Introduction
The coronavirus disease 2019 (COVID-19), which is caused by 2019 novel coronavirus (2019-nCoV), imposes a grand immediate challenge for global public health.[1,2]
A coronavirus closely related to 2019-nCoV was identified in a sample collected from Rhinolophus affinis bat in Yunnan in 2013, suggesting bats are likely the reservoirs of 2019-nCoV.[3] et al [4] Manis javanica ). Similarly, in October 2019, a viral metagenomic study of pangolins identified severe acute respiratory syndrome-coronavirus (SARS-CoV)-related sequences,[5] [6] [7] [5]
Due to its pathogenicity and transmissibility of 2019-nCoV, working with live 2019-nCoV requires high-level biocontainment facilities, which impedes the urgent needs for drug screening. Without prior experience of therapy, the current treatment of 2019-nCoV infection is mainly empirical and symptomatic, and a limited number of therapeutics in ongoing clinical trial were adopted from previous research on SARS-CoV and Middle East respiratory syndrome-coronavirus (MERS-CoV), which are only remotely related with 2019-nCoV. With low or no pathogenicity in humans and close genetic relationship with 2019-nCoV, our pangolin coronavirus provides an ideal alternative model for 2019-nCoV research. Our reasoning of this isolate having low or no pathogenicity in humans was based on the fact that, back in 2017, no suspected infections were found in those having close contacts with pangolins; and our pangolin coronavirus isolate was routinely cultured in biosafety level 2 facilities. Here we present a screening of clinically approved drugs for anti-coronavirus activity in this 2019-nCoVr model, and identify the potent inhibitors for pangolin coronavirus infection.
Methods
Cell lines, coronavirus, and key reagents
Vero E6 cells (American Type Culture Collection, Manassas, VA, USA) were grown in high-glucose-containing Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. 2019-nCoVr GX_P2V/pangolin/2017/Guangxi was isolated in Vero E6 cells from a dead smuggled pangolin in 2017, and its complete genome has been submitted to GenBank.[7] https://links.lww.com/CM9/A216
Plaque assay for determining virus titer
Confluent monolayer Vero E6 cells were infected with serially ten-fold diluted 2019-nCoVr, at a range of 10−1 to 10−6 . At 2 h post-infection (p.i.), the virus was removed and the cells were washed twice with phosphate-buffered saline. And then 3 mL 1% agarose overlay was added to each well to prevent cross-contamination. At 3 days p.i. or 5 days p.i., cells were fixed with 4% paraformaldehyde for 1 h at room temperature. Then the upper semi-solid agarose medium was removed. Fixed cells were stained with crystal violet for 10 min, and rinsed with water gently for several times. The number of plaques was counted and virus titers were calculated.
small interfering RNA (siRNA)-mediated silencing of angiotensin-converting enzyme 2 (ACE2)
Vero E6 cells were transfected with 0.8, 4, and 20 nmol/L angiotensin-converting enzyme 2-specific siRNApool (siACE2) or negative control siRNA (siNC) by using the RNAiMax transfection reagent (Invitrogen, Carlsbad, CA, USA) as previously described.[8]
Drug screening for 2019-nCoVr using approved drug library
Vero E6 cells were plated in 96 well plates at a density of 5000 cells/well. Cells were treated with 2019-nCoVr (MOI = 0.01) and different chemical drugs of the approved drug library with the final concentration of 10 μmol/L. At 2 h p.i., 2019-nCoVr and drugs were removed, and fresh culture medium containing 10 μmol/L drugs were added to each well. At 72 h p.i., the cytopathic effect (CPE) was observed using microscopy (Nikon, Cat:TS100 and TS2-S-SM, Tokyo, Japan). And the cells in the wells without obvious CPE were further analyzed.
Viral RNA extraction and real-time polymerase chain reaction (qRT-PCR)
Cell culture supernatants and Vero E6 cells were harvested for RNA extraction using the AxyPrep™ body fluid viral DNA/RNA Miniprep kit (Axygen, Cat No. AP-MN-BF-VNA-250, Hangzhou, Zhejiang, China) and AxyPrep™ multisource total RNA Miniprep kit (Axygene, Cat No. AP-MN-MS-RNA-250G) according to the manufacturer's instructions. Reverse transcription was performed with a Hifair II 1st Strand cDNA synthesis kit with gDNA digester (Yeasen Biotech, Cat:11121ES60, Shanghai, China), and qRT-PCR was performed using QuantStudio 1 Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with Hieff qPCRSYBR Green Master Mix (Yeasen Biotech, Cat:11202ES08, Shanghai, China) or two-step Taqman probe assay. The sequence information of primers used is listed in Supplementary Table 1, https://links.lww.com/CM9/A216 3 − 109 copies) of the plasmid. qRT-PCR amplification of SYBR Green method was performed as follows: 95°C for 5 min followed by 40 cycles consisting of 95°C for 10 s, 55°C for 20 s, and 72°C for 31 s. And the Taqman method was performed as follows: 50°C for 2 min, 95°C for 10 min followed by 40 cycles consisting of 95°C for 10 s, 60°C for 1 min.
Time-of-addition experiment of cepharanthine
The cepharanthine (CEP; 10 μmol/L) was used for the time of addition experiment. Vero E6 cells (5 × 104 cells/well) were plated in 12 well plates and treated with CEP at different stages of virus infection. The “Full time” treatment, “Entry” treatment, and “Post-entry” treatment experiments were performed according to a previous study.[9]
Statistical analysis
The data were analyzed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA) and presented as the mean ± standard deviation (normal distribution of data was checked by Kolmogorov-Smirnov test). Comparisons between the two groups were analyzed using the Student's t tests. A P value of <0.05 was considered statistically significant.
Results
Pangolin coronavirus isolate GX_P2V is as an alternative model for 2019-nCoV research
We reported the identification of 2019-nCoVr, which were composed of a lineage of 2019-nCoV and coronaviruses found in eight pangolin samples and three bat samples.[7] Figure 1 A]. Compared to the five critical residues in SARS-CoV, two in GX_P2V and four in 2019-nCoV are replaced with different residues.
Figure 1: Pangolin coronavirus GX_P2V has two residue changes among the five key residues for receptor binding when compared to SARS-CoV, but likely still utilizes ACE2 as the cell receptor. (A) Alignment of receptor binding domains of 2019-nCoV-related CoVs and SARS-CoV. Dots represent residues that are identical to those in SARS-CoV and hyphens represent gaps. Five key residues for binding between SARS-CoV RBD and ACE2 protein are indicated as underlined capital letters. (B) The ACE2 expressions and (C) viral RNA yields were significantly reduced in ACE2-specific siRNA treated cells (∗ P < 0.01). Vero cells were transfected with 0.8, 4, and 20 nmol/L ACE2-specific siRNAs or NSC siRNA by using the RNAiMax transfection reagent. At 48 h p.t., the cells were infected with pangolin CoV at an MOI of approximately 10. At 24 h p.i., CoV-infected cells were lysed in lysis buffer, and the RNA levels of ACE2, CoV, and β-actin were determined by qRT-PCR. SARS-CoV: Severe acute respiratory syndrome-coronavirus; ACE2: Angiotensin-converting enzyme 2; 2019-nCoV: 2019 Novel coronavirus; CoV: Coronavirus; RBD: Receptor binding domain; siRNAs: Small interfering RNA; NSC: Non-specific control; p.t.: Post-transfection; MOI: Multiplicity of infection; p.i.: Post-infection; qRT-PCR: Quantitative real-time polymerase chain reaction; mRNA: Messenger RNA; SiNC: siRNA of negative control; SiACE2: siRNA of ACE2; nM = nmol/L.
ACE2 is the cell receptor for both SARS-CoV, bat SARS-like coronavirus (CoV), and 2019-nCoV.[3,10,11] Figure 1 B] and ACE2 was also a receptor of 2019-nCoVr GX_P2V [Figure 1 C]. It appears that ACE2 is a conserved receptor of both SARS-CoV and 2019-nCoV-related viruses. It is noted, however, that having a possible receptor of ACE2 does not correlate to viral pathogenicity. In fact, no human infection relating to our pangolin CoV was identified or suspected, suggesting that CoV GX_P2V is nonpathogenic in humans. Altogether, the close relationship to 2019-nCoV, the shared receptor and non-pathogenicity, support that pangolin CoV GX_P2V can be used as an accessible in vitro model for developing therapies against 2019-nCoV.
Three drugs are potent inhibitors of 2019-nCoVr infection
In our 2019-nCoVr and Vero E6 cell model, we first screened a total of 2406 drugs and compounds for their inhibitory effects on viral infection-dependent CPE in 96 well plates [Figure 2 A]. Each drug or compound was added to 10 μmol/L at the starting time point of infection and all drugs were tested in duplicate. At 72 h p.i., cells were observed under phase microscopy. Infected cells without any drug treatment showed typical CPE-cell rounding with no obvious lysis. Importantly, three drugs, CEP, selamectin , and mefloquine hydrochloride , exhibited complete inhibition of CPE in infected cells [Supplementary Figure 1, https://links.lww.com/CM9/A217 −4 vs. 1.00 ± 0.12, t = 150.38, P < 0.001) [Figure 2 B]. Viral RNA quantification indicated that viral replication in other two drug-treated cells was also dramatically inhibited [Supplementary Figure 2, https://links.lww.com/CM9/A218
Figure 2: Screening of clinically approved drugs for their anti-viral activities against 2019-nCoV by observing CPE inhibition and relative quantification of viral RNA yields. (A) Vero E6 cells (5000 cells/well) were infected with pangolin CoV (MOI = 0.01) and were incubated in media containing different chemical drugs with a final concentration of 10 μmol/L. At 72 h p.i., the CPE was observed by using phase microscopy. (B) Infected cells with cepharanthine treatment had no obvious CPE and were further analyzed by detecting viral RNA level. ∗ P < 0.001. 2019n-CoV: 2019 Novel coronavirus; CPE: Cytopathic effect; CoV: Coronavirus; MOI: Multiplicity of infection; p.i.: Post-infection; mRNA: Messenger RNA.
Among the three drug candidates, CEP is of particular attention due to its profound anti-viral activity and previous reports of its inhibitory effects on both SARS-CoV and HCoV-OC43.[12,13] 50 ] = 0.98 μmol/L; cytotoxicity concentration 50% [CC50 ] = 39.30 μmol/L; selectivity index = 39.91) [Figure 3 A]. We next investigated this drug's anti-viral mechanism by conducting viral entry, post-entry, and full-time assays in 12 well plates [Figure 3 B]. In the viral entry assay, cells were incubated with media containing both viruses and CEP during the first 2 h of infection, then were washed with phosphate-buffered saline and were supplemented with media containing no drugs. In the viral post-entry assay, cells were incubated in media containing no CEP during the first 2 h of infection, then were supplemented with media containing CEP. In the viral full-time assay, cells were constantly incubated in media containing CEP. Viral RNA yields were determined by qRT-PCR at 48 h p.i. Compared to normal infections without drug treatment, the viral RNA yields in the entry, post-entry and full-time assays were 2.17-fold (0.46 ± 0.12 vs. 1.00 ± 0.37, t = 2.42, P < 0.01), 1618-fold ([6.18 ± 0.95] × 10−4 vs. 1.00 ± 0.43, t = 3.98, P <0.001), and 12,459-fold ([4.58 ± 1.27] × 10−5 vs. 1.00 ± 0.43, t = 4.03, P < 0.001) lower, respectively. Further plaque assays found no production of live viruses in the media containing 10 μmol/L CEP at 48 h p.i. [Figure 3 C]. Thus, our data suggest that CEP can potently inhibit coronavirus infection at viral entry and post-entry.
Figure 3: Anti-viral activities of cepharanthine against CoV in vitro . (A) Vero E6 cells were infected with CoV at an MOI of 0.05 in the treatment of different doses of cepharanthine for 48 h. The viral yield in the cell was then quantified by qRT-PCR and normalized by β-actin level. Cytotoxicity of these drugs to Vero E6 cells was measured by CellTiter-Blue assay. The left and right Y-axis of the graphs represent mean percentage of inhibition of virus yield and cytotoxicity of cepharanthine , respectively. The experiments were done in triplicates. (B) Time-of-addition experiment of cepharanthine . The detailed steps for time-of-addition experiment of cepharanthine were described in the method section, and virus and β-actin mRNA levels in the infected cell were quantified by qRT-PCR at 48 h p.i. ∗ P < 0.05 (C) Cepharanthine inhibited infectious virus production in the supernatant. The virus titers of supernatant of control cells (left) and cepharanthine treated cells (right) in the “full time” experiment were determined by plaque assay. CoV: Coronavirus; MOI: Multiplicity of infection; qRT-PCR: Quantitative real-time polymerase chain reaction; p.i.: Post-infection. EC50 : Concentration for 50% of maximal effect; CC50 : Cytotoxicity concentration 50%; SI: Selectivity index; mRNA: Messenger RNA; μM: μmol/L.
Discussion
Here we first described a 2019-nCoVr model for research of 2019-nCoV. This model is suitable for work at biosafety level-2. We then identified three clinically approved drugs (CEP, selamectin , and mefloquine hydrochloride ) that can inhibit a 2019-nCoVr infection, and suggested that these drugs be considered for further investigation in the treatment of 2019-nCoV infection.
Our finding of CEP as a potential drug for 2019-nCoV is especially instructive. This drug is an anti-inflammatory and anti-neoplastic alkaloid and is approved for leukopenia. It has multiple functions, such as inhibiting the efflux transporter ABCC10 of anti-tumor drugs,[14] [15] [16] [17,18]
Nonetheless, our finding of CEP and mefloquine as anti-2019-nCoVr agents was in agreement with previous studies in other coronaviruses of the genus Betacoronavirus . Two groups reported CEP as a drug candidate for SARS-CoV and HCoV-OC43, respectively.[12,13] [19] selamectin , which is marketed as a topical broad-spectrum parasiticide in cats and dogs to control fleas, heartworms, hookworms, roundworms, etc. The anti-viral mechanisms of these three drugs are unknown. We speculate that CEP and mefloquine are likely to target host cell pathways while selamectin might be a 2019-nCoVr-specific inhibitor.
The libraries of drugs used in this study contain 2406 compounds in total. Many of them have anti-viral activities against MERS-CoV and SARS-CoV.[19]
In conclusion, this is the first report of a 2019-nCoVr model. We suggest the three drugs (CEP, selamectin , and mefloquine hydrochloride ) be considered for further investigation to treat the 2019-nCoV infection. Due to its amenable nature, our 2019-nCoVr model could play a more important role in the development of therapies and vaccines against 2019-nCoV. With high homology to 2019-nCoV, this 2019-nCoVr isolate could be a potential live vaccine candidate. Cultured long before the outbreak of 2019-nCoV, our 2019-nCoVr isolate might play a significant role in the combat against COVID-19. Thus, our model in part reflects the importance of sustained coronavirus surveillance in wildlife.
Acknowledgements
The authors thank professor Guang-Xiang Luo from University of Alabama at Birmingham and Dr. Jun-Fen Fan from Xuanwu Hospital for their valuable suggestions.
Funding
This work was supported by a project from Ministry of Science and Technology of China (No. 2020YFC0840805), Key Project of Beijing University of Chemical Technology (No. XK1803-06), Fundamental Research Funds for Central Universities (No. BUCTRC201917), and a start-up funding for Dr. Yi-Gang Tong from Beijing Advanced Innovation Center for Soft Matter Science and Engineering.
Conflicts of interest
None.
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