1 INTRODUCTION

The emergence of novel infections is primarily caused by cross-species transmission, and it is also the defining process in the emergence of zoonotic disease.¹ Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), originating from zoonotic origins,² had a significant and negative effect on the health of the earth and human civilization. The evolutional analysis demonstrated that the main origin of SARS-CoV-2 might be due to bats or pangolins, but no animal reservoir has yet been confirmed.^{3, 4} Since the beginning of the novel coronavirus disease (COVID-19) pandemic in humans, spillover infections of SARS-CoV-2 have been detected in many animals, including nonhuman primates (NHP), hamsters, ferrets, cats, and bats,⁵ and the SARS-CoV-2 genome was found in hamsters, dogs, cats, and minks in the wild.^{6, 7} Cross-species transmission is the most challenging and least studied aspect of disease ecology. Current research on cross-species transmission of viruses mainly focuses on zoonotic spillovers.⁸ However, there is still much to learn about the intricate process of SARS-CoV-2's zoonotic spillover effects.

Most virus spillovers in nature entail interspecies interaction, effective replication in the new host, and efficient transmission between hosts.⁹ It is known that the current interspecies interaction and transmission of SARS-CoV-2 largely hinge on the binding level of the spike (S) protein to the host angiotensin-converting enzyme 2 (ACE2),^{3, 10} which constitutes a major interspecies barrier at the level of viral entry. For example, SARS-CoV-2 is most commonly detected in cell types with ACE2 expression,¹¹ and the S protein of SARS-CoV-2 has a low binding affinity to mouse ACE2, thus preventing the virus from entering mouse cells.^{3, 12} Additionally, it has been found that ACE2 from certain SARS-CoV-2-infected species, like cats and dogs, may withstand a range of amino acid alterations,¹³ offering a portion of the rationale behind SARS-CoV-2's broad occurrence across animals. However, many phenomena implied that the entry of SARS-CoV-2 into different species is not strictly limited to the “lock and key” interaction between S and ACE2. First, the conservation of ACE2 does not necessarily correlate with its role in facilitating viral entry. For example, compared with species infected by SARS-CoV-2, New World monkeys^{14, 15} or pigs^{13, 16} with higher hACE2 homology cannot effectively support SARS-CoV-2 S protein-driven entry. Second, viral infection susceptibility is not necessarily correlated with the binding affinity of S protein to ACE2. Mink ACE2 proved to have limited binding ability to the original SARS-CoV-2 spike protein,^{14, 17} but there was an outbreak of SARS-CoV-2 in mink farms.¹⁸ Last, the tissue tropism of SARS-CoV-2 infection is not fully elucidated by the ACE2 receptor alone, particularly in the respiratory tract.¹⁹ Many host receptors other than ACE2 have been discovered to mediate viral spread, such as host receptors AXL,²⁰ TMEM106B,²¹ and cofactors neuropilin-1.²² Furthermore, spike protein-mediated SARS-CoV-2 cell-to-cell transmission is a more effective route of transmission than spike protein-ACE2-mediated spread,²³ suggesting that complex cross-species transmission may be facilitated by a variety of mechanisms.

Based on our earlier research, extracellular vesicles (EVs) containing mature virus particles are produced when SARS-CoV-2 infection occurs. These EVs facilitate SARS-CoV-2 transmission into host cells without the need for particular viral receptors. Convincing evidence indicated that the SARS-CoV-2 envelope protein (2-E) accounts for the production of the EVs.²⁴ Notably, the sequence of the 2-E protein was highly conserved among the coronaviruses in some species that are related to SARS-CoV-2 cross-species transmission or origin, including mink-, bat- and pangolin-origin coronaviruses.^{18, 25} Thus, we hypothesized that the EVs induced by envelope proteins could serve as potential carriers of viral spillover, aiding the virus in evading interspecies barriers at the viral entry stage.

2 RESULTS

2.1 Two-way transmission on heterologous EVs induced by 2-E

It has been shown that 2-E can promote the development of EVs in a range of cell lines.²⁴ Compared with the Wuhan reference sequence NC_045512.2, the envelope (E) proteins amino acid sequence isolated from three species, Bat, Pangolin, and Mink, related to the SRAS-CoV-2 transmission shared 100% identity, indicating coronavirus of other species may also produce similar EVs via E proteins (Figure 1A). We first determined the ability of 2-E proteins to produce EVs in cell lines derived from various species and whether heterologous EVs could travel between cell lines from different species in vitro (Figure 1B). The NHP cells Vero E6 (African green monkey kidney) were initially selected as donor cells, and Tb 1 Lu (Tadarida brasiliensis (Mexican free-tailed bats) derived lung epithelial) were selected as recipient cells. After transfected with 2-E-mCherry plasmids, smooth and round EVs were secreted from these two cells (Figure S1). The microscopic examination showed the EVs were fluorescently labeled with mCherry, and the average size was approximately 3 (rank from 2 to 4) μm, which was consistent with our previous reports.²⁴

To track the EVs' location, the recipient cells (Tb 1 Lu) were labeled in green and incubated with the EVs produced by donor cells (Vero E6) for 24 h. 3D imaging was used to visualize the position of EVs in Tb 1 Lu. As shown in Figure 1C, the red fluorescence in the cytosol increased. Taking one of the EVs fields for 3D slice reconstruction, we observed the red dots wrapped inside the green fluorescence, supporting the presence of intact EVs in the cytoplasm rather than binding to the cell membrane. Radial intensity analysis of red and green fluorescence in different slices further confirmed that EVs secreted by Vero E6 (Monkey) cells entered bat cells (Figure 1C). Consistent with the images, 2-E-mCherry proteins were enriched in the EVs-treated cells compared with the untreated cells (Figure S2B). Likewise, the absorption of the heterologous EVs was seen in Vero E6 (Monkey) cells subsequent to a 24 h exposure to the EVs generated by Tb 1 Lu (Bat) (Figure 1D and Figure S2B).

To further verify the universality of EVs two-way transmission, more species cells that have been reported to be infected or carry the SARS-CoV-2 virus were chosen as recipient cells, including BHK-21 (Baby Hamster Kidney-21), F81 (Feline kidney fibroblast-like monolayer cells), MV 1 Lu (Mink lung epithelial cells), MDCK (Madin Darby Canine Kidney) (Figure S1). Fluorescence imaging and western blot results showed that multiple heterologous EVs could enter different cells bidirectionally (Figure 1E and Figure S2A,B). Furthermore, comparable outcomes were also seen in primary mouse lung cells (Figure S2C,D). Finally, we quantified the uptake efficiency of donor-derived EVs by different species via live-cell imaging assay. Under the identical EVs quantity treatment, F81 showed the highest uptake rate, while the uptake of EVs by Tb 1 Lu was relatively limited (Figure S2E). Overall, these observations suggest that 2-E triggers the formation of EVs in the cell lines of various species, and heterologous EVs can bidirectionally transport between species at the cellular level.

2.2 EVs induced by 2-E enter the host through macropinocytosis

Many modes are used in the endocytosis of EVs, which frequently varies based on the receiving cell type and the origin of the vesicles. Four cell lines were chosen to understand the endocytic pathways of EVs induced by 2-E (F81, high uptake efficiency of the EVs induced by 2-E; Vero E6, donor cells that produce EVs; SCC7, without classical receptors of SARS-COV-2 infection²⁴; HeLa, a reported effective recipient cell for EVs).²⁶ These cells were treated with a panel of endocytosis inhibitors before treatment with the EVs induced by 2-E, including chlorpromazine (CPZ, inhibitor of clathrin-mediated endocytosis), omeprazole (inhibitor of proton pump-related membrane fusion), genistein (inhibitor of caveolin-mediated endocytosis) and wortmannin (inhibitor of PI3 kinase pathway-related macropinocytosis). The effects of these inhibitors on the viability of four cell lines were first determined (Figure S3A), and the mCherry protein was quantified as a proxy measure for the EVs entry into cells by western blot. We found that the EVs uptake was significantly inhibited under CPZ and wortmannin treatment in three cell lines (Figure 2A,B). In addition，the uptake of EVs could be further inhibited

under the wortmannin combined CPZ treatments (Figure S3B-D). The above results indicated that clathrin-mediated endocytosis and macropinocytosis were involved in the EVs uptake. Remarkably, only wortmannin impaired the EVs uptake in all four cell lines (Figure 2B), and the wortmannin dose-dependently inhibited the EVs uptake in Hela (Figure 2C), suggesting the important role of macropinocytosis in their internalization. Similar results were obtained for another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (Ethylisopropylamiloride, Na⁺/H⁺-exchanger inhibitor) (Figure S3E-G).

To further comprehend the contribution of macropinocytosis to the internalization of EVs, we performed living cell imaging. Isolated EVs were incubated with cell tracker (green) stained Hela cells in the presence or absence of wortmannin treatment. The data showed that the ability of EVs infection was limited under the treatment of wortmannin (Figure 2D,E). All of these findings point to a significant role for macropinocytosis in the internalization of 2-E-induced EVs.

2.3 EVs from NHPs invade the zebrafish

Human- and animal-contaminated water is an important vector for the indirect transmission of viruses,²⁷ and SARS-CoV-2 has been detected in aquatic ecosystems worldwide,²⁸ emphasizing its potential route of transmission. We investigated the environmental persistence of the EVs induced by 2-E, derived from Vero E6, to understand their capacity as vehicles for waterborne infection. Samples were, upon exposure, realistic survival- and biological-environments at room temperature, including tap water and PBS (pH = 7.4) containing 10% fetal bovine serum (FBS), and quantified by confocal microscopy whole-hole scans at different time points. The data indicated that 36.16% and 44.49% of the EVs were persistent in tap water and PBS (PH = 7.4, 10%FBS) at 48 h (Figure 3A). High-magnification images also showed that the EVs remained intact for at least 48 h in both solutions (Figure S4A).

Next, the heterologous invasion capability of EVs induced by 2-E was investigated using zebrafish (Figure 3B), which were able to image some EVs in vivo with high spatiotemporal resolution.²⁹ Three experimental sets were conducted. First, the EVs derived from Vero E6 were injected into the common cardinal vein of transgenic zebrafish Tg (flk1:EGFP) larvae at about 3 days postfertilization (dpf). It was noticed that the undamaged EVs were still observed traversing the blood vessels of zebrafish after 1 h of injection, indicating their potential activity within the bloodstream (Figure 3C and Video S1). Second, the zebrafish at 2 dpf were immersed in a culture medium containing EVs to test whether the zebrafish could ingest the EVs in the environment. Pathogens are more likely to penetrate damaged skin and infect more sensitive host tissues during interspecies contact. To better simulate the process, the back brain skin of zebrafish larvae was removed using a glass electrode tip before the EVs therapy. Following that, we captured intact EVs in the forebrain of injured zebrafish larvae (Figure 3D and Figure S4B). The zebrafish were washed with PBS to remove the skin-bound EVs. The western blot analysis showed that the signal of 2-E-mCherry protein was detected in infected zebrafish (Figure 3E). These findings implied that the EVs were able to invade the zebrafish under conditions of skin damage and that the cargo carried by the EVs could potentially enter the cells of the zebrafish during this process.

Last, to investigate the potential of 2-E to trigger EVs production following in vivo colonization, we injected the plasmid (UAS-E1b-2-E-mcherry-p2a-H2B-BFP) into one-cell stage embryos of the transgenic lines Tg(HuC:GAL4-VP16) and Tg (4xnrUAS:GFP), in which neurons expressed 2-E were labeled by mCherry and the nucleus of this neuron was labeled by BFP, and other neurons could be visualized (Figure S4C). We observed the release of vesicles following 2-E expression in zebrafish larvae, as we previously described, at the cellular level (Figure 3F and Video S2).²⁴ The in vivo imaging substantiated that the SARS-CoV-2 envelope protein within the host organism could trigger EVs. The EVs produced by 2-E were shown to have excellent stability and heterologous infiltration ability as transmission vehicles, indicating that interspecific transfer may be possible.

2.4 EVs infiltrate the lungs of mice through nasal drops

In vivo studies have shown that SARS-CoV-2 can be transmitted among animals by airborne droplets.³⁰ Near-infrared fluorescent dyes (Cy5-NHS) labeled the EVs induced by 2-E derived from Vero E6 were intranasally administered to mice to evaluate their potential role as a carrier in respiratory transmission (Figure 4A). Controls consisting of PBS groups and Cy5-NHS groups were employed, and the dye groups were quantified to be equivalent to the EVs group (Figure S5A). Ex vivo tissue fluorescence imaging of the major organs was captured at selected time points. The fluorescence signal was limited to the lungs in the EVs group (Figure 4B and S5B). At the same time points, no significant signal was observed within the Cy5-NHS alone (dye control) and PBS (negative control) groups. Quantitative analyses of the lung tissue signal were performed at different time points, and the EVs group had a longer residence time in the lungs (Figure 4C). The findings above indicated that the EVs could be deposited in the lung through the respiratory tract. Another group of unlabeled EVs was euthanized 24 h after administration to study the effect of EVs after entering the lungs. In the EVs therapy, inflammatory cell infiltration, alveolar septal thickening, and unique vascular system damage were found, while PBS inoculate groups were healthy (Figure 4D and Figure S5D). In addition, EVs treatment led to cytokine production, including TNF-α, IL-1β, and IL-6, suggesting that EVs bring additional damage factors (Figure S5E). The damage induced by EVs could be reduced under endocytosis inhibitors treatment (Figure S5C-E), suggesting a potential inhibition of EVs endocytosis. These studies demonstrated that the 2-E-induced EVs could penetrate the lungs of mice and cause negative outcomes. SARS-CoV-2-induced heterologous EVs infect wild-type (WT) mice

Our previous study and the quantitative proteomic analysis revealed a high degree of relevance and similarity between the EVs produced by 2-E and the CoV-2-EVs, which are produced by SARS-CoV-2 infection and contain a lot of infectious virus particles (Figure S6).²⁴ The study demonstrated that the original SARS-CoV-2 strain could not infect WT mice due to the difference between hACE2 and mACE2.³¹ To explore if the EVs could carry SARS-CoV-2 across the interspecies barrier and establish infection in vivo, we administered heterologous CoV-2-EVs intranasally to WT mice lacking hACE2 expression and transduced Ad5-hACE2 (hACE2) mice were intranasally infected with free SARS-CoV-2 particles (2*10^5 plaque-forming units, PFU) or administrated the same dose of isolated CoV-2-EVs, respectively (Figure 5A). Ad5-hACE2 (hACE2) mice intranasally administrated PBS were used as control. All animals were monitored daily for body-weight changes and were killed on Day 2 postinfection (dpi). The viral replication of SARS-CoV-2 was measured by qRT-PCR and immunofluorescence assays. As reported, in free virus particle treatment, high levels of viral RNAs (>1*10^11 copies/g) were detected in the lungs of hACE2-expressing mice, while no viral RNA was detected in WT mice (Figure 5B). In contrast, under CoV-2-EVs treatment, high levels of viral RNAs (>1*10¹⁰ copies/g) were able to be detected in the lungs of both WT and hACE2-expressing mice. The expression of SARS-CoV-2 N protein was compared. Consistent with the qRT-PCR results, negligible expression of N protein was observed in WT mice treated with free virus particles. However, in WT mice that received CoV-2-EVs treatment and in hACE-2-expressing mice, significant N protein expression was observed in the tested lung sections. The colocalization of N proteins and nuclei validated the entry of the CoV-2-EVs encapsulated virus into the live cells (Figure 5C and S7A).

In addition, although fatalities were not observed, a significant loss of body weight was observed for all mice that received CoV-2-EVs but not free virus particles (Figure 5B). A histopathological examination was further performed. H&E and Masson's staining revealed inflammatory cell infiltration, alveolar septal thickening, and unique vascular system damage following CoV-2-EVs infection in all mice (Figure 5D,E and S7B,C). Interestingly, hACE2-expressing mice that received CoV-2-EVs developed more alveolar epithelial cell lesions and focal hemorrhages. In addition, CoV-2-EVs infection led to higher cytokine production, including IL-1β, IL-6, and TNF-α, in both WT and hACE-2 mice (Figure 5F). The above results underscored the importance of the EVs-mediated transmission of SARS-CoV-2 in vivo, indicating the potential of EVs as a vehicle for SARS-CoV-2 spillover.

3 DISCUSSION

Knowing the molecular source of SARS-CoV-2 and how it transcends interspecies barriers will be crucial for anticipating and averting future epidemics. Most viruses are specialists. They establish enduring relationships with preferred host species. As a result of the ongoing arms race between virus and host, coupled with human urbanization, an increasing number of viruses begin to “emerge” or “spilled” from the original host to the new host.³² The “lock and key” interaction between proteins provides a rational explanation for crossing species, but there are still many unresolved issues in the process of virus evolution and spread. We discovered a new SARS-CoV-2 infection pattern in our study, both in vitro and in vivo, which may benefit the spread and infection of SARS-CoV-2 in real-world environments.

The virus spreads through direct contact with natural hosts or exposure to environmental media such as water, soil, or food contaminated with urine, saliva, feces, or secretions from the animal host.³³ Pathogen-derived EVs are widely distributed in natural ecosystems,^{34, 35} potentially acting as carriers for viral transmission to new hosts via environmental media. It was discovered that the EVs generated by 2-E were able to move freely within several cell kinds. These EVs not only wrap mature virus particles but also have favorable stability and invasion functions, offering the foundation for their prospective role in the natural environment. When animals hunt, bite, or drink, EVs may encounter them via their blood and headwater. As we know, more than 70% of zoonotic infectious diseases originate from wild animals,³⁶ among which bats have been proven to be the natural reservoirs of many viruses, including SARS-CoV-2.³⁷ The lifestyle, food, and geographic location of these untamed animals may offer pathways for these EVs to infiltrate new hosts. Furthermore, the proliferation of intensive farming practices and both legal and illegal trade in live animals and their products contributes to the dissemination of zoonotic pathogens.³⁸ For instance, SARS-CoV-2 has led to infections in mink farms, with evidence suggesting that mutations originating from minks can be transmitted to humans.^{18, 39} Studying EVs within these contexts offers valuable insights into the mechanisms of viral transmission and the associated risks of inter-species viral exchange.

EVs may play a significant role in mediating communication and interaction among species in multispecies coexisting ecosystems. For example, they could facilitate the transfer of signaling molecules or genetic material between species, influencing their interactions and coexistence.^{40, 41} Exosomes have been demonstrated as a conservative mechanism for the horizontal transmission of arboviruses from insect vectors to plants, facilitating the establishment of the initial infection.⁴² It's important to note that we discovered WT mice devoid of receptor expression could contract the virus encased in EVs (Figure 5B,C). Although whether EVs could become a potential transmission mode of COVID-19 requires further exploration, consistent with our results, similar EVs have been observed in some animals and patients that have been infected with COVID-19.^{43, 44} It has been demonstrated that viruses carried by EVs are more contagious. This mode of transmission has been found to enhance both the replication efficiency and overall viral population fitness in comparison to infections involving comparable amounts of free virus particles.^{45, 46} Genetic background interactions between the infecting virus and the related host species drive the adaptive evolution of the virus, for instance, by ameliorating interactions with crucial host cell factors like the entry receptor ACE2. Previous studies have shown that VOC (variants of concern) containing the N501Y mutation that arose during the transmission of SARS-CoV-2 have been proven to contribute to the infection and pathogenesis of mice.⁴⁷ Virus-derived EVs use various mechanisms to evade the immune system and facilitate viral spread. They incorporate viral components like proteins and nucleic acids, mimicking host proteins to avoid immune recognition and antibody neutralization, and increase viral tropism.^{46, 48} These EVs also carry immunomodulatory molecules that suppress proinflammatory cytokines, and inhibit immune cell activation, creating an immunosuppressive environment conducive to viral persistence and replication.^49-51 Additionally, they downregulate MHC molecules on infected cells, impairing antigen presentation.^{49, 50} Viral EVs can directly target immune cells, inducing apoptosis or inhibiting proliferation and activation.^{46, 49, 52} For instance, EVs from HIV-1-infected cells carry viral microRNAs that prevent apoptosis in recipient cells, enhancing HIV infection.⁵³ In summary, the intricate strategies employed by virus-derived EVs to evade the immune system highlight the sophisticated mechanisms viruses have evolved to ensure their survival and propagation.

The viroporin nature of 2-E was proven by our previous research,⁵⁴ and the channel function induced the EVs.²⁴ Several coronavirus envelope proteins (E) have exhibited ion channel characteristics, suggesting that the E protein of these viruses may also have the ability to produce similar EVs.^54-56 It is interesting to note that the amino acid sequences of the virus derived from species linked to the spread of SARS-CoV-2 are identical (Figure 1A), indicating that the role of the E protein is essential and conserved throughout viral evolution or pandemic. The ability of the envelope protein to induce EVs production has also been validated in multiple mammalian cells (Figure S1). These observations implied that envelope protein-mediated EVs may be a conserved strategy for SARS-CoV-2 transmission. The EVs may also serve as a nonspecific strategy for the transmission of SARS-CoV-2. Different species may be infected at the cellular level by the EVs generated by 2-E (Figure 1). BHK-21 was previously proved to be nonsusceptible to SARS-CoV-2 spike protein-driven entry.¹⁰ We demonstrated that the EVs entered different species cells via macropinocytosis (Figure 2). A type of endocytotic pathway known as macropinocytosis is the nonselective intake of copious amounts of solute molecules, nutrients, and antigens in massive endocytic vesicles (0.2−10 μm) called macropinosomes,⁵⁷ this may be an unspecific tactic used by the virus to aid in the establishment of the infection. Macropinocytosis as the major pathway for EVs internalization differs from receptor-mediated endocytosis and phagocytosis in that it does not rely on receptor-ligand interactions and is immunologically inert.^{58, 59}

The MHC-I cross-species host compatibility of EVs is also a matter of discussion. 2-E-induced EVs may carry MHC-I molecules, crucial for immune response (Figure S8). Donor EVs carry preformed antigen-peptide/MHC complexes (pMHCs), aiding cross-dressing of recipient cells with pMHCs.^60-62 However, structural differences in MHC-I across species may limit cross-species compatibility.⁶³ If 2-E-induced EVs carry homologous MHC-I to recipient cells, they may be immunocompatible. Understanding cross-species compatibility is vital for transplantation and vaccine development. Further research on this could offer insights into 2-E-EVs' implications.

The cross-species transmission of viruses has been a long-term concern, but it is currently unclear how the virus fulfills this intricate process. Our study represents a potential vehicle for cross-species transmission of the SARS-CoV-2 or other viruses with similar properties. The main limitation is that we only focused on the contribution of the EVs in the viral entry process and the possibility as a transmission vehicle in vitro. However, the virus's host range or specificity is frequently constrained by a number of variables, including its potential to evade the host immune system and replicate and multiply within the host organism.⁶⁴ Because of the limitations of this study, we suggest using caution when interpreting the data.

Two host species must come into contact, either directly or indirectly, for cross-species transmission to occur. Here, we identify the ability of EVs to carry viral particles for in vivo infection using the authentic SARS-CoV-2 virus. Countless pathogens jump from one species to another every day, and these pathogens are taking advantage of new opportunities created by different hosts, reshaping the natural environment to “dance” with humans. Our results may be useful in directing future surveillance efforts to prevent emergent animal-human spillovers as best practices, thereby improving human health.

AUTHOR CONTRIBUTIONS

Zhaobing Gao, Shuangqu Li, and Bingqing Xia conceived the project. Zhaobing Gao, Shuangqu Li, Bingqing Xia, Jiulin Du, and Lei-Ke Zhang designed the experiments. Shuangqu Li and Qiguang Li carried out the cell-based assays. Jiwen Bu and Shuangqu Li carried out the zebrafish-related experiments. Xiaoli Zuo, Shuangqu Li, and Qiguang Li carried out the animal experiments with EVs produced by 2-E; Xiaoyan Pan performed antiviral assays in vivo and the virus assays in vitro. All authors analyzed and discussed the data. Zhaobing Gao, Shuangqu Li, and Bingqing Xia wrote the manuscript. All authors read and approved the manuscript.