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Establishment of a human organoid-based evaluation system for assessing interspecies infection risk of animal-borne coronaviruses
Associated Data
ABSTRACT
The COVID-19 pandemic presents a major threat to global public health. Several lines of evidence have shown that the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), along with two other highly pathogenic coronaviruses, SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV) originated from bats. To prevent and control future coronavirus outbreaks, it is necessary to investigate the interspecies infection and pathogenicity risks of animal-related coronaviruses. Currently used infection models, including in vitro cell lines and in vivo animal models, fail to fully mimic the primary infection in human tissues. Here, we employed organoid technology as a promising new model for studying emerging pathogens and their pathogenic mechanisms. We investigated the key host-virus interaction patterns of five human coronaviruses (SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV, HCoV-229E, and HCoV-OC43) in different human respiratory organoids. Five indicators, including cell tropism, invasion preference, replication activity, host response and virus-induced cell death, were developed to establish a comprehensive evaluation system to predict coronavirus interspecies infection and pathogenicity risks. Using this system, we further examined the pathogenicity and interspecies infection risks of three SARS-related coronaviruses (SARSr-CoV), including WIV1 and rRsSHC014S from bats, and MpCoV-GX from pangolins. Moreover, we found that cannabidiol, a non-psychoactive plant extract, exhibits significant inhibitory effects on various coronaviruses in human lung organoid. Cannabidiol significantly enhanced interferon-stimulated gene expression but reduced levels of inflammatory cytokines. In summary, our study established a reliable comprehensive evaluation system to analyse infection and pathogenicity patterns of zoonotic coronaviruses, which could aid in prevention and control of potentially emerging coronavirus diseases.
Introduction
The COVID-19 pandemic poses a significant threat to global public health and has resulted in immeasurable economic losses. To date, seven coronaviruses capable of human-to-human transmission have been identified: human coronavirus (HCoV)-229E, -NL63, -OC43, -HKU1, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome (SARS-CoV), and SARS-CoV-2 [1–3]. Among these strains, HCoV-229E, -OC43, -NL63, and -HKU1 cause mild respiratory tract infections and result in seasonal flu-like symptoms. In contrast, MERS-CoV and SARS-CoV are highly pathogenic with high mortality rates in humans. Moreover, SARS-CoV-2 has presented an unprecedented and widespread public health threat. After initial infection in the upper respiratory tract, these strains can further spread to bronchial epithelial cells, alveolar cells, and leading to severe or life-threatening respiratory diseases along with significant lung injuries [2,4–7].
It is generally believed that SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-NL63, and HCoV-229E may have originated from bats, while HCoV-OC43 and HCoV-HKU1 from rodents [8,9]. In recent years, numerous coronaviruses have been identified from wild animals, particularly bats [10]. Some of these animal-borne coronaviruses are highly similar to HCoV strains [11]. Since the first discovery of bat SARS-like coronavirus Rp3 in 2005, a series of bat SARS-CoV related coronaviruses (SARSr-CoVs) have been found [12]. Yet the potential threat of genetically diverse animal-borne coronaviruses to humans remains unclear. Current risk assessments for virus-associated interspecies infection primarily focus on receptor utilization, cell tropism, and animal infection models [13–17]. Several strains, such as WIV1 and RsSHC014, were identified to use human angiotensin converting enzyme 2 (ACE2) as cell receptor [18,19]. Pathogenicity testing in mouse models has shown their attenuated virulence compared to SARS-CoV [17]. Since the COVID-19 outbreak, SARS-CoV-2-related coronaviruses, most of which shown to utilize human ACE2 receptor [20,21], have been reported in wild-captured bats and pangolins [20,22–24]. Evaluation of two pangolin isolates, MpCoV-GX and-GD, in human ACE2 transgenic mice or hamsters, demonstrated their possible contact or aerosols transmission between animals [15,25]. These approaches, however, are not easy to obtain [26]. A better evaluation system is necessary to assess the potential risks of animal-borne coronaviruses.
The development of organoid culture technology provides an excellent new model for studying novel pathogens and their pathogenesis [27–32]. Human respiratory organoids are composed of different respiratory epithelial cells including basal, ciliated, goblet, club, and alveolar epithelial cells. The organoids provide a promising epithelium model for simulate human respiratory tissues in virus-host interaction studies, and for evaluating antiviral drugs [27].
The highly genetic diversity of coronaviruses brings challenges to the development of specific antiviral drugs. A broad-spectrum antiviral drug that can mitigate the pathological damage caused by inflammation is highly desirable. Cannabidiol (CBD), a natural compound primarily found in cannabis plants, exhibits various biological activities, including analgesic, anti-inflammatory, and antioxidant properties, without causing addiction or hallucinogenic effects [33]. A previous study reported that CBD, a non-psychoactive plant extract, can inhibit SARS-CoV-2 replication and upregulate the host’s antiviral response [34]. CBD directly interacts with host cells to bolster antiviral defenses, implying its potential as a broad-spectrum antiviral drug [35]. However, the anti-coronavirus capacity of CBD remains to be elucidated.
In this study, we established a comprehensive coronavirus evaluation system using human nasal organoids (hNOs) and human lung organoids (hLOs). We incorporated the infection characteristics of five HCoV strains (HCoV-229E, HCoV-OC43, MERS-CoV, SARS-CoV-2 original strain and Omicron BA.1) into a scoring system. With this system, we reliably evaluated the infection capacity of three animal-borne SARSr-CoVs. Our result further demonstrated that a plant extract, CBD, has the broad-spectrum antiviral potential in hLOs.
Results
Cultivation and characterization of human respiratory organoids
The human airway and nasal organoid culture system was previously established by Sachs et al. and Rajan et al. [36,37]. In this study, we made specific modifications to the system and successfully generated respiratory organoid models, including 3D cultures of hNOs and hLOs (Figure S1a–S1c). The respiratory organoid culture medium provided optimal conditions for robust growth and long-term propagation of human respiratory organoids, allowing continuous passaging for up to 6 months (Figure S1b, c).
The presence of diverse respiratory epithelial cell types in hNOs and hLOs was detected by the expression of specific cell markers, including KRT5 in basal cells, MUC 5AC in goblet cells, Acetylated α Tubulin (ACCTUB) in ciliated cells, CC10 in club cells and SFTPC in alveolar type II (AT2) cells (Figure S1d, e). Notably, the hLOs also expressed the ACE2 receptor, which is shown to be crucial for infection of certain coronaviruses (Figure S1e). These findings demonstrate that the organoid models we generated had a high degree of similarity in cellular composition and characteristics with corresponding human tissues.
Establishment of the comprehensive evaluation system
Using the organoid models generated above, we first assessed infection characteristics of five HCoVs (SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV, HCoV-229E, and HCoV-OC43) using, with hNOs representing the upper respiratory tract, and hLOs representing the lower respiratory tract. A comprehensive evaluation system for coronavirus pathogenicity was constructed based on five critical indicators: cell tropism, virus replication activity, invasion preference, host response, and cell apoptosis & death. We then used this evaluation system and assessed the interspecies infection risk of three animal-borne coronaviruses, bat coronavirus WIV1 and rRsSHC014S [38], and pangolin coronavirus MpCoV-GX [25] from various aspects (Figure 1(a)). At last, we tested the efficacy and efficiency of CBD upon viral infection in the organoid models and proposed a potential prevention and treatment strategy for diverse coronaviruses.
Cell tropism of HCoVs in human respiratory organoids. (a) Scheme of this study. (b) Cell tropism of five HCoVs in hNOs. (c) Cell tropism of five HCoVs in hLOs. hNOs and hLOs were infected with five HCoVs respectively (MOI = 1). Organoid samples were harvested at 48 hpi (n = 3). Immunofluorescence staining was performed. Cell nuclei (DAPI, blue), virus (NP, red), and epithelial cell markers (ciliated cells: ACCTUB, club cells: CC10, goblet cells: MUC5AC, alveolar type II cells: SFTPC, green) were stained. Scale bar = 50μm.
HCoVs have similar cellular tropism
The cellular tropism of a virus determines its infection and transmission route, and is closely associated with disease severity and host response. Our data indicated that SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV, and HCoV-OC43 were capable of infecting club, goblet, ciliated cells in hNOs, and club, goblet, ciliated, and AT2 cells in hLOs. By comparison, HCoV-229E failed to infect ciliated cells in both hNOs and hLOs (Figure 1(b,c), Table 1).
Table 1.
Comprehensive weighted score.
| SARS-CoV-2 | Omicron BA.1 | MERS-CoV | HCoV-229E | HCoV-OC43 | WIV1 | rRsSHC014S | MpCoV-GX | |
|---|---|---|---|---|---|---|---|---|
| Cell tropism (10%) | 10.00 | 10.00 | 10.00 | 7.15 | 10.00 | 10.00 | 10.00 | 7.15 |
| Virus invasion preference (15%) | 9.38 | 7.50 | 13.13 | 8.44 | 10.31 | 9.38 | 3.75 | 1.88 |
| Virus replication activity (20%) | 13.00 | 6.00 | 12.00 | 14.00 | 15.00 | 6.00 | 6.00 | 0.00 |
| Host response (30%) | 19.95 | 19.45 | 21.30 | 16.40 | 15.15 | 12.30 | 9.65 | 11.25 |
| Cell apoptosis & death (25%) | 17.50 | 17.50 | 17.50 | 11.25 | 11.25 | 7.50 | 11.25 | 10.00 |
| Overall score (Out of 100) | 69.83 | 60.45 | 73.93 | 57.24 | 61.71 | 45.18 | 40.65 | 30.28 |
Based on the minimal variations observed across different viral strains, we assigned 10% of the weightage to the indicator of cellular tropism and scores were given accordingly for different viruses in the evaluation system (Supplementary text). For example, SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV and HCoV-OC43 received an identical score in this regard, while HCoV-229E obtained a slightly lower score (Supplementary text, Table 1, Figure S5a).
Differential tissue tropism of HCoVs
The infectivity of various viruses towards human organoids displays significant variability, influenced by factors such as differences in virus receptor and entry co-factor expression, host innate immunity, and microenvironmental disparities. We assessed the viral replication and release of five HCoVs in organoids, with viral load within the organoids indicating replication ability. MERS-CoV exhibited the most extensive tissue tropism and replication capabilities as indicated by efficiently infecting and releasing in both hNOs and hLOs. Notably, its replication level was higher in the lower respiratory tract region of the lungs. While SARS-CoV-2 original strain infected both hNOs and hLOs, it displayed weaker release in the former and a stronger preference for the latter. Conversely, Omicron BA.1 showed weaker infection capacity in both types of organoids but more robust virus release capability in hNOs. HCoV-229E demonstrated a preference for infecting and releasing only in the upper respiratory tract region of the lungs and HCoV-OC43 can infect cells in both regions but has weaker virus release ability in hNOs (Figure 2(a)). These findings provide evidence for the idea that tissue tropism correlates directly with virus pathogenicity. Viruses that preferentially target upper respiratory tracts tend to exhibit relatively weaker pathogenicity compared to those targeting lower respiratory tracts.
Replication and invasion variability of HCoVs in human respiratory organoids. (a) Five HCoVs show distinct variability in hNOs and hLOs. hNOs and hLOs were infected with five HCoVs respectively (MOI = 0.1). Samples were harvested at 3, 24, 48 and 72 hpi. Viral load in culture supernatant or within cells was quantified by qRT-PCR on total viral RNA (NP gene as target) (n = 3). Dashed line in supernatant plot: effective release, virus amplification more than 100-fold; dashed line in intracellular plot: effective replication, virus amplification more than 10-fold. (b and c) Different HCoVs exhibit distinct invasion mechanisms in hNOs and hLOs. Organoids were pre-treated with 100 μM E-64D or camostat mesylate for 1 h (camostat mesylate, a TMPRSS2 inhibitor; E64-D, a CTSL/B inhibitor) before infected with five HCoVs respectively (MOI = 1). Organoid cell samples were collected at 24 hpi. Viral load in cells was quantified by qRT-PCR on total viral RNA (NP gene as target) (n = 3). Data are the mean ± SEM. Statistical significance is analysed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Differential invasion preference of HCoVs
Coronaviruses are enveloped viruses that predominantly invade host cells through two distinct pathways, direct membrane fusion and endocytosis. The viral spike protein is cleaved by TMPRSS2 for the former pathway, while proteases such as cathepsin B (CTSB) and cathepsin L (CTSL) cleave the viral spike protein and facilitate fusion with endosomal membranes for the latter pathway. However, it is unclear whether coronaviruses with different pathogenicity employ preferential invasion pathways into host cells.
To answer this question, we used two inhibitors, membrane inhibitor camostat mesylate and endocytosis inhibitor E64-D to block individual virus entry pathway. In hNOs, treatment with camostat mesylate but not E64-D, led to a significant decrease in viral loads of the two SARS-CoV-2 strains and MERS-CoV while virus load of HCoV-229E and HCoV-OC43 decreased significantly after treatment with both camostat mesylate and E64-D (Figure 2(b)). Similarly, in hLOs, the viral loads of the SARS-CoV-2 original strain and MERS-CoV were significantly diminished following treatment with camostat mesylate but not E64-D. Additionally, both Omicron BA.1 and HCoV-OC43 exhibited a substantial reduction in viral loads after treatment with camostat mesylate and E64-D. By contrast, HCoV-229E was only affected by E64-D treatment (Figure 2(c)). These findings suggest that highly pathogenic viruses tend to utilize the direct membrane fusion pathway for cell invasion in both the upper and lower respiratory tracts, whereas low pathogenic viruses prefer the endocytic pathway.
Therefore, we considered viral invasion preference as the second indicator within the comprehensive evaluation system, accounting for a weight of 15%. This indicator encompassed both tissue tropism and invasion pathway preference. According to the scoring criteria (Supplementary text), the ranking of the five HCoVs, from highest to lowest, was as follows: MERS-CoV, HCoV-OC43, SARS-CoV-2 original strain, HCoV-229E, Omicron BA.1 (Supplementary text, Table 1, Figure S5a).
Differential replication activity of HCoVs
To probe variations in the replication activity among different HCoVs, we quantified virus subgenomic RNA (sgRNA) levels in organoid cells by qRT-PCR and virus titres in culture supernatants by plaque assays. Due to the low overall infection rate, sgRNA expression levels are generally low in organoid cells. Notably, Omicron BA.1 was detectable in both hNOs and hLOs as early as 3 hpi, indicating its rapid release, which was consistent with findings observed in other cell models [39] (Figure 3(a,b)). In hNOs, both HCoV-229E and -OC43 showed viral titres higher than Omicron BA.1 in the supernatant, in agreement with higher levels of actively replicating viruses within organoid cells. HCoV-OC43 was detectable at 3 hpi, while HCoVs-229E maintained constantly high viral titres. In contrast, both SARS-CoV-2 original strain and MERS-CoV displayed relatively slow replication rates with viral titres detectable only after 48 hpi (Figure 3(c)). For hLOs, SARS-CoV-2 original strain demonstrated the highest viral titre and the most rapid replication rate, with MERS-CoV following closely behind (Figure 3(d)). Both viruses can constantly sustain high viral titres, underscoring their pronounced tissue affinity for the lower respiratory tract. In comparison, the other three HCoVs displayed lower viral titres (Figure 3(d)). The overall pattern indicates that highly pathogenic coronaviruses tend to infect hLOs, while low pathogenic coronaviruses tend to infect hNOs.
Human respiratory organoids support replication of HCoVs. (a) Differential sgRNA replication activity of five HCoVs in hNO cells. (b) Differential sgRNA replication activity of five HCoVs in hLO cells. (c) Variation in the viral titres of five HCoVs in the culture supernatant of hNO cells. Dashed line: the lowest limit of detection threshold. (d) Variation in the viral titres of five HCoVs in the culture supernatant of hLO cells. Dashed line: the lowest limit of detection threshold. hNOs and hLOs were infected with five HCoVs respectively (MOI = 0.1). Samples were harvested at 3, 24, 48 and 72 hpi (n = 3). Viral load in organoid cells was quantified by qRT-PCR and viral load in culture supernatant was quantified by plaque assay. Error bars represent the mean ± SEM.
Based on the strength of viral replication as well as viral titre rankings across all five viruses, we assigned the third indicator as virus replication activity, which accounted for 20% of the weight, and calculated a composite index (Supplementary text). As regards to the virus replication activity, the five viruses ranked in descending order as follows: HCoV-OC43, HCoV-229E, SARS-CoV-2 original strain, MERS-CoV, Omicron BA.1 (Supplementary text, Table 1, Figure S5a).
Robust host response elicited by HCoV infection
Upon coronavirus infection, organoids promptly exhibit immediate non-specific host responses. The innate immunity plays a crucial role as an early defense mechanism by swiftly inducing interferon responses. We categorized the host response into two parts: initiation of interferon expression and acute local inflammation marked by interleukin expression as well as widespread inflammation associated with cytokine and chemokine expression. We evaluated representative host factors in each part and found that organoids exhibit differential responses to various HCoVs (Figure 4(a)). SARS-CoV-2 original strain, MERS-CoV, and Omicron BA.1 elicited much robust host responses characterized by an initially weak then increasing or continuously increasing interferon response pattern. Moreover, these three viruses induced higher levels of overall inflammatory cytokines/chemokines expression, suggesting a more pronounced inflammatory response compared to the other two viruses (Figure 4(a)).
Host responses to HCoV infection in hLOs. (a) Induction of antiviral and inflammatory responses by infection of five HCoVs (MOI = 0.1). Organoid cell samples were harvested at 24, 48 and 72 hpi (n = 3). Gene expression was quantified by qRT-PCR. (b) Proportion of infected cells in hLOs after viral infection (MOI = 1). (c) Cell death and apoptosis rates induced by virus infection (MOI = 1). Organoid samples were digested into single cells, stained with apoptosis and death probe, fixed, and labelled with virus NP antibodies for flow cytometry analysis (n = 3). Data are the mean ± SEM. Statistical significance is analysed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).
Host response, which plays a pivotal role in virus pathogenicity, served as the fourth evaluation indicator and carried a substantial weight of 30%. The strength of host responses was evaluated by analysing the trend of the response and calculating the area under the curve (AUC) using relative gene expression levels as indicators. Scores were ranked in descending order as follows: MERS-CoV, SARS-CoV-2 original strain, Omicron BA.1, HCoV-229E, -OC43 (Supplementary text, Table 1, Figure S5a).
Differential induction of cell death by HCoVs
To investigate the cell apoptotic and death responses induced by different coronaviruses, we employed the Apotracker™ Green probe to indicate cell apoptosis and the Zombie AquaTM probe to detect cell death. At 24 hpi, infection of SARS-CoV-2 original strain significantly increased cell death, while HCoV-229E infection led to a marked increase in both cell apoptosis and death. In contrast, HCoV-OC43 infection resulted primarily in a significant increase in cell apoptosis. Notably, at 72 hpi, no significant changes in apoptosis or death were observed for most viruses, except for Omicron BA.1, which was associated with suppressed cell apoptosis (Figure 4(b,c), S3). Therefore, regarding indicator of host cell apoptosis and death, which accounted for 25% of the evaluation, we assigned identical scores to SARS-CoV-2 original strain, Omicron BA.1, and MERS-CoV, and lower scores to HCoV-229E and -OC43 (Supplementary text, Table 1, Figure S5a).
Based on the five criteria mentioned above, we developed the comprehensive coronavirus evaluation system and calculated the total weighted score for individual HCoV (Supplementary text, Table 1, Figure S5a). The five HCoVs were ranked in descending order as follows: MERS-CoV, SARS-CoV-2 original strain, HCoV-OC43, Omicron BA.1 and HCoV-229E (Figure S5c). These results align well with clinical data for the infection capacity of five human coronaviruses, as a proof of concept for the reliability of using this system to predict clinical outcomes following coronavirus infection.
Infection characteristics of animal SARSr-CoVs
With the comprehensive coronavirus evaluation system, we further examined the interspecies infection and pathogenicity capacity of three animal-borne SARSr-CoVs. With regard to cell tropism, WIV1 and rRsSHC014S were capable of infecting all tested human respiratory epithelial cell types, whereas MpCoV-GX failed to infect goblet cells and AT2 cells in hLOs (Figure S2a,b, Table S1).
To investigate tissue tropism of these viruses, we infected both the respiratory organoids and intestine organoids with three animal-borne SARSr-CoVs. WIV1 demonstrated high replication in hLOs, human small intestinal organoids (hIOs), and human colonic organoids (hCOs), but successful virus release was only observed in the hCOs. rRsSHC014S replicated in hNOs but failed to release in all four types of organoids. MpCoV-GX displayed relatively low replication capacity in all four types of organoids but released only in hLOs (Figure 5(a)). These findings suggest that WIV1 has a broader tissue tropism than the other two viruses. rRsSHC014S shows a preference for infecting upper respiratory tissue. MpCoV-GX exhibits weaker overall replication ability with a stronger preference for gastrointestinal tissues. Overall, the three animal SARSr-CoVs exhibited lower infection capacity to respiratory and digestive tract compared to SARS-CoV-2 original strain (Figure 2(a), S4).
Tissue Tropism and Invasion Mechanism Preferences of animal-borne coronaviruses. (a) Replication levels of three animal-borne coronaviruses in hNOs, hLOs, hIOs and hCOs (MOI = 0.1, n = 3). Dashed line in supernatant plot: effective release, virus amplification more than 100-fold; dashed line in intracellular plot: effective replication, virus amplification more than 10-fold. (b) Impact of protease inhibitors on three animal-borne coronaviruses in hNOs and hLOs (MOI = 1, n = 3). (c) SgRNA levels of three animal-borne coronaviruses in hNO and hLO cells (MOI = 0.1, n = 3). (d) Virus titres of three animal-borne coronaviruses in hNO and hLO culture supernatants (MOI = 0.1, n = 3). Dashed line: the lowest limit of detection threshold. Data are the mean ± SEM. Statistical significance is analysed by Student’s t-test (**p < 0.01; ****p < 0.0001).
As for invasion preference, treatment with camostat mesylate resulted in a significant reduction in WIV1 viral loads in hNOs and hLOs. In contrast, rRsSHC014S exhibited comparable reliance on both direct membrane fusion and endocytic pathways in hNOs but no significant inhibitory effect was observed in hLOs. MpCoV-GX showed no significant changes upon inhibitor treatment in both hNOs and hLOs, which may be attributed to its low levels of viral replication (Figure 5(b)).
We next examined the replication activity of the three animal-borne SARSr-CoVs. Within the organoid cells, both WIV1 and rRsSHC014S displayed higher sgRNA replication levels compared to MpCoV-GX in hNOs. WIV1 and MpCoV-GX exhibited stronger sgRNA replication activity in hLOs than rRsSHC014S (Figure 5(c)). In the supernatant of organoid culture, WIV1 and rRsSHC014S were detectable in hNOs only at 48 hpi, whereas in hLOs, they were readily detected from 24 hpi to 72 hpi, indicating that WIV1 and rRsSHC014S replicated more rapidly and demonstrated greater infectivity in the lower respiratory tract. Live virus titres of MpCoV-GX were undetectable in both hNOs and hLOs (Figure 5(d)).
Host response assays revealed that, during the early stages of infection, rRsSHC014S and MpCoV-GX induced a robust upregulation of IFNλ1 expression, while WIV1 infection resulted in downregulation of IFNB expression. In the mid-stage of infection, MpCoV-GX continued to upregulate IFNλ1 expression, whereas WIV1 infection sustained the downregulation of IFNB expression. Moreover, MpCoV-GX infection triggered the upregulation of tumour necrosis factor-α (TNFα), C-X-C motif chemokine ligand 10 (CXCL10), and monocyte chemotactic protein 1 (MCP1) expression. As the infection progressed to the late stage, while IFNB downregulation and IL1β upregulation persisted with WIV1 infection, CXCL10 expression was significantly reduced, indicating controlled inflammation. rRsSHC014S infection was associated with downregulation of most genes, suggesting that the infection was under control without a remarkable host response. MpCoV-GX infection induced an upregulation of certain inflammatory cytokines and chemokines indicating its potential pathogenicity (Figure 6(a)).
Host responses to animal-borne coronavirus infection in hLOs. (a) Induction of antiviral and inflammatory responses by infection of three animal-borne coronaviruses (MOI = 0.1). Organoid cell samples were harvested at 24, 48 and 72 hpi (n = 3). Gene expression was quantified by qRT-PCR. (b) Proportion of infected cells in hLOs after viral infection (MOI = 1). (c) Cell death and apoptosis rates induced by infection (MOI = 1). Organoid samples were digested into single cells, stained with apoptosis and death probe, fixed, and labelled with virus NP antibodies for flow cytometry analysis (n = 3). Data are the mean ± SEM. Statistical significance is analysed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
As regards to virus-induced cell death, varying degrees of host cell apoptosis or death were detected with different animal coronaviruses. At 24 hpi, all three coronaviruses induced significant cell apoptosis and death. By 72 hpi, however, the cell viability largely recovered and cells infected by rRsSHC014S showed a significant decrease in apoptosis (Figure 6(b,c)).
Finally, we applied the comprehensive coronavirus evaluation system to score the infection characteristics of three animal-borne coronaviruses and compared their scores to those of the five HCoVs (Supplementary text, Table 1, Figure S5b,c). In terms of infection capacity, the viruses were ranked in descending order as follows: MERS-CoV, SARS-CoV-2 original strain, HCoV-OC43, Omicron BA.1, HCoV-229E, WIV1, rRsSHC014S, MpCoV-GX (Figure S5c).
CBD as a broad-spectrum antiviral candidate against coronaviruses
By use of the organoid models, we tested the direct impact and antiviral effect of the cannabis extract CBD on hLOs. The results showed that 10 μM CBD did not have any negative impact on organoid formation (Figure S6a). And no significant changes were observed in the expression levels of coronavirus receptors and proteases, apart from an increased expression of the HCoV-229E receptor aminopeptidase N (APN) (Figure S6b). To assess the antiviral effectiveness of CBD against the evolving SARS-CoV-2 virus, we utilized the newly emerged variant, Omicron BA.5. In the supernatant of hLO culture, CBD prominently suppressed the levels of SARS-CoV-2 original strain, Omicron BA.5, MERS-CoV, and HCoV-OC43 (Figure 7(a)). Within hLO cells, replication of Omicron BA.5, MERS-CoV, HCoV-OC43, and WIV1 was significantly inhibited by CBD, as indicated by viral load (Figure 7(b)) as well as sgRNA levels (Figure S7). However, CBD did not show inhibitory effects on HCoV-229E replication, possibly due to the enhanced expression of its receptor, APN (Figure S6b). Additionally, the inhibitory effects of CBD on rRsSHC014S and MpCoV-GX were not as apparent, presumably due to the low infection levels of these two viruses in hLOs (Figure 7(a,b)).
CBD as a potential broad-spectrum inhibitor of coronaviruses (a) Changes in viral load in hLO culture supernatant after CBD treatment. (b) Changes in viral load in hLO cells after CBD treatment. (c) Changes in ISG response in hLOs after CBD treatment. (d) Changes in inflammatory response in hLO after CBD treatment. HLOs were pre-treated with 10 μM CBD for 12 h before infection with eight coronaviruses respectively (MOI = 1). After infection, the hLOs were cultured with 10 μM CBD for 24 h (n = 3) before harvest. Viral load or related gene expression was then quantified by qRT-PCR. Data are the mean ± SEM. Statistical significance is analysed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). In figure c and d, pairwise comparisons were performed between the CBD-treated and DMSO-treated groups for each set of experiments.
Upon viral infection, CBD significantly upregulated the expression of interferon-stimulated genes, including IFIT3, ISG15, and SOCS1 (Figure 7(c)). Additionally, CBD effectively reduced the release of inflammatory cytokine TNFα (Figure 7(d)). To test whether this effect is attributed to CBD’s ability to enhance ER stress response, we examined ER stress-related gene expression in hLOs after CBD treatment and revealed only a mild influence on ER stress by CBD (Figure S8). In summary, our results supported the general antiviral effect of CBD possibly independent from its role in regulating ER stress.
Discussion
Here, we have for the first time established a comprehensive coronavirus evaluation system using human organoid model to score the infection and pathogenicity risks of HCoVs and animal-borne coronaviruses. Our findings demonstrated that bat SARSr-CoV WIV1, rRsSHC014S, and pangolin MpCoV-GX pose lower risks compared to HCoVs. Additionally, our research highlighted CBD as a promising therapeutic candidate for coronavirus-associated diseases. This work provides a valuable and adaptable framework that enhances preparedness and improves response to future coronavirus outbreaks.
When conducting risk assessments, the conventional approaches usually include receptor usage tests, infectivity tests in passaged cell lines and pathogenicity tests in animal models [15,40]. However, the culture properties of cell lines differ significantly from those of mucosal epithelial cells. The use of animal infection models often encounters the challenge of ineffective utilization of homologous receptors. Besides, immunological differences between human and animals may result in disparities in clinical symptoms [41]. Previous studies have demonstrated that the human organoids possess superior performance in accurately mimicking the post-infection responses of corresponding organs [42]. Multiple research teams have utilized human organoid models to investigate the pathogenesis of SARS-CoV-2 original strain. Importantly, the characteristics of infected organoids highly correlate with the lung pathology observed in COVID-19 patients and the single-cell transcriptomic data obtained from patients’ bronchoalveolar lavage fluid [43]. Clinically, SARS-CoV-2 original strain and Omicron BA.1 both infect the upper and lower respiratory tracts. SARS-CoV-2 original strain has a higher viral load in the lungs, whereas Omicron BA.1 predominantly affects the upper respiratory tract, especially the nose, windpipe, and throat. Omicron BA.1, exhibits faster replication and higher transmissibility, while causing milder symptoms. Our experimental data align with these clinical observations (Figures 2(a) and and3(a))3(a)) [44]. Previous research demonstrated that the Omicron BA.5 and B.1.1.529 variants exhibited higher infectivity and replication capacity compared to SARS-CoV-2 original strain in 2D hNOs and airway organoid monolayers [45–47]. In this study using 3D alveolar organoids, the viral titre of Omicron BA.5 was notably lower than that of SARS-CoV-2 original strain and Omicron B.1.1.529 [47]. This deviation may be attributed to differences in experimental models and viral strains. Typically, 3D organoids resemble the in vivo environment with their spherical structure, while 2D organoid monolayers show enhanced differentiation maturity and susceptibility to infection due to their flat layout. However, the abundance of ciliated cells in differentiated 2D organoids poses challenges for fully observing viral infection features [46].
In this study, we pioneered the establishment of a comprehensive coronavirus evaluation system. We paid special attention to the mucosal layer of the human respiratory tract which serves as the first line of defense against coronavirus infections (Figure 1(a)). Our results are consistent with the clinical coronavirus infection manifestations which mainly reflect the correlation of the pathogenicity and tissue tropism (Figures 2(a) and and3).3). Comparing our data on invasion preference to those of prior researches, we found that highly pathogenic HCoVs are more likely to use TMPRSS2 in both upper and lower respiratory tract, while other strains prefer cathepsins (Figure 2(b,c)). We also found that HCoVs are able to infect the vast majority of cell types in respiratory tract (Figure 1(b,c)), which ties well with previous studies. Given the ineffective infection of ciliated cells in HCoV-229E [6], one possibility is that diverse cell tropisms are closely associated with the pro-viral expression profiles specific to different epithelial cell types. The correlations between virus infectivity and gene expression profiles need to be further investigated.
Albeit their genetic diversity, HCoVs, whether highly pathogenic or not have been reported to exhibit similar virus-host interaction patterns [48]. Comparative transcriptomic analysis of cells infected with three highly pathogenic HCoVs revealed a significant overlap in the cellular response, including the robust activation of innate immune signalling pathways, and inhibition of the glutathione metabolism pathway, both of which have a direct impact on the host’s oxidative balance [49]. In our study, infection-associated interferon response and inflammation changes in hLOs were refined and conducted as an important pathogenicity indicator. MERS-CoV and SARS-CoV-2 original strain induced more robust host response than other strains, which corresponds to the difference of their pathogenicity (Figure 4(a)). In general, host cells undergo apoptosis and/or other programmed death after coronavirus infection, which is considered a critical innate immune mechanism to limit pathogen propagation [50]. Our results are consistent with the previous findings that highly pathogenic strains induce limited cell apoptosis while others cause significantly increased apoptosis in the early infection stage (Figure 4(c)). To better understand the cell type specific anti-coronavirus responses in the future, advanced technologies such as single-cell transcriptomics would be useful to investigate key host response patterns, which are correlated with coronavirus pathogenicity.
In contrast to HCoVs, the limited infectivity of three animal-derived SARSr-CoVs in hNOs and hLOs is evident. Tissue tropism analysis revealed that WIV1 has a pulmonary preference, rRsSHC014S has a nasal preference, and MpCoV-GX has a colonic preference (Figure 5(a)). In prior study involving MpCoV-GX-infected hamsters, both direct contact and aerosol routes enabled virus transmission among animals [15]. Besides, WIV1 and rRsSHC014S show comparable replication efficiency as SARS-CoV-1 in Vero cells [17]. The differences between these experimental findings and our results may be attributed to distinct infected cell types as well as different infection-associated gene expression patterns between human and other animals. Although the comprehensive risk scores of these strains are lower than that of HCoVs, their infectivity in human organoids reminds us that once viral adaptation occurs, it may lead to the emergence of a pathogenic coronavirus in humans. Further surveillance of animal-borne coronaviruses is still needed.
Vaccines, antibodies, and antiviral drugs are potent tools in the fight against infectious diseases. However, during the SARS-CoV-2 pandemic, viral evolution and immune evasion have presented significant challenges [51]. The narrow therapeutic window following symptom onset underscores the importance of controlling excessive immune responses in later stages. An ideal antiviral countermeasure should possess dual functions of antiviral activity and immune regulatory effects. A previous study reported that CBD, a non-psychoactive plant extract, can inhibit SARS-CoV-2 replication and upregulate the host’s antiviral response [34]. In our study, CBD was found to inhibit most HCoVs’ replication in hLOs while enhancing the interferon signalling pathway and reducing inflammatory cytokine expression (Figure 7). However, we did not observe CBD-induced upregulation of ER stress in HCoVs infection except for SARS-CoV-2 original strain (Figure S8). These results highlight that CBD may serve as a potential broad-spectrum anti-coronavirus candidate, and improve preparedness for other coronaviruses with pandemic potential. Further exploration is required to delve into the antiviral mechanisms of CBD and its potential cellular side effects, thus laying a theoretical groundwork for the development of antiviral medications.
So far, the majority of organoid models utilized in virology research solely encompass epithelial cells that represent host tissues, lacking vascular systems, immune cells, and inter-organ communication. In subsequent investigations, the incorporation of co-cultured vascular endothelial cells and pericytes into organoids could facilitate the development of vascularized organoid models. Furthermore, by establishing immune-vascular-epithelial organoid models that incorporate host epithelial cells, immune cells, and vascular cells, a more advanced platform can be optimized for studying the infection of newly emerging viruses. This would enable a more accurate simulation of physiological and pathological conditions within the human body. Additionally, our study only covers a limited range of coronaviruses, leaving larger and more diverse sample sets for future research endeavours, including data from animal models and clinical patients. These studies together will improve and refine the evaluation system, leading to a more comprehensive understanding and prediction of characteristics and potential threats posed by various coronaviruses.
Supplementary Material
Acknowledgements
We thank Dr. Tao Du and Mr. Jin Xiong at the BSL-3 laboratory, Wuhan Institute of Virology. We thank Mr. Ding Gao and Ms. Juan Min from the WIV core facility for their help with producing flow cytometry and confocal imaging. We thank Yuanlin Song, Zhenju Song, Shanghai Key Laboratory of Lung Inflammation and Injury, and Zhongshan Hospital for technical assistance and support. Q.-C.G. and R.-D.J. designed and conducted the experiments and wrote the manuscript; Q.-C.G., R.-D.J. and L.-N.J. performed the experiments; H.-F.L., Z.-S.G. and M.-Q.L. helped with performing BSL3 virus infection; Y.Y. and J.C. helped with virus culture. S.-Z.X., T.-T.J., X.W., P.Z. and X.-L.Y. helped with organoid culture. Q.L. helped with flow cytometry. Q.W. and W.H. helped with data analysis. X.-F.T. revised and refined the language. X.-H.L. and Z.-L.S. conceived the project and improved the manuscript. All authors have read and approved the article.
Funding Statement
This work was supported by National Key R&D Program of China [2023YFC2605500 to Z.-L.S.], the Guangzhou Laboratory [SRPG22-001 to Z.-L.S.], the Key project of the Chinese Academy of Sciences [2020YJFK-Z-0149 and KJZD-SW-L11 to Z.-L.S.], the National Key Research and Development Program of China [2022YFA0806200 to X.-H.L.], the National Natural Science Foundation of China [32192400 to X.-H.L.], the fellowship of China National Postdoctoral Program for Innovative Talents [BX2021076 to R.-D.J.], and the fellowship of China Postdoctoral Science Foundation [2022M720794 to R.-D.J., 2023M740691 to G.-Q.C.].
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