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Post by Admin on Apr 3, 2020 2:30:20 GMT
Structural determination of the ACE2-B0AT1 complex Full-length human ACE2 and B0AT1, with Strep and FLAG tags on their respective N termini, were coexpressed in human embryonic kidney (HEK) 293F cells and purified through tandem affinity resin and size exclusion chromatography. The complex was eluted in a single monodisperse peak, indicating high homogeneity (Fig. 1A). Details of cryo-sample preparation, data acquisition, and structural determination are given in the materials and methods section of the supplementary materials. A three-dimensional (3D) reconstruction was obtained at an overall resolution of 2.9 Å from 418,140 selected particles. This immediately revealed the dimer of heterodimers’ architecture (Fig. 1B). After applying focused refinement and C2 symmetry expansion, the resolution of the extracellular domains improved to 2.7 Å, whereas the TM domain remained at 2.9-Å resolution (Fig. 1B, figs. S1 to S3, and table S1). Fig. 1 Overall structure of the ACE2-B0AT1 complex. (A) Representative size exclusion chromatography purification profile of full-length human ACE2 in complex with B0AT1. UV, ultraviolet; mAU, milli–absorbance units; MWM, molecular weight marker. (B) Cryo-EM map of the ACE2-B0AT1 complex. The map is generated by merging the focused refined maps shown in fig. S2. Protomer A of ACE2 (cyan), protomer B of ACE2 (blue), protomer A of B0AT1 (pink) and protomer B of B0AT1 (gray) are shown. (C) Cartoon representation of the atomic model of the ACE2-B0AT1 complex. The glycosylation moieties are shown as sticks. The complex is colored by subunits, with the PD and CLD in one ACE2 protomer colored cyan and blue, respectively. (D) An open conformation of the ACE2-B0AT1 complex. The two PDs, which contact each other in the closed conformation, are separated in the open conformation. The high resolution supported reliable model building. For ACE2, side chains could be assigned to residues 19 to 768, which contain the PD (residues 19 to 615) and the CLD (residues 616 to 768), which consists of a small extracellular domain, a long linker, and the single TM helix (Fig. 1C). Between the PD and TM helix is a ferredoxin-like fold domain; we refer to this as the neck domain (residues 616 to 726) (Fig. 1C and fig. S4). Homodimerization is entirely mediated by ACE2, which is sandwiched by B0AT1. Both the PD and neck domains contribute to dimerization, whereas each B0AT1 interacts with the neck and TM helix in the adjacent ACE2 (Fig. 1C). The extracellular region is highly glycosylated, with seven and five glycosylation sites on each ACE2 and B0AT1 monomer, respectively. During classification, another subset with 143,857 particles was processed to an overall resolution of 4.5 Å. Whereas the neck domain still dimerizes, the PDs are separated from each other in this reconstruction (Fig. 1D and fig. S1, H to K). We therefore define the two classes as the open and closed conformations. Structural comparison shows that the conformational changes are achieved through rotation of the PD domains, with the rest of the complex left nearly unchanged (movie S1). Homodimer interface of ACE2 Dimerization of ACE2 is mainly mediated by the neck domain, with the PD contributing a minor interface (Fig. 2A). The two ACE2 protomers are hereafter referred to as A and B, with residues in protomer B followed by a prime symbol. Extensive polar interactions are mapped to the interface between the second (residues 636 to 658) and fourth (residues 708 to 717) helices of the neck domain (Fig. 2B). Arg652 and Arg710 in ACE2-A form cation-π interactions with Tyr641′ and Tyr633′ in ACE2-B. Meanwhile, Arg652 and Arg710 are respectively hydrogen-bonded (H-bonded) to Asn638′ and Glu639′, which also interact with Gln653, as does Asn636′. Ser709 and Asp713 from ACE2-A are H-bonded to Arg716′. This extensive network of polar interactions indicates stable dimer formation. Fig. 2 Dimerization interface of ACE2. (A) ACE2 dimerizes through two interfaces, the PD and the neck domain. The regions enclosed by the cyan and red dashed lines are illustrated in detail in (B) and (C), respectively. (B) The primary dimeric interface is through the neck domain in ACE2. Polar interactions are represented by red dashed lines. (C) A weaker interface between PDs of ACE2. The only interaction is between Gln139 and Gln175′, which are highlighted as spheres. The polar residues that may contribute to the stabilization of Gln139 are shown as sticks. (D) The PDs no longer contact each other in the open state. Single-letter abbreviations for the amino acid residues used in the figures are as follows: C, Cys; D, Asp; E, Glu; F, Phe; H, His; K, Lys; L, Leu; M, Met; N, Asn; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr. The PD dimer interface appears much weaker, with only one pair of interactions between Gln139 and Gln175′ (Fig. 2C). Gln139 is in a loop that is stabilized by a disulfide bond between Cys133 and Cys141 as well as multiple intraloop polar interactions (Fig. 2C). The weak interaction is consistent with the ability to transition to the open conformation, in which the interface between the neck domains remains the same while the PDs are separated from each other by ~25 Å (Fig. 2D and movie S1).
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Post by Admin on Apr 3, 2020 6:59:26 GMT
Overall structure of the RBD-ACE2-B0AT1 complex To gain insight into the interaction between ACE2 and SARS-CoV-2, we purchased 0.2 mg of recombinantly expressed and purified RBD-mFc of SARS-CoV-2 (for simplicity, hereafter referred to as RBD; mFc, mouse Fc tag) from Sino Biological Inc., mixed it with our purified ACE2-B0AT1 complex at a stoichiometric ratio of ~1.1 to 1, and proceeded with cryo-grid preparation and imaging. Finally, a 3D EM reconstruction of the ternary complex was obtained. In contrast to the ACE2-B0AT1 complex—which has two conformations, open and closed—only the closed state of ACE2 was observed in the dataset for the RBD-ACE2-B0AT1 ternary complex. The structure of the ternary complex was determined to an overall resolution of 2.9 Å from 527,017 selected particles. However, the resolution for the ACE2-B0AT1 complex was substantially higher than that for the RBDs, which are at the periphery of the complex (Fig. 3A). To improve the local resolution, focused refinement was applied; this allowed us to reach a resolution of 3.5 Å for the RBD, supporting reliable modeling and analysis of the interface (Fig. 3, figs. S5 to S7, and table S1). Fig. 2 Dimerization interface of ACE2. (A) ACE2 dimerizes through two interfaces, the PD and the neck domain. The regions enclosed by the cyan and red dashed lines are illustrated in detail in (B) and (C), respectively. (B) The primary dimeric interface is through the neck domain in ACE2. Polar interactions are represented by red dashed lines. (C) A weaker interface between PDs of ACE2. The only interaction is between Gln139 and Gln175′, which are highlighted as spheres. The polar residues that may contribute to the stabilization of Gln139 are shown as sticks. (D) The PDs no longer contact each other in the open state. Single-letter abbreviations for the amino acid residues used in the figures are as follows: C, Cys; D, Asp; E, Glu; F, Phe; H, His; K, Lys; L, Leu; M, Met; N, Asn; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr. The PD dimer interface appears much weaker, with only one pair of interactions between Gln139 and Gln175′ (Fig. 2C). Gln139 is in a loop that is stabilized by a disulfide bond between Cys133 and Cys141 as well as multiple intraloop polar interactions (Fig. 2C). The weak interaction is consistent with the ability to transition to the open conformation, in which the interface between the neck domains remains the same while the PDs are separated from each other by ~25 Å (Fig. 2D and movie S1). Overall structure of the RBD-ACE2-B0AT1 complex To gain insight into the interaction between ACE2 and SARS-CoV-2, we purchased 0.2 mg of recombinantly expressed and purified RBD-mFc of SARS-CoV-2 (for simplicity, hereafter referred to as RBD; mFc, mouse Fc tag) from Sino Biological Inc., mixed it with our purified ACE2-B0AT1 complex at a stoichiometric ratio of ~1.1 to 1, and proceeded with cryo-grid preparation and imaging. Finally, a 3D EM reconstruction of the ternary complex was obtained. In contrast to the ACE2-B0AT1 complex—which has two conformations, open and closed—only the closed state of ACE2 was observed in the dataset for the RBD-ACE2-B0AT1 ternary complex. The structure of the ternary complex was determined to an overall resolution of 2.9 Å from 527,017 selected particles. However, the resolution for the ACE2-B0AT1 complex was substantially higher than that for the RBDs, which are at the periphery of the complex (Fig. 3A). To improve the local resolution, focused refinement was applied; this allowed us to reach a resolution of 3.5 Å for the RBD, supporting reliable modeling and analysis of the interface (Fig. 3, figs. S5 to S7, and table S1). Fig. 3 Overall structure of the RBD-ACE2-B0AT1 complex. (A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. The overall reconstruction of the ternary complex at 2.9 Å is shown on the left. The inset shows the focused refined map of RBD. The color scheme is the same as that in Fig. 1B, with the addition of red and gold, which represent RBD protomers. (B) Overall structure of the RBD-ACE2-B0AT1 complex. The color scheme is the same as that in Fig. 1C. The glycosylation moieties are shown as sticks. Interface between the RBD and ACE2 As expected, each PD accommodates one RBD (Fig. 3B). The overall interface is similar to that between SARS-CoV and ACE2 (7, 8), mediated mainly through polar interactions (Fig. 4A). An extended loop region of the RBD spans the arch-shaped α1 helix of the ACE2-PD like a bridge. The α2 helix and a loop that connects the β3 and β4 antiparallel strands, referred to as loop 3-4, of the PD also make limited contributions to the coordination of the RBD. Fig. 4 Interactions between SARS-CoV-2-RBD and ACE2. (A) The PD of ACE2 mainly engages the α1 helix in the recognition of the RBD. The α2 helix and the linker between β3 and β4 also contribute to the interaction. Only one RBD-ACE2 is shown. (B to D) Detailed analysis of the interface between SARS-CoV-2-RBD and ACE2. Polar interactions are indicated by red dashed lines. NAG, N-acetylglucosamine. The contact can be divided into three clusters. The two ends of the bridge interact with the N and C termini of the α1 helix as well as small areas on the α2 helix and loop 3-4. The middle segment of α1 reinforces the interaction by engaging two polar residues (Fig. 4A). At the N terminus of α1, Gln498, Thr500, and Asn501 of the RBD form a network of H-bonds with Tyr41, Gln42, Lys353, and Arg357 from ACE2 (Fig. 4B). In the middle of the bridge, Lys417 and Tyr453 of the RBD interact with Asp30 and His34 of ACE2, respectively (Fig. 4C). At the C terminus of α1, Gln474 of the RBD is H-bonded to Gln24 of ACE2, whereas Phe486 of the RBD interacts with Met82 of ACE2 through van der Waals forces (Fig. 4D). Comparing the SARS-CoV-2 and SARS-CoV interfaces with ACE2 Superimposition of the RBD in the complex of SARS-CoV (SARS-CoV-RBD) and ACE2-PD [Protein Data Bank (PDB) 2AJF] with the RBD in our ternary complex shows that the SARS-CoV-2 RBD (SARS-CoV-2-RBD) is similar to SARS-CoV-RBD with a root mean square deviation (RMSD) of 0.68 Å over 139 pairs of Cα atoms (Fig. 5A) (8). Despite the overall similarity, a number of sequence variations and conformational deviations are found in their respective interfaces with ACE2 (Fig. 5 and fig. S8). At the N terminus of α1, the variations Arg426→Asn439, Tyr484→Gln498, and Thr487→Asn501 at equivalent positions are observed between SARS-CoV-RBD and SARS-CoV-2-RBD (Fig. 5B). More variations are observed in the middle of the bridge. The most prominent alteration is the substitution of Val404 in the SARS-CoV-RBD with Lys417 in the SARS-CoV-2-RBD. In addition, from SARS-CoV-RBD to SARS-CoV-2-RBD, the substitution of interface residues Tyr442→Leu455, Leu443→Phe456, Phe460→Tyr473, and Asn479→Gln493 may also change the affinity for ACE2 (Fig. 5C). At the C terminus of α1, Leu472 in the SARS-CoV-RBD is replaced by Phe486 in the SARS-CoV-2-RBD (Fig. 5D).
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Post by Admin on Apr 3, 2020 19:40:08 GMT
Fig. 5 Interface comparison between SARS-CoV-2-RBD and SARS-CoV-RBD with ACE2. (A) Structural alignment for the SARS-CoV-2-RBD and SARS-CoV-RBD. The structure of the ACE2-PD and the SARS-CoV-RBD complex (PDB 2AJF) is superimposed on our cryo-EM structure of the ternary complex relative to the RBDs. The regions enclosed by the purple, blue, and red dashed lines are illustrated in detail in (B) to (D), respectively. SARS-CoV-2-RBD and the PD in our cryo-EM structure are colored orange and cyan, respectively; SARS-CoV-RBD and its complexed PD are colored green and gold, respectively. (B to D) Variation of the interface residues between SARS-CoV-2-RBD (labeled in brown) and SARS-CoV-RBD (labeled in green). Discussion Although ACE2 is a chaperone for B0AT1, our focus is on ACE2 in this study. With the stabilization by B0AT1, we elucidated the structure of full-length ACE2. B0AT1 is not involved in dimerization, suggesting that ACE2 may be a homodimer even in the absence of B0AT1. Further examination suggests that a dimeric ACE2 can accommodate two S protein trimers, each through a monomer of ACE2 (fig. S9). The trimeric structure of the S protein of SARS-CoV-2 was recently reported, with one RBD in an up conformation and two in down conformations (PDB 6VSB) (14). The PD clashes with the rest of the S protein when the ternary complex is aligned to the RBD of the down conformation. There is no clash when the complex is superimposed on RDB in the up conformation, with a RMSD of 0.98 Å over 126 pairs of Cα atoms, confirming that an up conformation of RDB is required to bind to the receptor (fig. S9) (14). Cleavage of the S protein of SARS-CoV is facilitated by cathepsin L in endosomes, indicating a mechanism of receptor-mediated endocytosis (10). Further characterization is required to examine the interactions between ACE2 and the viral particle as well as the effect of cofactors on this process (25, 33). It remains to be investigated whether there is clustering between the dimeric ACE2 and trimeric S proteins, which may be important for invagination of the membrane and endocytosis of the viral particle, a process similar to other types of receptor-mediated endocytosis. Cleavage of the C-terminal segment, especially residues 697 to 716 (fig. S4), of ACE2 by proteases, such as transmembrane protease serine 2 (TMPRSS2), enhances the S protein–driven viral entry (34, 35). Residues 697 to 716 form the third and fourth helices in the neck domain and map to the dimeric interface of ACE2. The presence of B0AT1 may block the access of TMPRSS2 to the cutting site on ACE2. The expression distribution of ACE2 is broader than that of B0AT1. In addition to kidneys and intestine, where B0AT1 is mainly expressed, ACE2 is also expressed in lungs and heart (27). It remains to be tested whether B0AT1 can suppress SARS-CoV-2 infection by blocking ACE2 cleavage. Enteric infections have been reported for SARS-CoV, and possibly also for SARS-CoV-2 (36, 37). B0AT1 has also been shown to interact with another coronavirus receptor, aminopeptidase N (APN or CD13) (38). These findings suggest that B0AT1 may play a regulatory role for the enteric infections of some coronaviruses. Comparing the interaction interfaces of SARS-CoV-2-RBD and SARS-CoV-RBD with ACE2 reveals some variations that may strengthen the interactions between SARS-CoV-2-RBD and ACE2 and other variations that are likely to reduce the affinity compared with SARS-CoV-RBD and ACE2. For instance, the change from Val404 to Lys317 may result in a tighter association because of the salt bridge formation between Lys317 and Asp30 of ACE2 (Figs. 4C and 5C). The change from Leu472 to Phe486 may also result in a stronger van der Waals contact with Met82 (Fig. 5D). However, replacement of Arg426 with Asn439 appears to weaken the interaction by eliminating one important salt bridge with Asp329 on ACE2 (Fig. 5B). Our structural work reveals the high-resolution structure of full-length ACE2 in a dimeric assembly. Docking the S protein trimer onto the structure of the ACE2 dimer with the RBD of the S protein bound suggests simultaneous binding of two S protein trimers to an ACE2 dimer. Structure-based rational design of binders with enhanced affinities to either ACE2 or the S protein of the coronaviruses may facilitate development of decoy ligands or neutralizing antibodies for suppression of viral infection.
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Post by Admin on Apr 6, 2020 7:14:59 GMT
Presumed Asymptomatic Carrier Transmission of COVID-19 Yan Bai, MD1; Lingsheng Yao, MD2; Tao Wei, MD3; et al
A novel coronavirus has resulted in an ongoing outbreak of viral pneumonia in China.1-3 Person-to-person transmission has been demonstrated,1 but, to our knowledge, transmission of the novel coronavirus that causes coronavirus disease 2019 (COVID-19) from an asymptomatic carrier with normal chest computed tomography (CT) findings has not been reported.
Methods In January 2020, we enrolled a familial cluster of 5 patients with fever and respiratory symptoms who were admitted to the Fifth People’s Hospital of Anyang, Anyang, China, and 1 asymptomatic family member. This study was approved by the local institutional review board, and written informed consent was obtained from all patients. A detailed analysis of patient records was performed.
All patients underwent chest CT imaging. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests for COVID-19 nucleic acid were performed using nasopharyngeal swabs (Novel Coronavirus PCR Fluorescence Diagnostic Kit, BioGerm Medical Biotechnology).
Results Patient 1 (presumed asymptomatic carrier), a 20-year-old woman, lives in Wuhan and traveled to Anyang on January 10, 2020. She initially met with patients 2 and 3 on January 10. On January 13, she accompanied 5 relatives (patients 2 through 6) to visit another hospitalized relative in Anyang District Hospital (Figure). There was no report of COVID-19 at this hospital. After development of disease in her relatives, patient 1 was isolated and observed. As of February 11, she had no elevated temperature measured or self-reported fever and no gastrointestinal or respiratory symptoms, including cough and sore throat, reported or observed by the physicians. Chest CT images on January 27 and 31 showed no significant abnormalities. Her C-reactive protein level and lymphocyte count were normal (Table). Results of RT-PCR testing were negative on January 26, positive on January 28, and negative on February 5 and 8.
Patients 2 through 6 developed COVID-19. Four were women, and ages ranged from 42 to 57 years. None of the patients had visited Wuhan or been in contact with any other people who had traveled to Wuhan (except patient 1).
Patients 2 through 5 developed fever and respiratory symptoms between January 23 and January 26 and were admitted to the hospital on the same day. All patients had RT-PCR test results positive for COVID-19 within 1 day. Patient 6 developed fever and sore throat on January 17 and went to the local clinic for treatment. There was no report of COVID-19 at the clinic. Her symptoms improved over the next few days but worsened on January 24, when she was admitted to the hospital and confirmed to have COVID-19 on January 26. Two patients developed severe pneumonia; the other infections were moderate.
All symptomatic patients had multifocal ground-glass opacities on chest CT, and 1 also had subsegmental areas of consolidation and fibrosis. All the symptomatic patients had increased C-reactive protein levels and reduced lymphocyte counts (Table).
Discussion A familial cluster of 5 patients with COVID-19 pneumonia in Anyang, China, had contact before their symptom onset with an asymptomatic family member who had traveled from the epidemic center of Wuhan. The sequence of events suggests that the coronavirus may have been transmitted by the asymptomatic carrier. The incubation period for patient 1 was 19 days, which is long but within the reported range of 0 to 24 days.4 Her first RT-PCR result was negative; false-negative results have been observed related to the quality of the kit, the collected sample, or performance of the test. RT-PCR has been widely deployed in diagnostic virology and has yielded few false-positive outcomes.5 Thus, her second RT-PCR result was unlikely to have been a false-positive and was used to define infection with the coronavirus that causes COVID-19.
One previous study reported an asymptomatic 10-year-old boy with COVID-19 infection, but he had abnormalities on chest CT.6 If the findings in this report of presumed transmission by an asymptomatic carrier are replicated, the prevention of COVID-19 infection would prove challenging. The mechanism by which asymptomatic carriers could acquire and transmit the coronavirus that causes COVID-19 requires further study.
Published Online: February 21, 2020. doi:10.1001/jama.2020.2565
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Post by Admin on Apr 13, 2020 7:56:41 GMT
The FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro Abstract Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 hours post infection with SARS-CoV-2 able to effect ∼5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans. Ivermectin is an FDA-approved broad spectrum anti-parasitic agent1 that in recent years we, along with other groups, have shown to have anti-viral activity against a broad range of viruses2, 3, 4, 5 in vitro. Originally identified as an inhibitor of interaction between the human immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the importin (IMP) α/β1 heterodimer responsible for IN nuclear import6, Ivermectin has since been confirmed to inhibit IN nuclear import and HIV-1 replication5. Other actions of ivermectin have been reported7, but ivermectin has been shown to inhibit nuclear import of host (eg.8,9) and viral proteins, including simian virus SV40 large tumour antigen (T-ag) and dengue virus (DENV) non-structural protein 55, 6. Importantly, it has been demonstrated to limit infection by RNA viruses such as DENV 1-44, West Nile Virus10, Venezuelan equine encephalitis virus (VEEV)3 and influenza2, with this broad spectrum activity believed to be due to the reliance by many different RNA viruses on IMPα/β1 during infection11,12. Ivermectin has similarly been shown to be effective against the DNA virus pseudorabies virus (PRV) both in vitro and in vivo, with ivermectin treatment shown to increase survival in PRV-infected mice13. Efficacy was not observed for ivermectin against Zika virus (ZIKV) in mice, but the authors acknowledged that study limitations justified re-evaluation of ivermectin’s anti-ZIKV activity14. Finally, ivermectin was the focus of a phase III clinical trial in Thailand in 2014-2017, against DENV infection, in which a single daily oral dose was observed to be safe and resulted in a significant reduction in serum levels of viral NS1 protein, but no change in viremia or clinical benefit was observed (see below)15. The causative agent of the current COVID-19 pandemic, SARS-CoV-2, is a single stranded positive sense RNA virus that is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV). Studies on SARS-CoV proteins have revealed a potential role for IMPα/β1 during infection in signal-dependent nucleocytoplasmic shutting of the SARS-CoV Nucleocapsid protein16, 17, 18, that may impact host cell division19,20. In addition, the SARS-CoV accessory protein ORF6 has been shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMPα/β1 on the rough ER/Golgi membrane21. Taken together, these reports suggested that ivermectin’s nuclear transport inhibitory activity may be effective against SARS-CoV-2. To test the antiviral activity of ivermectin towards SARS-CoV-2, we infected Vero/hSLAM cells with SARS-CoV-2 isolate Australia/VIC01/2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM ivermectin. Supernatant and cell pellets were harvested at days 0-3 and analysed by RT-PCR for the replication of SARS-CoV-2 RNA (Fig. 1A/B). At 24 h, there was a 93% reduction in viral RNA present in the supernatant (indicative of released virions) of samples treated with ivermectin compared to the vehicle DMSO. Similarly a 99.8% reduction in cell-associated viral RNA (indicative of unreleased and unpackaged virions) was observed with ivermectin treatment. By 48h this effect increased to an ∼5000-fold reduction of viral RNA in ivermectin-treated compared to control samples, indicating that ivermectin treatment resulted in the effective loss of essentially all viral material by 48 h. Consistent with this idea, no further reduction in viral RNA was observed at 72 h. As we have observed previously3, 4, 5, no toxicity of ivermectin was observed at any of the timepoints tested, in either the sample wells or in parallel tested drug alone samples. Figure 1. Ivermectin is a potent inhibitor of the SARS-CoV-2 clinical isolate Australia/VIC01/2020. Vero/hSLAM cells were in infected with SARS-CoV-2 clinical isolate Australia/VIC01/2020 (MOI = 0.1) for 2 h prior to addition of vehicle (DMSO) or Ivermectin at the indicated concentrations. Samples were taken at 0-3 days post infection for quantitation of viral load using real-time PCR of cell associated virus (A) or supernatant (B). IC50 values were determined in subsequent experiments at 48 h post infection using the indicated concentrations of Ivermectin (treated at 2 h post infection as per A/B). Triplicate real-time PCR analysis was performed on cell associated virus (C/E) or supernatant (D/F) using probes against either the SARS-CoV-2 E (C/D) or RdRp (E/F) genes. Results represent mean ± SD (n=3). 3 parameter dose response curves were fitted using GraphPad prism to determine IC50 values (indicated). G. Schematic of ivermectin’s proposed antiviral action on coronavirus. IMPα/β1 binds to the coronavirus cargo protein in the cytoplasm (top) and translocates it through the nuclear pore complex (NPC) into the nucleus where the complex falls apart and the viral cargo can reduce the host cell’s antiviral response, leading to enhanced infection. Ivermectin binds to and destabilises the Impα/β1 heterodimer thereby preventing Impα/β1 from binding to the viral protein (bottom) and preventing it from entering the nucleus. This likely results in reduced inhibition of the antiviral responses, leading to a normal, more efficient antiviral response. To further determine the effectiveness of ivemectin, cells infected with SARS-CoV-2 were treated with serial dilutions of ivermectin 2 h post infection and supernatant and cell pellets collected for real-time RT-PCR at 48 h (Fig. 1C/D). As above, a >5000 reduction in viral RNA was observed in both supernatant and cell pellets from samples treated with 5 μM ivermectin at 48 h, equating to a 99.98% reduction in viral RNA in these samples. Again, no toxicity was observed with ivermectin at any of the concentrations tested. The IC50 of ivermectin treatment was determined to be ∼2μM under these conditions. Underlining the fact that the assay indeed specifically detected SARS-CoV-2, RT-PCR experiments were repeated using primers specific for the viral RdRp gene (Fig. 1E/F) rather than the E gene (above), with nearly identical results observed for both released (supernatant) and cell-associated virus. Taken together these results demonstrate that ivermectin has antiviral action against the SARS-CoV-2 clinical isolate in vitro, with a single dose able to control viral replication within 24-48 h in our system. We hypothesise that this is likely through inhibiting IMPα/β1-mediated nuclear import of viral proteins (Fig. 1G), as shown for other RNA viruses4,5,10; confirmation of this mechanism in the case of SARS-CoV-2, and identification of the specific SARS-CoV-2 and/or host component(s) impacted (see10) is an important focus future work in this laboratory. Ultimately, development of an effective anti-viral for SARS-CoV-2, if given to patients early in infection, could help to limit the viral load, prevent severe disease progression and limit person-person transmission. Benchmarking testing of ivermectin against other potential antivirals for SARS-CoV-2 with alternative mechanisms of action22, 23, 24, 25, 26 would thus be important as soon as practicable. This Brief Report raises the possibility that ivermectin could be a useful antiviral to limit SARS-CoV-2, in similar fashion to those already reported22, 23, 24, 25, 26; until one of these is proven to be beneficial in a clinical setting, all should be pursued as rapidly as possible. Ivermectin has an established safety profile for human use1,12,27, and is FDA-approved for a number of parasitic infections1,27. Importantly, recent reviews and meta-analysis indicate that high dose ivermectin has comparable safety as the standard low-dose treatment, although there is not enough evidence to make conclusions about the safety profile in pregnancy28,29. The critical next step in further evaluation for possible benefit in COVID-19 patients will be to examine a multiple addition dosing regimen that mimics the current approved usage of ivermectin in humans. As noted, ivermectin was the focus of a recent phase III clinical trial in dengue patients in Thailand, in which a single daily dose was found to be safe but did not produce any clinical benefit. However, the investigators noted that an improved dosing regimen might be developed, based on pharmacokinetic data15. Although DENV is clearly very different to SARS-CoV-2, this trial design should inform future work going forward. Altogether the current report, combined with a known-safety profile, demonstrates that ivermectin is worthy of further consideration as a possible SARS-CoV-2 antiviral. Methods Cell culture, viral infection and drug treatment Vero/hSLAM cells30 were maintained in Earle’s Minimum Essential Medium (EMEM) containing 7% Fetal Bovine Serum (FBS) (Bovogen Biologicals, Keilor East, AUS) 2 mM L-Glutamine, 1 mM Sodium pyruvate, 1500 mg/L sodium bicarbonate, 15 mM HEPES and 0.4 mg/ml geneticin at 37°C, 5% CO2. Cells were seeded into 12-well tissue culture plates 24 h prior to infection with SARS-CoV-2 (Australia/VIC01/2020 isolate) at an MOI of 0.1 in infection media (as per maintenance media but containing only 2% FBS) for 2 h. Media containing inoculum was removed and replaced with 1 mL fresh media (2% FBS) containing Ivermectin at the indicated concentrations or DMSO alone and incubated as indicated for 0-3 days. At the appropriate timepoint, cell supernatant was collected and spun for 10 min at 6,000g to remove debris and the supernatant transferred to fresh collection tubes. The cell monolayers were collected by scraping and resuspension into 1 mL fresh media (2% FBS). Toxicity controls were set up in parallel in every experiment on uninfected cells. Generation of SARS-CoV-2 cDNA RNA was extracted from 200 μL aliquots of sample supernatant or cell suspension using the QIAamp 96 Virus QIAcube HT Kit (Qiagen, Hilden, Germany) and eluted in 60 μl. Reverse transcription was performed using the BioLine SensiFAST cDNA kit (Bioline, London, United Kingdom), total reaction mixture (20 μl), containing 10 μL of RNA extract, 4 μl of 5x TransAmp buffer, 1μl of Reverse Transcriptase and 5 μl of Nuclease free water. The reactions were incubated at 25°C for 10 min, 42°C for 15 min and 85°C for 5 min. Detection of SARS-CoV-2 using a TaqMan Real-time RT-PCR assay. TaqMan RT-PCR assay were performed using 2.5 μl cDNA, 10 μl Primer Design PrecisonPLUS qPCR Master Mix 1 μM Forward (5’- AAA TTC TAT GGT GGT TGG CAC AAC ATG TT-3’), 1 μM Reverse (5’- TAG GCA TAG CTC TRT CAC AYT T-3’) primers and 0.2 μM probe (5’-FAM- TGG GTT GGG ATT ATC-MGBNFQ-3’) targeting the BetaCoV RdRp (RNA-dependent RNA polymerase) gene or Forward (5’-ACA GGT ACG TTA ATA GTT AAT AGC GT -3’), 1 μM Reverse (5’-ATA TTG CAG CAG TAC GCA CAC A-3’) primers and 0.2 μM probe (5’-FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-286 NFQ-3’) targeting the BetaCoV E-gene31. Real-time RT-PCR assays were performed on an Applied Biosystems ABI 7500 Fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA) using cycling conditions of 95°C for 2 min, 95°C for 5 s, 60°C for 24 s. SARS-CoV-2 cDNA (Ct∼28) was used as a positive control. Calculated Ct values were converted to fold-reduction of treated samples compared to control using the ΔCt method (fold changed in viral RNA = 2ˆΔCt) and expressed as % of DMSO alone sample. IC50 values were fitted using 3 parameter dose response curves in GraphPad prism. doi.org/10.1016/j.antiviral.2020.104787
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