through the cell plasma membrane (e.g., Ebola virus, vaccinia virus, flaviviruses, arena viruses, etc.) $(34,55-57)$$(34,55-57)$(34,55-57)(34,55-57). On the other hand, the reports regarding ApoBDs as a form of viral transmission vehicles are scarce.
对周围环境的影响 $(46,47)$$(46,47)$(46,47)(46,47) 。虽然许多病毒常常利用凋亡模拟来增强病毒的进入和传播 $(33,34,52-54)$$(33,34,52-54)$(33,34,52-54)(33,34,52-54) ，但在大多数报告的案例中，这一机制是通过在病毒从细胞质膜出芽时将磷脂酰丝氨酸（PS）包装到病毒包膜中以子代病毒的形式进行的（例如，埃博拉病毒、牛痘病毒、黄病毒、沙粒病毒等） $(34,55-57)$$(34,55-57)$(34,55-57)(34,55-57) 。另一方面，关于 ApoBDs 作为病毒传播载体的报告很少。
One recent study documented that avian influenza virus (AIV) can utilize the monocyte apoptotic bodies as vehicles for viral propagation (58), but it lacks intuitive evidence, such as using live-cell imaging, to prove AIV transmission via ApoBDs. In this study, we provide strong
一项最近的研究记录了禽流感病毒（AIV）可以利用单核细胞凋亡小体作为病毒传播的载体（58），但缺乏直观证据，例如使用活细胞成像来证明 AIV 通过 ApoBDs 传播。在本研究中，我们提供了强有力的证据。

Fig. 7. ApoBD-mediated viral transmission is fully resistant to swine sera to ASFV. (A) IFA analysis of the titer of swine anti-ASFV serum in PAMs with the antibody to p 30 as a control. ( $B-D$$B-D$B-DB-D ) Intracellular virions, extracellular virions, and ASFV-containing ApoBDs were incubated with negative swine serum (-) or ASFV antibody positive swine serum (+) at a volume ratio of $1:1$$1:1$1:11: 1 at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} for 1 h . The mixtures were then incubated with naive PAMs at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} for 1 h followed by washes and addition of culture medium. (B) At 12 hpi , the cells were fixed and stained with antibodies to p30 for IFA.
图 7. ApoBD 介导的病毒传播对 ASFV 的猪血清完全耐受。(A) 在 PAM 中使用针对 p30 的抗体作为对照进行的猪抗 ASFV 血清的 IFA 分析。( $B-D$$B-D$B-DB-D ) 细胞内病毒颗粒、细胞外病毒颗粒和含 ASFV 的 ApoBD 与阴性猪血清(-)或 ASFV 抗体阳性猪血清(+)以 $1:1$$1:1$1:11: 1 的体积比在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 下孵育 1 小时。然后将混合物在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 下与天真 PAM 孵育 1 小时，随后洗涤并添加培养基。(B) 在 12 小时后，细胞被固定并用针对 p30 的抗体进行 IFA 染色。
© The infection efficiency of three virion forms was expressed as proportion of p30-positive cells by comparing sera ( + ) group with sera (-) group, respectively. (D) The same as above, but the cells were harvested at 24 hpi for viral titration by ${\mathrm{TCID}}_{50}$${\mathrm{TCID}}_{50}$TCID_(50)\mathrm{TCID}_{50}. Data information: Error bars indicate means $\pm$$\pm$+-\pm SDs.
© 三种病毒颗粒形式的感染效率通过比较血清（+）组与血清（-）组中 p30 阳性细胞的比例来表示。(D) 同上，但细胞在 24 小时感染后被收集以进行病毒滴度测定。数据说明：误差条表示均值 $\pm$$\pm$+-\pm 标准差。
evidence to show that that ASFV makes use of ApoBDs for virus spread between macrophages (Figs. 2-4).
证据表明非洲猪瘟病毒利用 ApoBDs 在巨噬细胞之间传播（图 2-4）。

The establishment of ApoBDs as an efficient transmission route is supported by three lines of evidence. The confocal time-elapse
ApoBDs 作为一种高效传输途径的建立得到了三条证据的支持。共聚焦时间延迟
cell imaging provides the real-time, direct evidence to show the dynamic process of apoptotic induction and cell-to-cell transmission by using a GFP-tagged virus as a model organism (Movie S2).
细胞成像提供了实时、直接的证据，展示了使用 GFP 标记病毒作为模型生物的凋亡诱导和细胞间传播的动态过程（电影 S2）。
The second line of evidence comes from characterization of the in vitro purified ApoBDs. Its identity and purity were confirmed by A5-Alexa568 staining, TEM, IFA, and dot blotting (Figs. $1F,2B$$1F,2B$1F,2B1 F, 2 B, and $3C-F$$3C-F$3C-F3 C-F ). Moreover, a modified dilution assay was used to further distinguish ApoBD-containing virions from the extracellular viruses (Fig. $3G$$3G$3G3 G and $H$$H$HH ). The dilutions led to a decrease titer of extracellular free viruses but not the infectivity of virions within ApoBDs (Fig. $3G$$3G$3G3 G and $H$$H$HH ). The transmission was further verified by live-cell imaging (Movies S2-S4). The third piece of evidence comes from pharmacological and genetics approaches.
第二条证据来自对体外纯化的 ApoBDs 的表征。其身份和纯度通过 A5-Alexa568 染色、透射电子显微镜（TEM）、免疫荧光分析（IFA）和点杂交（dot blotting）得到了确认（图 $1F,2B$$1F,2B$1F,2B1 F, 2 B 和 $3C-F$$3C-F$3C-F3 C-F ）。此外，使用了一种改进的稀释实验，以进一步区分含 ApoBD 的病毒颗粒与细胞外病毒（图 $3G$$3G$3G3 G 和 $H$$H$HH ）。稀释导致细胞外自由病毒的滴度下降，但不影响 ApoBD 内病毒颗粒的感染性（图 $3G$$3G$3G3 G 和 $H$$H$HH ）。通过活细胞成像进一步验证了传播（电影 S2-S4）。第三条证据来自药理学和遗传学方法。
Treatment with either Annexin V or anti-TIM4 antibody significantly suppressed the ApoBD-mediated ASFV infection, but not on the infections by virions devoid of the outer membrane (Figs. 5 and $6A-C$$6A-C$6A-C6 A-C and SI Appendix, Fig. S8 $B$$B$BB and $C$$C$CC ). Likewise, the swine sera to ASFV exerted no effect on the ApoBD-mediated transmission, but could partially act on the single-membrane virions (Fig. 7 and SI Appendix, Fig. S8A).
使用 Annexin V 或抗 TIM4 抗体的治疗显著抑制了 ApoBD 介导的 ASFV 感染，但对缺乏外膜的病毒感染没有影响（图 5 和 $6A-C$$6A-C$6A-C6 A-C 以及 SI 附录，图 S8 $B$$B$BB 和 $C$$C$CC ）。同样，猪血清对 ASFV 对 ApoBD 介导的传播没有影响，但对单膜病毒可以部分起作用（图 7 和 SI 附录，图 S8A）。
These findings suggest that the entry mechanism of single-membrane virions is quite different from ApoBD-associated and the extracellular viruses (double membrane).
这些发现表明，单膜病毒颗粒的进入机制与 ApoBD 相关的病毒和细胞外病毒（双膜）截然不同。

The finding of ApoBD-mediated virus spread provides further insight into the ASFV tropism to macrophages.
ApoBD 介导的病毒传播的发现进一步揭示了非洲猪瘟病毒对巨噬细胞的嗜性。
It has been reported that ASFV prefers to infect the monocyte-derived macrophages at an intermediate to late stage of differentiation and that macrophages are about five times more susceptible to ASFV infection than monocytes $(21,22,59)$$(21,22,59)$(21,22,59)(21,22,59). The high expression level of certain surface markers (e.g., PS, etc.) renders macrophages a higher capacity of phagocytosing apoptotic cells for clearance (22, 60, 61).
据报道，非洲猪瘟病毒（ASFV）更倾向于感染在分化中期到晚期的单核细胞来源的巨噬细胞，并且巨噬细胞对 ASFV 感染的易感性约是单核细胞的五倍 $(21,22,59)$$(21,22,59)$(21,22,59)(21,22,59) 。某些表面标记物（例如，PS 等）的高表达水平使巨噬细胞具有更高的吞噬凋亡细胞以进行清除的能力（22, 60, 61）。
The utilization of PS-positive ApoBDs for ASFV infection and cell-cell transmission is in line with these reports and explains well at least in part why ASFV prefers macrophages as a primary tropism. In accordance with the tempo-dynamics of virus-induced apoptosis (Fig. $1A$$1A$1A1 A and SI Appendix, Fig. S1C), the ApoBDs were induced at late stage of ASFV infection (SI Appendix, Fig. S4 and Fig. 5A), and the associated viral titer reached to a level comparable to the amount of intracellular virions (Fig. 4D).
利用 PS 阳性的 ApoBDs 进行非洲猪瘟病毒（ASFV）感染和细胞间传播与这些报告一致，并在一定程度上解释了为什么 ASFV 更倾向于巨噬细胞作为主要嗜性。根据病毒诱导的细胞凋亡的时间动态（图 $1A$$1A$1A1 A 和 SI 附录，图 S1C），ApoBDs 在 ASFV 感染的晚期被诱导（SI 附录，图 S4 和图 5A），相关的病毒滴度达到了与细胞内病毒颗粒数量相当的水平（图 4D）。
Thus, this process is tailored in a manner ready for the easy phagocytic uptake by macrophages for further cellcell transmission of progeny virions. As for receptor usages, several receptor candidates, i.e., CD163, CD45, MHC-I, etc., have been proposed as entry receptors $(15,22,57$$(15,22,57$(15,22,57(15,22,57 ), but no certain receptor for ASFV entry has yet been indispensable, indicating the complexity of ASFV entry and transmission.
因此，这一过程被调整为便于巨噬细胞轻松吞噬，从而进一步进行子代病毒颗粒的细胞间传播。至于受体的使用，已经提出了几个受体候选者，即 CD163、CD45、MHC-I 等，作为进入受体，但尚未确定非必需的非洲猪瘟病毒（ASFV）进入受体，这表明 ASFV 进入和传播的复杂性。
As compared to the receptor-mediated endocytosis, the ApoBD-mediated spread of ASFV represents a rather less restricted, complementary entry mechanism conducive to virus spread in the presence of selective immune pressures.
与受体介导的内吞作用相比，ApoBD 介导的非洲猪瘟病毒传播代表了一种相对不受限制的互补进入机制，有利于在选择性免疫压力下病毒的传播。

We also found that ASFV exists in three forms during infection (Fig. $3A$$3A$3A3 A ) and that both extracellular and ApoBD-containing virions, but not the single-membrane virions, were resistant to antibody neutralization (Fig. 7 and SI Appendix, Fig. S8A).
我们还发现 ASFV 在感染过程中存在三种形式（图 $3A$$3A$3A3 A ），并且细胞外病毒颗粒和含 ApoBD 的病毒颗粒对抗体中和具有抵抗力，但单膜病毒颗粒则不然（图 7 和 SI 附录，图 S8A）。
Thus, the hijack of the cellular apoptotic uptake pathway provides an efficient means of immune evasion (e.g., inflammation induction, antibody neutralization, etc.) and a quick and economical way of virus spread (no need of outer membrane).
因此，劫持细胞凋亡摄取途径提供了一种有效的免疫逃避手段（例如，炎症诱导、抗体中和等）以及一种快速且经济的病毒传播方式（无需外膜）。
The findings provide an explanation on why the sera from animals infected with ASFV often lack neutralizing activity and thus have important implications in ASFV control and vaccine development.
研究结果解释了为什么感染非洲猪瘟病毒（ASFV）的动物血清通常缺乏中和活性，因此对 ASFV 的控制和疫苗开发具有重要意义。
In addition, the results provide insight into transmission of large DNA viruses, especially giant viruses.
此外，结果为大型 DNA 病毒的传播，特别是巨型病毒，提供了见解。

Fig. 8. A proposed model for ASFV transmission. ASFV induces cell apoptosis to shed the virion-loaded ApoBDs at late stage of infection via caspase-3 activation.
图 8. ASFV 传播的拟议模型。ASFV 诱导细胞凋亡，通过 caspase-3 激活在感染后期释放载有病毒颗粒的 ApoBDs。
The PS-positive ApoBDs carrying single-membrane virions are then phagocytosed by neighboring PAMs via PS interaction with efferocytosis receptors (e.g., TIM4, MFG-E8, etc.). Meanwhile, ASFV particles can directly bud from plasma membrane to acquire the PS-positive outer membrane.
携带单膜病毒颗粒的 PS 阳性 ApoBDs 随后通过 PS 与吞噬细胞凋亡受体（如 TIM4、MFG-E8 等）的相互作用被邻近的 PAM 吞噬。同时，ASFV 颗粒可以直接从质膜出芽以获得 PS 阳性的外膜。
These extracellular virions can enter into recipient PAMs by means of either efferocytosis, clathrin-mediated endocytosis, or macropinocytosis.
这些细胞外病毒颗粒可以通过凋亡细胞吞噬、网格蛋白介导的内吞作用或大吞噬作用进入受体 PAMs。

Reagents. Primary PAMs from 1-mo-old SPF piglets and WSL cells were maintained in RPMI-1640 medium. Type II ASFV strain CADC_HN09 (GenBank accession no: MZ614662.1) was used a model organism in this study.
试剂。来自 1 个月大 SPF 小猪的初级 PAM 和 WSL 细胞在 RPMI-1640 培养基中维持。类型 II 非洲猪瘟病毒株 CADC_HN09（GenBank 登录号：MZ614662.1）被用作本研究的模型生物。
The commercial antibodies and chemicals are from various sources, and the plasmids were engineered by standard recombinant DNA procedures.
商业抗体和化学品来自不同来源，质粒是通过标准重组 DNA 程序工程化的。

ApoBD Isolation. ApoBDs were purified from ASFV-infected PAMs via differential centrifugation. Briefly, the samples were centrifuged at 300 g to obtain the ApoCells-enriched fraction, whereas the supernatant containing ApoBDs was centrifuged at 3,000 g to pellet ApoBDs.
ApoBD 分离。ApoBD 从 ASFV 感染的 PAM 中通过差速离心纯化。简而言之，样品在 300 g 下离心以获得富含 ApoCells 的部分，而含有 ApoBD 的上清液在 3,000 g 下离心以沉淀 ApoBD。
Purified-ApoBD samples were validated by DIC microscopy and transmission electron microscopy.
纯化的 ApoBD 样品通过差示干涉对比显微镜和透射电子显微镜进行了验证。
Blocking Assay. The isolated ApoBDs were incubated with Annexin Vor swine sera for 1 h prior to infection of recipient PAMs. To block the function of efferocytosis receptor on recipient PAMs, the cells were incubated with anti-TIM4 antibody prior to incubation with ApoBDs.
阻断实验。分离的 ApoBDs 在感染受体 PAMs 之前与猪血清中的 Annexin V 孵育 1 小时。为了阻断受体 PAMs 上吞噬凋亡细胞受体的功能，细胞在与 ApoBDs 孵育之前与抗 TIM4 抗体孵育。
The unbound viruses or ApoBDs were washed off with serum-free RPMI1640, and the cells were cultured in maintenance medium containing 2% FBS. ASFV replication was measured at 12 or 24 hpi by western analysis and IFA.
未结合的病毒或 ApoBDs 用无血清的 RPMI1640 洗涤，细胞在含有 2% FBS 的维持培养基中培养。ASFV 复制在 12 或 24 小时后通过西方印迹分析和免疫荧光分析进行测定。

Annexin V Conjugates for Apoptosis Detection. PAMs were washed with annexin-binding buffer and incubated with the Alexa Fluor ${}^{\circledR }568$${}^{\circledR }568$^(®)568{ }^{\circledR} 568 conjugated Annexin $V$$V$VV for 15 min before being washed three times and observed with a fluorescence microscope.
用于凋亡检测的 Annexin V 结合物。PAMs 用 Annexin 结合缓冲液洗涤后，与 Alexa Fluor ${}^{\circledR }568$${}^{\circledR }568$^(®)568{ }^{\circledR} 568 结合的 Annexin $V$$V$VV 孵育 15 分钟，然后洗涤三次，并用荧光显微镜观察。

Construction of Recombinant ASFV. The GFP was inserted to replace the loci MGF360-18R under the control of p72 promoter. The recombinant virus
重组非洲猪瘟病毒的构建。GFP 被插入以替代在 p72 启动子控制下的 MGF360-18R 位点。重组病毒

ASFV-GFP was generated via homologous recombination, followed by successive rounds of plaque assay in PAMs, and confirmed by PCR.
ASFV-GFP 是通过同源重组生成的，随后在 PAM 中进行了连续的斑块测定，并通过 PCR 确认。

Live-Cell Imaging. PAMs on coverslip-bottomed dishes were infected with ASFVGFP or purified ApoBDs and imaged for time-lapse and DIC microscopy with a Nikon A1 confocal microscope.
活细胞成像。覆盖玻璃底的培养皿中的 PAMs 被 ASFVGFP 或纯化的 ApoBDs 感染，并使用尼康 A1 共聚焦显微镜进行时间推移和差示干涉对比显微成像。

Transmission Electron Microscopy. PAMs or isolated ApoBDs were fixed with 0.1 M phosphate buffer containing 4% paraformaldehyde and 2% glutaraldehyde. The pellet was then enrobed in low melting point agarose and post-fixed in $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% osmium tetroxide in cacodylate buffer and en bloc stained with $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% uranyl acetate. Following dehydration with acetone, it was embedded in epoxy (TAAB 812 resin). After polymerization, 80-nm-thick (ultrathin) sections were obtained and stained with uranyl acetate and lead citrate. Imaging was performed in a HITACHI HT7700 electron microscope.
透射电子显微镜。PAMs 或孤立的 ApoBDs 用含有 4%多聚甲醛和 2%戊二醛的 0.1 M 磷酸盐缓冲液固定。然后将沉淀物包裹在低熔点琼脂糖中，并在 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% 醋酸铀中后固定于砷酸盐缓冲液中，并用 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% 醋酸铀进行整体染色。经过丙酮脱水后，嵌入环氧树脂（TAAB 812 树脂）。聚合后，获得 80 纳米厚（超薄）切片，并用醋酸铀和柠檬酸铅染色。成像是在 HITACHI HT7700 电子显微镜中进行的。

Statistical Analysis. Statistical significance was analyzed by two-tailed unpaired Student’s $t$$t$tt test. Significance symbols are defined as follows: NS, no significance; $\ast P<0.05$$\ast P<0.05$**P < 0.05* P<0.05; ** $P<0.01;\ast \ast \ast P<0.001$$P<0.01;\ast \ast \ast P<0.001$P < 0.01;******P < 0.001P<0.01 ; * * * P<0.001. Error bars indicate means $\pm$$\pm$+-\pm SD. Detailed descriptions are provided in SI Appendix, Materials and Methods.
统计分析。统计显著性通过双尾非配对学生 t 检验进行分析。显著性符号定义如下：NS，无显著性； $\ast P<0.05$$\ast P<0.05$**P < 0.05* P<0.05 ；** $P<0.01;\ast \ast \ast P<0.001$$P<0.01;\ast \ast \ast P<0.001$P < 0.01;******P < 0.001P<0.01 ; * * * P<0.001 。误差条表示均值 $\pm$$\pm$+-\pm SD。详细描述见 SI 附录，材料与方法。

Data, Materials, and Software Availability. All study data are included in the article and/or supporting information.
数据、材料和软件可用性。所有研究数据均包含在文章和/或支持信息中。

ACKnOwLEDGMENTs. 致谢。
This study was supported by the National Key Research and Development Program of China (2021YFD1800100), the National Natural Science foundation of China (32025035), and China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs (CARS-35).
本研究得到了中国国家重点研发计划（2021YFD1800100）、国家自然科学基金（32025035）以及财政部和农业农村部的中国农业研究系统（CARS-35）的支持。
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