Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-D-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-D-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
In the past 20 years, three novel human CoVs have emerged. The group 2b SARS-like CoV represent an existential and future threat to global health as evidenced by the emergence of SARS-CoV and SARS-CoV-2. Zoonotic SARS-like bat-CoV strains can use human angiotensin-converting enzyme 2 (ACE2) receptors, grow well in primary human airway cells, and vary by as much as 25% in key therapeutic and vaccine gene targets. Thus, to address the current public health emergency of COVID-19 and to maximize pandemic preparedness in the future, broad-based vaccines and therapeutics, which are active against the higher-risk RNA virus families prone to emergence, are desperately needed. Small-molecule antivirals can exert their antiviral effect through multiple mechanisms including blocking viral entry, inhibiting a virally encoded enzyme, blocking virus particle formation, or targeting a host factor required for replication. Multiple direct acting antivirals are currently under evaluation in randomized control trials to treat COVID-19 including hydroxychloroquine, RDV, and lopinavir/ritonavir. Here, we report the broad-spectrum antiviral activity of NHC and its orally bioavailable prodrug EIDD-2801 against SARS-CoV, MERS-CoV, and the current pandemic strain SARS-CoV-2 in primary HAE cells. In addition to CoV, NHC is broadly active against multiple genetically distinct viruses including VEEV, influenzas A and B, Ebola, and Chikungunya viruses. Here, we show that prophylactic and therapeutic EIDD-2801 significantly reduced lung viral loads and improved pulmonary function in mouse models of both SARS-CoV and MERS-CoV pathogenesis. Although the improvement in both SARS-CoV and MERS-CoV outcomes diminished with the delay of treatment initiation time, it is important to note that the kinetics of disease in mice are compressed as compared to that in humans. Whereas SARS-CoV and MERS-CoV lung titers peak on 1 to 2 dpi in mice concurrent with the onset of clinical signs and notable damage to the lung epithelium, in humans this occurs 7 to 10 days after the onset of symptoms. Thus, in mice, the window within which to treat emerging CoV infection before peak replication is compressed (e.g., 24 to 48 hours). As with oseltamivir treatment for influenza, which fails to provide a protective effect if administered >5 days after the onset of symptoms, the window in when to treat COVID-19 patients before peak virus replication is likely during the first week of symptoms when pharyngeal shedding is at its highest. However, virus replication and shedding may continue for several weeks in the most severe COVID-19 patients. Thus, early intervention with an antiviral-like EIDD-2801 is likely to provide the most clinical benefit, although there may be opportunities in severe patients where the duration of virus replication may be extended. Our current study is clearly limited by the lack of in vivo efficacy testing with SARS-CoV-2. Currently, robust mouse models that recapitulate the SARS-CoV-2 pathogenesis observed in humans do not yet exist because of a noted virus spike glycoprotein and mouse ACE2 receptor incompatibility complicating the evaluation of medical countermeasures. In addition, SARS-CoV, MERS-CoV, and SARS-CoV-2 disease severity increases with increasing age. Our studies are limited by the lack of drug efficacy testing in CoV-aged mouse models that recapitulate the age-related increase in pathogenesis observed in humans. The data provided in this manuscript suggest that EIDD-2801 should be quickly evaluated in primate models of human disease using immediate models for MERS-CoV and SARS-CoV pathogenesis or newly described cynomolgus and rhesus macaque models for SARS-CoV-2.
For VEEV and influenza, NHC/EIDD-2801 exerts its antiviral activity on the RdRp, leading to error catastrophe by inducing an error rate of replication that surpasses the error threshold allowed to sustain a virus population. This process occurs when NHC is incorporated during RNA synthesis and then subsequently misread, thus increasing mutation rates. Therefore, for CoV, the NHC MOA would appear less likely to be affected by the RNA proofreading activity encoded by the nsp14 exonuclease function that otherwise limits misincorporation. Here, we present data using Primer ID NGS to quantitate the frequency and identity of the mutational spectra in the MERS-CoV genome in both drug-treated primary human airway cells and in mice at single genome resolution. Because CoV are positive-sense RNA viruses that replicate through a negative-sense RNA intermediate, NHC incorporation as a C or a U can occur in both polarities of RNA. We found increased nucleotide transitions (A-to-G, G-to-A, C-to-U, and U-to-C) consistent with those reported after influenza and VEEV infections. Under identical conditions, RDV did not alter the mutation rate in MERS-CoV genomic RNA, supporting its reported MOA as a chain terminator of viral RNA synthesis. In primary human lung cell cultures and mice infected with MERS-CoV, the NHC mutation rates inversely correlated with a reduction in infectious virus. In addition, we found a positive correlation between increased mutation rates and the frequency of nonsynonymous mutations and the degree of therapeutic efficacy in mice. To explore the potential off-target effect in host mRNA, which may contribute to drug toxicity, we also examined the impact of NHC treatment on ISG15 transcripts, a gene highly induced after MERS-CoV infection. Although ISG15 transcripts are present in great abundance, an accumulation of mutations was not observed in ISG15 in this model even at 500 mg/kg dosing. These data also support previous studies using RNA-seq to demonstrate that the model CoV MHV displayed increased mutation frequencies after NHC treatment in vitro. With regard to nucleic acid specificity, ribonucleotides are efficiently removed from eukaryotic cell DNA; therefore, treating a viral infection with a mutagenic ribonucleoside analog should show a selectivity for incorporation into the viral genome and not be efficient at being incorporated into and inducing mutations into host cell DNA. Together, these data strongly support the notion that EIDD-2801 and its active nucleoside analog NHC exert their antiviral effect through the induction of error catastrophe in the targeted virus. While our data suggest that the MERS-CoV nsp14 proofreading activity appeared ineffective against NHC in vitro and EIDD-2801 in vivo, future studies should investigate the antiviral activity of NHC in the presence or absence of the nsp14 proofreading activity, as loss of this activity increased the sensitivity of MHV and SARS-CoV replication to RDV treatment.
Together, our data support the continued development of EIDD-2801 as a potent broad-spectrum antiviral that could be useful in treating contemporary, newly emerged and emerging CoV infections of the future.
MATERIALS AND METHODS Study design The primary goal of this study was to determine the antiviral activity of the nucleoside analog NHC (EIDD-1931) against multiple emerging CoV in vitro and antiviral efficacy of its prodrug, EIDD-2801, in mouse models of CoV pathogenesis. In coupling cell lines and primary HAE cell cultures, we evaluated the antiviral activity of NHC against the three most recently emerged human CoV: SARS-CoV, MERS-CoV, and SARS-CoV-2. For both SARS-CoV and MERS-CoV, the data presented for HAE studies are representative of those from two to three separate human donors. For SARS-CoV-2, the HAE were from a single human donor. We evaluated drug cytotoxicity in both Calu-3 2B4 and HAE cell cultures. Calu-3 and the SARS-CoV and MERS-CoV HAE studies were performed in biological triplicate. HAE studies with SARS-CoV-2 and the SARS- and MERS-like bat-CoV were performed with two wells per condition. Drug effects were measured relative to vehicle controls in vitro, and comparisons in vivo were performed to vehicle controls. We also aimed to determine the antiviral efficacy of EIDD-2801 in mouse models of CoV pathogenesis. These studies were intended to provide the preclinical data to justify nonhuman primate studies and human clinical trials. Mice were age- and sex-matched and randomly assigned into groups before infection and treatment. Pathology was scored blinded by a board-certified veterinary pathologist. Primary data for all studies are provided in data file S1.
Ethics regulation of laboratory animals Efficacy studies were performed in animal biosafety level 3 facilities at University of North Carolina (UNC) Chapel Hill. All work was conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at UNC Chapel Hill (Institutional Animal Care and Use Committee protocol no. 16-284) according to guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.
Compounds The parental compound NHC (all in vitro studies) and its prodrug EIDD-2801 (all in vivo studies) were supplied by the EIDD. NHC was supplied as a 10 mM stock in dimethyl sulfoxide (DMSO) and EIDD-2801 as a solid and solubilized in vehicle containing 10% PEG-400 and 2.5% Cremophor RH 40 in water (10, 2.5, or 87.5%, all v/v) before use. RDV was solubilized in 100% DMSO and provided by Gilead Sciences Inc. as previously described.
Cell cultures At UNC, the human lung epithelial cell line Calu-3 2B4 cells was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco), 20% fetal bovine serum (FBS; Hyclone), and 1× antibiotic/antimycotic (Gibco). At Vanderbilt University Medical Center (VUMC), Calu-3 2B4 cells were propagated in DMEM supplemented with 20% FBS (Gibco), penicillin and streptomycin (100 U/ml; Gibco), and 0.25 μM amphotericin B (Corning). At VUMC, Vero E6 cells were cultured in DMEM supplemented with 10% FBS (Gibco), 100 U/ml penicillin and streptomycin (Gibco), and 0.25 μM amphotericin B (Corning). At UNC, Vero E6 cells were cultured in DMEM supplemented with 10% FetalClone II (Hyclone) and 1× antibiotic/antimycotic (Gibco). Murine delayed brain tumor cells were maintained in DMEM supplemented with 10% FBS (Gibco), penicillin and streptomycin (100 U/ml; Gibco), and 0.25 μM amphotericin B (Corning). Primary HAE cell cultures were obtained from the Tissue Procurement and Cell Culture Core Laboratory in the Marsico Lung Institute/Cystic Fibrosis Research Center at UNC and are described more thoroughly below (52).
Virus strains Except for SARS-CoV-2, all viruses used for these studies were derived from infectious clones and isolated as previously described. SARS-CoV-2 clinical isolate was obtained at VUMC and UNC from the U.S. Centers for Disease Control and Prevention (2019-nCoV/USA-WA1/2020 strain, GenBank accession no. MN985325.1) and passaged twice in Vero E6 cells at each respective institution to create a passage 5 working stock. Virus strains for in vitro experiments include SARS-CoV expressing the green fluorescent protein (GFP) in place of ORF7a/b (SARS-GFP), bat spike receptor-binding domain (bat-SRBD), a chimeric CoV strain derived from the HKU3 SARS-like bat-CoV genomic sequence that has the wild-type (Urbani SARS-CoV strain) RBD in the HKU3 spike gene to allow for virus replication in nonhuman primate cell lines and HAE cultures, SHC014 SARS-like bat-CoV (11), MERS-nLUC in the place of ORF3, and MERS-CoV expressing the red fluorescent protein (RFP) gene in the place of ORF 5 (MERS-RFP). The virus stock used for MERS-CoV in vivo studies was derived from a plaque-purified isolate of the mouse-adapted MERS-CoV p35C4 strain. The virus stock used for SARS-CoV in vivo studies was derived from the infectious clone of the mouse-adapted SARS-MA15 strain. All work with MHV was performed using the recombinant wild-type strain MHV-A59 (GenBank accession no. AY910861).
In vitro antiviral activity experiments MERS-nLUC in Calu-3. At 48 hours before infection, Calu-3 2B4 cells were plated in a 96-well black-walled clear bottom plate at 5 × 104 cells per well. A 10 mM stock of NHC was serially diluted in 100% DMSO in threefold increments to obtain a 10-point dilution series. MERS-nLUC was diluted in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic to achieve a multiplicity of infection (MOI) of 0.08. Cells were infected and concurrently treated with NHC in triplicate per drug dilution for 1 hour, after which viral inoculum was aspirated, cultures were rinsed once, and fresh medium containing drug or vehicle was added. At 48 hpi, nanoluciferase expression as a surrogate for virus replication was quantitated on a SpectraMax plate reader (Molecular Devices) according to the manufacturer’s instructions (Promega, Nano-Glo). For the 100% inhibition control, diluted MERS-nLUC was exposed to short-wave ultraviolet light (UVP LLC) for 6 min to inhibit the ability of the virus to replicate. For the 0% inhibition control, cells were infected in the presence of vehicle only. DMSO was kept constant in all conditions at 0.05%. Values from triplicate wells per condition were averaged and compared to controls to generate a percent inhibition value for each drug dilution. The IC50 value was defined as the concentration at which there was a 50% decrease in luciferase expression. Data were analyzed using GraphPad Prism 8.0. The IC50 values were calculated by nonlinear regression analysis using the dose-response (variable slope) equation (four parameter logistic equation): Y = bottom + (top − bottom)/(1 + 10((LogIC50 − X)*HillSlope)). To measure cell viability to determine whether there was any NHC-induced cytotoxicity, Calu-3 2B4 cells were plated and treated with NHC only as described above. Cells were exposed to the same 10-point dilution series created for the in vitro efficacy studies. As above, 0.05% DMSO-treated cells served as the 0% cytotoxicity control. Wells without cells served as the 100% cytotoxic-positive control. After 48 hours, cell viability was measured on a SpectraMax (Molecular Devices) via CellTiter-Glo assay (Promega) according to the manufacturer’s protocol. Similar data were obtained in three independent experiments.
SARS-CoV-2 in Calu-3. Calu-3 2B4 cells were adsorbed with an MOI of 0.1 PFU per cell of SARS-CoV-2 (2019-nCoV/USA-WA1/2020 strain) at 37°C. Plates were manually rocked every 10 min to redistribute the inoculum. After 30 min, virus inoculum was removed, cells were washed with phosphate-buffered saline (PBS) once to remove unbound virus, medium containing NHC or vehicle control (DMSO) was added back onto the cells, and cells were incubated for 72 hours at 37°C.
SARS-CoV-2 in Vero E6. Vero E6 cells were plated at 20,000 cells per well in a 96-well plate. Twenty-four hours later, medium containing a dose response of NHC was added concurrent with SARS-CoV-2 (2019-nCoV/USA-WA1/2020 strain) at an MOI of 0.05. At 48 hpi, cell viability was measured by CellTiter-Glo assay.
SARS-CoV, MERS-CoV, and SARS-CoV-2 in HAE. Human tracheobronchial epithelial cells provided by S. Randell were obtained from airway specimens resected from patients undergoing surgery under UNC Institutional Review Board–approved protocols (no. 03-1396) by the Cystic Fibrosis Center Tissue Culture Core. Primary cells were expanded to generate passage 1 cells, and passage 2 cells were plated at a density of 250,000 cells per well on Transwell-COL (12 mm in diameter) supports (Corning). HAE cultures were generated by provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures that resembled in vivo pseudostratified mucociliary epithelium..At 48 hours before infection, the apical surface of the culture was washed with 500 μl of PBS for 1.5 hours at 37°C, and the cultures were moved into fresh air liquid interface media. Immediately before infection, apical surfaces were washed twice to remove accumulated mucus with 500 μl of PBS with each wash lasting 30 min at 37°C, and HAE cultures were moved into air liquid interface media containing various concentrations of NHC ranging from 10 to 0.0016 μM as indicated for each experiment (final % DMSO < 0.05%). Upon removing the second PBS wash, 200 μl of viral inoculum (SARS-GFP, MERS-RFP, or 2019-nCoV/USA-WA1/2020 strain) at an MOI of 0.5 was added to the apical surface, and HAE cultures were incubated for 2 hours at 37°C. Viral inoculum was then removed, and the apical surface of the cultures was washed three times with 500 μl of PBS and then incubated at 37°C until 48 hpi. For all HAE cultures, infectious virus produced was collected by washing the apical surface of the culture with 100 μl of PBS. Apical washes were stored at −80°C until analysis and titered by plaque assay as previously described.
qRT-PCR approach to assess cytotoxicity Total RNA was isolated using the Zymo Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s directions. Cells were treated with 1 μM staurosporine (Sigma-Aldrich) as a positive control. First-strand complementary DNA (cDNA) was generated using SuperScript III Reverse Transcriptase (Life Technologies). For quantification of cellular markers of toxicity/apoptosis, real-time PCR was performed using commercially validated TaqMan-based primer-probe sets (table S1) and TaqMan Universal PCR Mix (Life Technologies). Results were then normalized as described above.
MERS-CoV genomic RNA qRT-PCR Mouse lungs were stored in RNAlater (Thermo Fisher Scientific) at −80°C until processed via homogenization in TRIzol (Invitrogen). Total RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research). Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA [targeting orf1a: forward, 5′- GCACATCTGTGGTTCTCCTCTCT-3′; probe (6-FAM/ZEN/IBFQ), 5′- TGCTCCAACAGTTACAC-3′; reverse, 5′-AAGCCCAGGCCCTACTATTAGC-3′]. qRT-PCR was performed using 100 ng of total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) on a QuantStudio 3 (Applied Biosystems).
Quantification of SARS-CoV-2 viral RNA genome copy number by qRT-PCR Cell supernatants were harvested in TRIzol LS reagent (Invitrogen), and RNA was purified after phase separation by chloroform as recommended by the manufacturer. The RNA in the aqueous phase was collected and further purified using the PureLink RNA Mini Kits (Invitrogen) according to the manufacturer’s protocol. Viral RNA was quantified using one-step qRT-PCR on a StepOnePlus Real-Time PCR system (Applied Biosystems) by TaqMan Fast Virus 1-Step Master Mix chemistry (Applied Biosystems). SARS-CoV-2 N gene RNA was amplified using forward (5′-GACCCCAAAATCAGCGAAAT-3′) and reverse (5′-TCTGGTTACTGCCAGTTGAATCTG-3′) primers and probe (5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′) designed by the U.S. Centers for Disease Control and Prevention (oligonucleotides produced by IDT, catalog no. 10006606). Copy numbers were interpolated from a standard curve produced with dilutions of N gene RNA. In brief, SARS-CoV-2-N–positive control plasmid DNA (IDT, catalog no. 10006625) was amplified using forward (5′-TAATACGACTCACTATAGGGATGTCTGATAATGGACCCCA-3′) and reverse (5′- TTAGGCCTGAGTTGAGTCAG-3′) primers, resulting in a 1280 nucleotide fragment containing a T7 promoter. The PCR product was purified by column (Promega) and in vitro transcribed using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to the manufacturer’s protocol. Transcribed RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, and serial 10-fold dilutions were quantified as described above.
Primer ID and deep sequencing Primer ID NGS is designed to specifically identify and remove RT-PCR mutations while facilitating highly accurate sequence determination of single RNA molecules, because each cDNA is created with a barcoded degenerate primer (N10, 410 combinations) from which Illumina-indexed libraries are made. We used a multiplexed Primer ID library prep approach and MiSeq sequencing to investigate the presence of mutations in the viral genomes and murine mRNA. We designed cDNA primers targeting multiple regions on the viral genome and murine mRNA, each with a block of random nucleotides (11-bp long) as the Primer ID (table S2). Viral RNA was extracted using a QIAamp Viral RNA kit. A preamplification titration of templates was performed to estimate the amount of template to use. We used SuperScript III to make cDNA with multiplexed cDNA primers based on the regions to be sequenced. We used 41R_PID11 for the pilot sequencing and titration determination. For the MERS-CoV sequencing, we multiplexed nsp10_PID11, nsp12_PID11, and nsp14_PID11 for the cDNA reaction; for the murine mRNA sequencing, we used mixed primers of nsp10_PID11, ifit3_PID11, and isg15_PID11. After bead purification, we amplified the cDNA with a mixture of forward primers (based on the described schemes) and a universal reverse primer, followed by another round of PCR to incorporate Illumina sequencing adaptors and barcodes in the amplicons. After gel purification and quantification, we pooled 24 libraries for MiSeq 300-bp paired-end sequencing. The TCS pipeline version 1.38 (https://github.com/SwanstromLab/PID) was used to process the Primer ID sequencing data and construct TCSs to represent each individual input templates, and the sequences of each region in the pool were demultiplexed. The Ruby package viral_seq version 1.0.6 (https://rubygems.org/gems/viral_seq) was used to calculate the mutation rate at each position. National Center for Biotechnology Information (NCBI) sequence read archive (SRA) accession numbers for sequence data are as follows: PRJNA613261 and PRJNA613454.
In vivo experiments
We performed four mouse studies to evaluate the in vivo efficacy of the NHC prodrug (EIDD-2801). First, we performed prophylactic dose escalation studies for both SARS-CoV and MERS-CoV to determine the most efficacious dose of EIDD-2801 per virus. For SARS-CoV, in cohorts of equivalent numbers of male and female 20- to 29-week-old specific pathogen–free C57BL/6J (stock 000664, the Jackson laboratory) mice (n = 10 per dose group), we administered vehicle (10% PEG and 2.5% Cremophor RH 40 in water) or EIDD-2801 (50, 150, or 500 mg/kg) by oral gavage 2 hours before intranasal infection with 1 × 104 PFU mouse-adapted SARS-MA15 in 50 μl. Mice were anesthetized with a mixture of ketamine/xylazine before intranasal infection. Vehicle or drug was administered every 12 hours for the remainder of the study. Body weight and pulmonary function by WBP were measured daily. On 5 dpi, animals were sacrificed by isoflurane overdose, lungs were scored for lung hemorrhage, and the inferior right lobe was frozen at −80°C for viral titration via plaque assay. In brief, 500,000 Vero E6 cells per well were seeded in six-well plates. The following day, medium was removed, and serial dilutions of clarified lung homogenate were added per plate (10−1 to 10−6 dilutions) and incubated at 37°C for 1 hour, after which wells were overlaid with 1× DMEM, 5% FetalClone II Serum, 1× antibiotic/antimycotic, and 0.8% agarose. Two days after, plaques were enumerated to generate a plaque per milliliter value. Lung hemorrhage is a gross pathological phenotype readily observed by the naked eye driven by the degree of virus replication where the coloration of the lung changes from pink to dark red. The large left lobe was placed in 10% buffered formalin and stored at 4°C for 1 to 3 weeks until histological sectioning and analysis. For MERS-CoV, the prophylactic dose escalation studies were performed similarly as done for SARS-CoV with our recently developed mouse model for MERS-CoV, which has an hDPP4 receptor. We performed all in vivo studies with EIDD-2801 in equivalent numbers of 10- to 14-week-old female and male C57BL/6J hDPP4 mice. Second, we intranasally infected mice with 5 × 104 PFU mouse-adapted MERS-CoV strain M35C4 in 50 μl. Third, to titer lungs by plaque assay, Vero CCL81 cells were used, and plaques were enumerated 3 dpi.
To determine the time at which therapeutic administration of EIDD-2801 would fail to improve outcomes with SARS-CoV or MERS-CoV infection, we performed therapeutic efficacy studies in mice where we initiated treatment 2 hours before infection or 12, 24, or 48 hpi. As 500 mg/kg provided the most complete protection from disease in prophylactic SARS-CoV studies, this dose was used for both therapeutic efficacy studies. Vehicle or EIDD-2801 was given via oral gavage twice daily after initiation of treatment. For both SARS-CoV and MERS-CoV, the infectious dose for the therapeutic studies and the mouse strains were the same as that used in the prophylactic studies. The numbers of mice per group for the SARS-CoV studies were as follows: vehicle (n = 10), −2 hours (n = 10), +12 hours (n = 10), +24 hours (n = 10), and +48 hours (n = 10). The numbers of mice per group for the MERS-CoV therapeutic studies were as follows: vehicle (n = 9), −2 hours (n = 9), +12 hours (n = 9), +24 hours (n = 7), and +48 hours (n = 10). As described above, each day mouse body weight and pulmonary function were quantitated. On 5 dpi for SARS-CoV and 6 dpi for MERS-CoV, animals were humanely euthanized, and tissues were harvested and analyzed as described above. In addition, for the MERS-CoV study, lung tissue was harvested and stored in RNAlater (Thermo Fisher Scientific) at −80°C, thawed, and homogenized in TRIzol reagent (Invitrogen), and total RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research). This total RNA was then used for Primer ID and qRT-PCR.
Whole-body plethysmography Pulmonary function was monitored once daily via WBP (Buxco respiratory solutions, DSI Inc.). Mice intended for this analysis were randomly chosen before the initiation of the study. In brief, after a 30-min acclimation time in the plethysmograph, data for 11 parameters were recorded every 2 s for 5 min. ALI histological assessment tools
Two different and complementary quantitative histologic tools were used to determine whether antiviral treatments diminished the histopathologic features associated with lung injury. Both analyses and scoring were performed by a board-certified veterinary pathologist who was blinded to the treatment groups. American Thoracic Society lung injury scoring tool. To help quantitate histological features of ALI observed in mouse models and increase their translation to the human condition, we used the American Thoracic Society scoring tool. In a blinded manner, we chose three random diseased fields of lung tissue at high power (60×), which were scored for the following: (A) neutrophils in the alveolar space (none, 0; one to five cells, 1; >5 cells, 2), (B) neutrophils in the interstitial space/septae (none, 0; one to five cells, 1; >5 cells, 2), (C) hyaline membranes (none, 0; one membrane, 1; >1 membrane, 2), (D) proteinaceous debris in air spaces (none, 0; one instance, 1; >1 instance, 2), and (E) alveolar septal thickening (<2× mock thickness, 0; 2 to 4× mock thickness, 1; >4× mock thickness, 2). To obtain a lung injury score per field, the scores for A to E were then put into the following formula, which contains multipliers that assign varying levels of importance for each phenotype of the disease state: score = [(20 × A) + (14 × B) + (7 × C) + (7 × D) + (2 × E)]/100. The scores for the three fields per mouse were averaged to obtain a final score ranging from 0 to and including 1. DAD tool. The second histological tool to quantitate lung injury was reported by Schmidt et al. (64). DAD is the pathological hallmark of ALI. Three random diseased fields of lung tissue were scored at high power (60×) for the following in a blinded manner: 1, absence of cellular sloughing and necrosis; 2, uncommon solitary cell sloughing and necrosis (one to two foci per field); 3, multifocal (3 + foci) cellular sloughing and necrosis with uncommon septal wall hyalinization; or 4, multifocal (>75% of field) cellular sloughing and necrosis with common and/or prominent hyaline membranes. The scores for the three fields per mouse were averaged to get a final DAD score per mouse.
nsp12 phylogenetic analysis and conservation modeling CoV RdRp (nsp12) protein sequence alignments and phylogenetic trees were generated using Geneious Tree Builder in Geneious Prime (version 2020.0.5) and visualized using EvolView (www.evolgenius.info/evolview/). Protein similarity scores were calculated using BLOSUM62 matrix. The accession numbers used were as follows: PDCoV (KR265858), AIBV (NC_001451), HCoV-229E (JX503060), PEDV (NC_003436), MHV (AY700211), HCoV-HKU1 (DQ415904), HCoV-NL63 (JX504050), HCOV-OC43 (AY903460), HKU5-1 (NC_009020), MERS-CoV (JX869059), HKU9-4 (EF065516), 2019-nCoV (MN996528), HKU3-1 (DQ022305), SHC014 (KC881005), WIV1 (KF367457), and SARS-CoV (AY278741). Amino acid conservation scores of CoV RdRp were generated using ConSurf Server (https://consurf.tau.ac.il/) using the protein alignment described above and visualized on the SARS-CoV RdRp structure (Protein Data Bank: 6NUR) in PyMol (version 1.8.6.0).
Statistical analysis
All statistical data analyses were performed in GraphPad Prism 8. Statistical significance for each end point was determined with specific statistical tests. In general, for metrics with multiple treatment groups with longitudinal data (e.g., mouse weight loss or pulmonary function over time), two-way ANOVA was performed with the suggested multiple comparison test as advised by Prism. For comparative data with for a single time point (e.g., lung titer), Kruskal-Wallis test or one-way ANOVA was performed with the suggested multiple comparison test. For each test, P < 0.05 was considered significant. Specific tests are noted in each figure legend.
Reference & source information: https://stm.sciencemag.org/
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