Broad-spectrum antivirals are desirable, particularly in the context of emerging zoonotic infections for which specific interventions do not yet exist. Sheahan et al. tested the potential of a ribonucleoside analog previously shown to be active against other RNA viruses such as influenza and Ebola virus to combat coronaviruses. This drug was effective in cell lines and primary human airway epithelial cultures against multiple coronaviruses including SARS-CoV-2. Mouse models of SARS and MERS demonstrated that early treatment reduced viral replication and damage to the lungs. Mechanistically, this drug is incorporated into the viral RNA, inducing mutations and eventually leading to error catastrophe in the virus. In this manner, inducing catastrophe could help avoid catastrophe by stemming the next pandemic.

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-N 4 -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-N 4 -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.

Currently, there are no approved therapies specific for any human CoV. β-d-N 4 -hydroxycytidine (NHC; EIDD-1931) is an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various unrelated RNA viruses including influenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV) ( 13 – 16 ). For VEEV, the mechanism of action (MOA) for NHC has been shown to be through lethal mutagenesis where deleterious transition mutations accumulate in viral RNA ( 14 , 17 ). Thus, we sought to determine NHC’s breadth of antiviral activity against multiple emerging CoV, its MOA for CoV, and its efficacy in mouse models of CoV pathogenesis.

The genetically diverse Orthocoronavirinae [coronavirus (CoV)] family circulates in many avian and mammalian species. Phylogenetically, CoVs are divided into four genera: alpha (group 1), beta (group 2), gamma (group 3), and delta (group 4). Three new human CoV have emerged in the past 20 years with severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012, and now SARS-CoV-2 in 2019 ( 1 – 3 ). All human CoV are thought to have emerged originally as zoonoses ( 4 – 6 ). The ongoing SARS-CoV-2 pandemic [referred to as CoV disease 2019 (COVID-19)] has caused more than 500,000 infections and more than 25,000 deaths in 199 countries. Like SARS-CoV and MERS-CoV, the respiratory disease caused by SARS-CoV-2 can progress to acute lung injury (ALI), an end-stage lung disease with limited treatment options and very poor prognoses ( 3 , 7 , 8 ). This emergence paradigm is not limited to humans. A novel group 1 CoV called swine acute diarrhea syndrome CoV (SADS-CoV) recently emerged from bats causing the loss of more than 20,000 pigs in Guangdong Province, China ( 9 ). More alarmingly, many group 2 SARS-like and MERS-like CoVs are circulating in bat reservoir species that can use human receptors and replicate efficiently in primary human lung cells without adaptation ( 9 – 12 ). The presence of these “preepidemic” zoonotic strains foreshadow the emergence and epidemic potential of additional SARS-like and MERS-like viruses in the future. Given the diversity of CoV strains in zoonotic reservoirs and a penchant for emergence, broadly active antivirals are clearly needed for rapid response to new CoV outbreaks in humans and domesticated animals.

RESULTS

NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication To determine whether NHC blocks the replication of highly pathogenic human CoV, we performed antiviral assays in cell lines with MERS-CoV and the newly emerging SARS-CoV-2. We first assessed the antiviral activity of NHC against MERS-CoV in the human lung epithelial cell line Calu-3 2B4 (“Calu-3” cells). Using a recombinant MERS-CoV expressing nanoluciferase (MERS-nLUC) (18), we measured virus replication in cultures exposed to a dose range of drug for 48 hours. NHC was potently antiviral with an average median inhibitory concentration (IC 50 ) of 0.15 μM and no observed cytoxicity in similarly treated uninfected cultures across the dose range [50% cytotoxic concentration (CC 50 ) of >10 μM; Fig. 1A]. The therapeutic index for NHC was >100. Using a clinical isolate of SARS-CoV-2 (2019-nCoV/USA-WA1/2020), we performed antiviral assays in African green monkey kidney (Vero) cells and found that NHC was potently antiviral with an IC 50 of 0.3 μM and CC 50 of >10 μM (Fig. 1B). We then determined the antiviral activity of NHC against SARS-CoV-2 in the Calu-3 cells through the measurement of infectious virus production and viral genomes. We observed a dose-dependent reduction in virus titers (Fig. 1C) with an IC 50 of 0.08 μM. Viral genomic RNA was quantitated in clarified supernatants by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 1D). Like the effect on infectious titers, we found a dose-dependent reduction in viral genomic RNA and a similar calculated IC 50 of 0.09 μM. Collectively, these data demonstrate that NHC is potently antiviral against two genetically distinct emerging CoV. Fig. 1 NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (MERS-nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via CellTiter-Glo assay. Data are combined from three independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by CellTiter-Glo assay. Cytotoxicity was measured as in (A). Data are combined from two independent experiments. (C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with SARS-CoV-2 at an MOI of 0.1 for 30 min, washed, and exposed to a dose response of NHC in triplicate per condition. At 72 hpi, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in (C). Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For (A) to (D), the symbol is at the mean, and the error bars represent the SD.

NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures To determine whether NHC would be similarly antiviral in primary human cells, we performed a series of studies in primary human airway epithelia (HAE) cell cultures. HAE models the architecture and cellular complexity of the conducting airway and is readily infected by multiple human and zoonotic CoV, including SARS-CoV and MERS-CoV (19). We first assessed the cytotoxicity of NHC in HAE treated with an extended dose range for 48 hours using quantitative PCR of cell death–related gene transcripts as our metric. NHC treatment did not appreciably alter gene expression even at doses up to 100 μM (fig. S1). We then sought to determine whether NHC would inhibit clinical isolate SARS-CoV-2 replication in HAE. We observed a dose-dependent reduction in SARS-CoV-2 infectious virus production (Fig. 2A). In MERS-CoV–infected HAE, NHC substantially reduced virus production with maximal titer reduction of >5 logs at 10 μM (average IC 50 = 0.024 μM), which correlated with reduced genomic open reading frame 1 (ORF1) and subgenomic [ORF nucleocapsid (ORFN)] RNA in paired samples (Fig. 2B). We observed similar trends in titer reduction (>3 log at 10 μM, average IC 50 = 0.14 μM) and in copies of genomic and subgenomic RNA in SARS-CoV–infected HAE (Fig. 2C). Thus, NHC was potently antiviral against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human epithelial cell cultures without cytotoxicity. Fig. 2 NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary HAE cell cultures. (A) Human airway epithelia (HAE) cultures were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate, after which the virus was removed, and cultures were washed in, incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to (A). qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in (C) for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units; l.o.d., limit of detection. (C) Studies performed as in (A) but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean, and the error bars represent the SD.

NHC is effective against remdesivir-resistant virus and multiple distinct zoonotic CoV CoV are taxonomically divided into multiple genogroups (alpha, beta, gamma, and delta), but human-infecting CoV are found only in the alpha and beta subgroups thus far (Fig. 3A). There is high sequence conservation in the RNA-dependent RNA polymerase [RdRp; nonstructural protein 12 (nsp12)] across CoV (Fig. 3A). For example, the RdRp of SARS-CoV-2 has 99.1% similarity and 96% amino acid identity to that of SARS-CoV (Fig. 3A). To gain insight into structural conservation of RdRp across the CoV family, we modeled the variation reflected in the RdRp dendrogram in Fig. 3A onto the structure of the SARS-CoV RdRp (Fig. 3B) (20). The core of the RdRp molecule and main structural motifs that all RdRp harbor (Fig. 3B and fig. S2) is highly conserved among CoV including SARS-CoV-2. We previously reported that CoV resistance to another broad-spectrum nucleoside analog, remdesivir (RDV), was mediated by RdRp residues F480L and V557L in a model CoV mouse hepatitis virus (MHV) and in SARS-CoV, resulting in a fivefold shift in IC 50 (Fig. 3C) (21). Consequently, we tested whether RDV resistance mutations in MHV conferred cross-resistance to NHC. The two RDV resistance mutations, alone or together, conferred increased sensitivity to inhibition by NHC (Fig. 3D). As our previous studies have demonstrated a high genetic barrier to NHC for VEEV, influenza, and CoV (14–16), the lack of cross-resistance further suggests that NHC and RDV may select for exclusive and mutually sensitizing resistance pathways. Fig. 3 Remdesivir resistance mutations in the highly conserved RdRp increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heat map adjacent to each neighbor-joining tree depicts percent amino acid identity (% amino acid similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in (A) was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in (A) at known resistance alleles to antiviral drug remdesivir (RDV). (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph. To explore the breadth of antiviral efficacy against zoonotic CoV, we performed antiviral assays in HAE with three zoonotic bat-CoV: SHC014, HKU3, and HKU5. Closely related to the beta 2b SARS-CoV, bat-CoV SHC014 is capable of replicating human cells without adaptation (11), suggesting its potential for zoonotic emergence into humans. The more distantly related SARS-like beta 2b CoV, recombinant bat-CoV HKU3, has a modified receptor-binding domain to facilitate growth in cell culture (22). Last, bat-CoV HKU5 is a MERS-like beta 2c CoV (23). NHC diminished infectious virus production and the levels of genomic/subgenomic viral RNA in HAE in a dose-dependent manner for all three bat-CoVs (Fig. 4). Therefore, the antiviral activity of NHC was not limited by natural amino acid variation in the RdRp, which, among the group 2b and group 2c CoV, can vary by almost 20%. Moreover, these data suggest that if another SARS- or MERS-like virus was to spill over into humans in the future, they would likely be susceptible to the antiviral activity of NHC. Fig. 4 NHC is effective against multiple genetically distinct bat-CoV. (Top) Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3 and SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, the virus produced was titrated via plaque assay. Each data point represents the titer per culture. (Bottom) qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock treated. Representative data from two separate experiments with two different cell donors are displayed.

NHC antiviral activity is associated with increased viral mutation rates It has recently been shown that NHC treatment increases the mutation rate in viral genomic RNA of Rous sarcoma virus (24), VEEV (14), and influenza (24), and our previous study used RNA sequencing (RNA-seq) to show that overall transition mutation frequency is increased during NHC treatment of MHV and MERS-CoV during infection in continuous cell lines (16). We sought to determine whether NHC would increase the mutation frequency during MERS-CoV infection in human primary HAE. Using MERS-CoV–infected HAE treated with either vehicle or a dose range of NHC or RDV, we show that both drugs reduced virus titers in a dose-dependent manner (Fig. 5A). We then used a highly sensitive, high-fidelity deep-sequencing approach [Primer ID next-generation sequencing (NGS)], which uses barcoded degenerate primers and Illumina-indexed libraries to determine accurate mutation rates on viral RNA production (25). Using this approach, we analyzed a 538–base pair (bp) region of viral genomic RNA in nsp15. The error rates (number of mutations per 10,000 bases) in vehicle-treated (0.01) or RDV-treated (0.01) cultures were very low. RDV is reported to act via chain termination of nascent viral RNA, and thus, the low error rates in RDV-treated cultures are in line with the proposed MOA (26). In contrast, the error rate was significantly increased in NHC-treated MERS-CoV RNA in a dose-dependent manner {two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test; 10-fold increase at 10 μM [P < 0.0001 at 24 and 48 hours post-infection (hpi)] and fivefold increase at 1 μM (P < 0.0001 at 24 hpi and P = 0.0015 at 48 hpi; Fig. 5C}. The magnitude of the error rate in NHC-treated cultures correlated with virus titer reduction. At 48 hpi, the respective error rate and virus titer was 0.015 × 106 and 3.96 × 106 plaque-forming units (PFU)/ml for vehicle treatment, 0.045 × 104 and 2.86 × 104 PFU/ml with 1 μM NHC, and 0.090 × 102 and 1.5 × 102 PFU/ml with 10 μM NHC. Thus, with 1 μM NHC, a threefold increase in error rate resulted in a 138-fold decrease in virus titer, whereas with 10 μM NHC, a sixfold increase in error rate resulted in a 26,000-fold decrease in virus titer. Fig. 5 NHC antiviral activity is associated with increased viral mutation rates. (A) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, and the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in (A) as determined by Primer ID. Error rate values are number of mutations per 10,000 bases. Asterisk indicates significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive- and negative-sense viral RNA. (E) Nucleotide transitions in cDNA were derived from MERS-CoV genomic RNA. We then examined the mutational spectra induced by NHC, which can be incorporated into viral RNA as a substitution for either cytosine (C) or uracil (U). RNA-mutagenic antivirals may incorporate in both nascent negative- and positive-sense RNA during genome replication (Fig. 5D). Adenine-to-guanine (A-to-G) and U-to-C transitions were enriched in MERS-CoV genomic RNA in an NHC dose-dependent manner (Fig. 5E). Collectively, these data used high-fidelity sequence analysis to demonstrate a specific enrichment for A:G and C:U transitions in MERS-CoV RNA after NHC treatment of primary HAE cell cultures.

Therapeutic EIDD-2801 reduces SARS-CoV replication and pathogenesis Given the promising antiviral activity of NHC in vitro, we next evaluated its in vivo efficacy using EIDD-2801, an orally bioavailable prodrug of NHC (β-d-N4-hydroxycytidine-5′-isopropyl ester), designed for improved in vivo pharmacokinetics and oral bioavailability in humans and nonhuman primates (15). The plasma profiles of NHC and EIDD-2801 are similar in mice after oral delivery (15). We first performed a prophylactic dose escalation study in C57BL/6 mice where we orally administered vehicle [10% polyethylene glycol (PEG) and 2.5% Cremophor RH 40 in water] or EIDD-2801 (50, 150, or 500 mg/kg) 2 hours before intranasal infection with 5 × 104 PFU of mouse-adapted SARS-CoV (SARS-MA15) and then vehicle or drug every 12 hours thereafter. Beginning at 3 days post-infection (dpi) and through the end of the study, body weight loss compared to vehicle treatment was significantly diminished (50 mg/kg) or prevented (150 and 500 mg/kg) with EIDD-2801 prophylaxis (two-way ANOVA with Dunnett’s multiple comparison test, P < 0.0001; fig. S3A). Lung hemorrhage was also significantly reduced 5 dpi with EIDD-2801 (500 mg/kg) treatment (Kruskal-Wallis test, P = 0.010; fig. S3B). There was a dose-dependent reduction in SARS-CoV lung titer (median titers: 50 mg/kg = 7 × 103 PFU/ml, 150 mg/kg = 2.5 × 103 PFU/ml, 500 mg/kg = 50 PFU/ml, and vehicle = 6.5 × 104 PFU/ml) with significant differences (Kruskal-Wallis with Dunn’s multiple comparisons test) among the vehicle, 150 mg/kg (P = 0.03), and 500 mg/kg (P = 0.006) groups. Thus, prophylactic orally administered EIDD-2801 was robustly antiviral and able to prevent SARS-CoV replication and disease. Since only the 500 mg/kg group significantly diminished weight loss, hemorrhage, and reduced lung titer to near undetectable levels, we tested this dose under therapeutic treatment conditions to determine whether EIDD-2801 could improve the outcomes of an ongoing CoV infection. As a control, we initiated oral vehicle or EIDD-2801 2 hours before infection with 1 × 104 PFU SARS-MA15. For therapeutic conditions, we initiated EIDD-2801 treatment at 12, 24, or 48 hpi. After initiating treatment, dosing for all groups was performed every 12 hours for the duration of the study. Both prophylactic treatment initiated 2 hours before infection and therapeutic treatment initiated 12 hpi significantly (two-way ANOVA with Tukey’s multiple comparison test) prevented body weight loss after SARS-CoV infection on 2 dpi and thereafter (−2 hours, P = 0.0002 to <0.0001; +12 hours, P = 0.0289 to <0.0001) as compared to vehicle-treated animals (Fig. 6A). Treatment initiated 24 hpi also significantly reduced body weight loss (3 to 5 dpi, P = 0.01 to <0.0001), although not to the same degree as the earlier treatment initiation groups. When initiated 48 hpi, body weight loss was only different from vehicle on 4 dpi (P = 0.037; Fig. 6A). Therapeutic EIDD-2801 significantly (Kruskal-Wallis with Dunnett’s multiple comparison test) reduced lung hemorrhage when initiated up to 24 hpi (−2, +12, and + 24 hours, P < 0.0001), mirroring the body weight loss phenotypes (Fig. 6B). All EIDD-2801–treated mice had significantly (Kruskal-Wallis with Dunnett’s multiple comparison test) reduced viral loads in the lungs even in the +48-hour group (All P < 0.0001; Fig. 6C), which experienced the least protection from body weight loss and lung hemorrhage. We also measured pulmonary function via whole-body plethysmography (WBP). In Fig. 6D, we show that the WBP enhanced pause (PenH) metric, which is a surrogate marker for bronchoconstriction or pulmonary obstruction (27), was significantly (two-way ANOVA with Dunnett’s multiple comparison test) improved throughout the course of the study if treatment was initiated up to 12 hpi [−2 hours, P < 0.0001 to 0.019 (2 to 5 dpi); +12 hours, P < 0.0001 to 0.0192 (2 to 5 dpi)], although the +24-hour group showed sporadic improvement as well (3 dpi, P = 0.002; Fig. 6D). Last, we blindly evaluated hematoxylin and eosin–stained lung tissue sections for histological features of ALI using two different and complementary scoring tools (18), which show that treatment initiated up to +12 hours significantly reduced ALI [Kruskal-Wallis with Dunn’s multiple comparison test; American Thoracic Society lung injury score: −2 hours, P = 0.0004; +12 hours, P = 0.0053; diffuse alveolar damage (DAD) score: −2 hours, P = 0.0015; +12 hours, P = 0.0004; Fig. 6E]. Together, therapeutic EIDD-2801 was potently antiviral against SARS-CoV in vivo, but the degree of clinical benefit was dependent on the time of initiation after infection. Fig. 6 Prophylactic and therapeutic EIDD-2801 reduces SARS-CoV replication and pathogenesis. Equivalent numbers of 25- to 29-week-old male and female C57BL/6 mice were administered vehicle (10% PEG and 2.5% Cremophor RH 40 in water) or NHC prodrug EIDD-2801 beginning at −2, +12, +24, or + 48 hpi and every 12 hours thereafter by oral gavage (n = 10 per group). Mice were intranasally infected with 1 × 104 PFU mouse-adapted SARS-CoV MA15 strain. (A) Percent starting weight. Asterisks indicate differences from vehicle treated by two-way ANOVA with Tukey’s multiple comparison test. (B) Lung hemorrhage in mice from (A) scored on a scale of 0 to 4, where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from (A) as determined by plaque assay. Asterisks in both (B) and (C) indicate differences from vehicle by one-way ANOVA with a Dunnett’s multiple comparison test. (D) Pulmonary function by whole-body plethysmography was performed daily on five animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with a Dunnett’s multiple comparison test. (E) The histological features of acute lung injury (ALI) were blindly scored using an American Thoracic Society lung injury scoring system and a DAD scoring system. Three randomly chosen high-power (60×) fields of diseased lung were assessed per mouse. The numbers of mice scored per group: vehicle, n = 7; −2 hours, n = 9; +12 hours, n = 9; +24 hours, n = 10; +48 hours, n = 9. Asterisks indicate statistical significance compared to vehicle by Kruskal-Wallis with a Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, and the whiskers represent the range. *, −2 and +12 hours compared to vehicle; **, +24 hours compared to vehicle; ***, +48 hours compared to vehicle.