Controversies' cIarification regarding ribavirin efficacy in measIes and coronav

George D Liatsos

George D Liatsos, Department of Internal Medicine, "Hippokration" General Hospital, Athens 11527, Attiki, Greece

Abstract BACKGROUND Ribavirin is a broad-spectrum nucleoside antiviral drug with multimodal mechanisms of action, which supports its longevity and quality as a clinical resource. It has been widely administered for measles and coronavirus infections.Despite the large amount of data concerning the use of ribavirin alone or in combination for measles, severe acute respiratory syndrome, Middle East respiratory syndrome, and coronavirus disease 2019 (COVID-19) outbreaks, the conclusions of these studies have been contradictory. Underlying reasons for these discrepancies include possible study design inaccuracies and failures and misinterpretations of data, and these potential confounds should be addressed.AIM To determine the confounding factors of ribavirin treatment studies and propose a therapeutic scheme for COVID-19.METHODS PubMed database was searched over a period of five decades utilizing the terms“ribavirin” alone or combined with other compounds in measles, severe acute respiratory syndrome, Middle East respiratory syndrome, and COVID-19 infections. The literature search was performed and described according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.Articles were considered eligible when they reported on ribavirin dose regimens and/or specified outcomes concerning its efficacy and/or possible adverseeffects. In vitro and animal studies were also retrieved. A chapter on ribavirin’s pharmacology was included as well.RESULTS In addition to the difficulties and pressures of an emerging pandemic, there is the burden of designing and conducting well-organized, double-blind, randomized controlled trials. Many studies have succumbed to specific pitfalls, one of which was identified in naturally ribavirin-resistant Vero cell lines in in vitro studies.Other pitfalls include study design inconsistent with the well-established clinical course of disease; inappropriate pharmacology of applied treatments; and the misinterpretation of study results with misconceived generalizations. A comprehensive treatment for COVID-19 is proposed, documented by thorough, longterm investigation of ribavirin regimens in coronavirus infections.CONCLUSION A comprehensive treatment strictly tailored to distinct disease stages was proposed based upon studies on ribavirin and coronavirus infections.

Key Words: COVID-19; Ribavirin; Severe acute respiratory syndrome-associated coronavirus; Middle East respiratory syndrome coronavirus; Measles; Treatment

INTRODUCTION

Ribavirin (RBV) was synthesized in 1972 in an attempt to identify ribonucleosides with the potential to affect enzymatic processes common to all viruses[1]. It is a broadspectrum antiviral agent that exerts inhibitory activity against deoxyribonucleic acid(DNA) and ribonucleic acid (RNA) viruses both in tissue cultures and in animal models. RBV has been used clinically for almost five decades in numerous viral infections but its efficacy has only been established for chronic hepatitis C virus (HCV)infection[2-4], chronic hepatitis E virus (HEV) infection in transplant recipients[5],respiratory syncytial virus infection in infants and immunocompromised elderly patients[6] and for some of the large group of hemorrhagic fever viruses, mainly for Lassa and Crimean-Congo hemorrhagic fever virus[7].

During the 2018 measles outbreak, a number of adult cases suffering measles pneumonitis were hospitalized and treated with RBV. Because of the lack of specific guidelines on severe measles disease treatment in adults, we reviewed the literature on RBV dosing regimens and outcomes in any infectious disease. The most amount of clinical data available was for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), where RBV was widely utilized. There were,however, conflicting data on its efficacy due to the lack of randomized controlled trials(RCTs) thus probably resulting in suboptimal targeting and efficacy. While preparing the measles/RBV study for publication, the new coronavirus disease-19 (COVID-19)outbreak emerged, prompting us to focus heavily on COVID-19 treatment with RBV alone or in combination with other compounds. These conclusions are based principally on data already available regarding other coronaviruses. Since then,COVID-19 has emerged as a global health issue with the highest priority.

MATERIALS AND METHODS

PubMed (https://pubmed.ncbi.nlm.nih.gov/) was queried with the following search term combinations: (“measles”, or “SARS”, or “MERS”, or “COVID-19”, or “viral infection”) AND (“ribavirin treatment”) between 1971 and October 15, 2020.In vitro,animal and clinical studies, reviews, and meta-analyses in English language only were considered for data extraction. Each coronavirus was searched separately with the general term “treatment”, retrieving a large amount of results. Furthermore, all review articles referring to COVID-19 treatment were searched, regardless of whether“ribavirin” was included in key words. Because those two last searches retrieved a very large number of relevant articles, it was not possible to read them from beginning to end. In downloaded files, we applied the computer software order “find on page” to locate instantly specific words within article's body and to assess evidence of our interest. After an exhaustive work-up of the retrieved literature, we limited studies to those reporting on RBV treatment regimens in coronaviruses. All the references within each eligible article were also evaluated carefully and downloaded if relevant. We considered eligible those manuscripts referring to RBV treatment alone or in combination and/or those reporting on its dose regimens, adverse effects, or outcomes. The literature search was performed and described according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. After a thorough and repetitive revision of all relevant literature throughout five decades of RBV utilization, we identified six specific pitfalls into which bothin vitroandin vivostudies fell; these result in misinterpreted conclusions and contradictory outcomes and thus misleading the scientific community and creating misconceptions about the true efficacy of RBV.

RESULTS

Rbv clinical pharmacology

RBV inhibits some DNA[8-10] and several RNA viruses[11-31] (Table 1). The degree of inhibition varies with the virus, the cell line used (RBV-resistant cell lines), and the parameters of antiviral activity examined. The mechanisms of action of RBV comprise direct antiviral effects[32-50] and indirect, immunomodulatory effects[51-71] (Table 2).RBV is accumulated and concentrated intracellularly through specific transporter proteins, including the equilibrative nucleoside transporters (ENTs) 1 and 2, concentrative nucleoside transporter 2, and multidrug resistance proteins 4 and 5[72-75].Adenosine kinase converts RBV to RBV monophosphate (RMP), and subsequent phosphorylation of RMP yields the di- and triphosphorylated nucleotides, with RBV triphosphate (RTP) being the predominant metabolite[76].

TabIe 1 Ribavirin's antiviraI activity and cIinicaI uses

Table 2 Ribavirin multimodal mechanisms of action with distinct examples

ATP: Adenosine triphosphate; dGTP: Deoxyguanosine triphosphate; GMP: Guanosine monophosphate; GTP: Guanosine triphosphate; HCV: Hepatitis C virus; HEV: Hepatitis E virus; HIV: Human immunodeficiency virus; IFN: Interferon; IL: Interleukin; IMPDH: Inosine monophosphate dehydrogenase; IP-10: IFN-γ inducible protein 10; MHV-3: Mouse hepatitis virus strain-3; MPA: Mycophenolic acid; RBV: Ribavirin; RdRp: RNA-dependent RNA polymerase.

RBV may act by perturbing intracellular nucleoside triphosphate pools. RBV is a structural analogue of guanosine, and RMP acts as a potent competitive inhibitor of the enzyme inosine monophosphate dehydrogenase, leading to reduced guanosine monophosphate biosynthesis and depletion of the guanosine triphosphate (GTP) pool[51-52]. Guanosine monophosphate is converted to the guanine metabolites GTP and deoxy-GTP, which are essential precursors for RNA and DNA synthesis, respectively[53]. GTP depletion has a major impact on host cell and viral gene expression as well as on viral replication[54-58]. Another direct mechanism is interference with the formation of the 5’ cap structure of viral mRNA (capping activity) [39]. The 5’-end of most cellular RNAs and some viral RNAs contains a 7-methylguanosine cap structure essential for RNA stability and translation[39]. RBV has the potential to interact with enzymes responsible for “capping” cellular mRNAs and viral genomic RNAs[48].Another direct mechanism is the inhibition of RNA dependent RNA polymerase(RdRp) through direct interaction with RTP[32]. RBV also increases viral mutation ratesviaits misincorporation into the genome, leading to population extinction[39].

RBV exerts important immunomodulatory effects that seem to be mediated by enhancing T helper (Th)1 over Th2 responses or upregulating the interferon (IFN)-stimulated response element[61-63]. Years before SARS emerged, the first coronavirus animal model for acute and chronic liver disease was induced by mouse hepatitis virus strain-3[67]. Viral infection of macrophages leads to a marked inflammatory response and is associated with a Th2 cellular immune response and production of nonneutralizing antibodies. In hepatocellular necrosis (viral, toxins,etc.) resident macrophages (Kupffer cells) are activated and release a number of inflammatory mediators. Inactivation of Kupffer cells prevents hepatic necrosis. RBV has minimal inhibitory effects on replication of mouse hepatitis virus strain-3in vitroeven at highconcentrations. However, at concentrations achievablein vivo, RBV almost totally inhibits the production of proinflammatory mediators tumor necrosis factor alpha,interleukin (IL)-1, and procoagulant activity in macrophagesin vitro[67]. Similar Th1 and Th2 responses have been associated with susceptibility/resistance in murine models of leishmaniasis, candidiasis, and listeriosis[77,78]. Therefore, the beneficial effect of RBV may be related to its ability to reduce markedly macrophage activation and diminish Th2 cytokine production while preserving Th1 cytokine production. The cellular mechanisms involved in enhancement of IFN signaling by RBV are mediated by stimulation of mammalian target of rapamycin, which interacts and activates p53,which in turn stimulates the transcription of IFN regulatory factor-9[70]. RBV may also stimulate extracellular signal-regulated kinase 1/2 pathway with subsequent enhanced antiviral response of IFN-a + RBV against HCV, suggesting that mammalian target of rapamycin signaling might interact with extracellular signal-regulated kinase 1/2 signaling in some way[71].

Oral RBV is rapidly absorbed and distributed, with a bioavailability of 40%-50% (±22%)[79,80] compared with intravenous administration. Plasma protein binding is negligible, whereas the plasma elimination of RBV occurs in two phases; the first has a relatively short half-life of 2 h and the second has a much longer terminal half-life of 16-164 h or a mean half-life of 37 ± 14 h[80]. Due to large distribution volume and elimination dependent on renal function, RBV may require more than 4 wk to reach steady-state concentrations[79,81]. The active metabolite of the drug, RTP, concentrates in erythrocytes and leaches out slowly, with a half-life of 40 d. In nucleated cells, RMP is rapidly hydrolyzed to RBV by 5-nucleotidase or alkaline phosphatase. Recently, RTP was reported to be dephosphorylated intracellularly to RMP by inosine triphosphate pyrophosphatase (ITPase)[53]. As RBV and RMP, but not RTP, can be transported across the plasma membrane through transporters, it is not surprising that reduced ITPase activity is associated both with higher intracellular RTP levels[53] and lower plasma RBV concentrations[82]. Notably, ITPase gene variants associated with reduced enzymatic activity that are naturally occurring in approximately one-third of humans have been demonstrated to protect against RBV-induced hemolytic anemia during RBV therapy in combination with pegylated (peg)-IFN-α[83,84] for HCV. This improved efficacy was associated with a reduced relapse risk in spite of lower RBV plasma concentrations[82]. Unchanged RBV and its major metabolite are excreted in the urine. Urinary metabolites, however, may be up to 5-fold higher after oralvsintravenous administration, suggesting a major role for gastrointestinal or, more likely, hepatic metabolism when given orally[79].

RBV’s principal toxicity is the development of a dose-dependent, reversible anemia.This anemia is due to a combination of shortened erythrocyte half-life because of hemolysis and bone marrow suppression. Hemolysis is hypothesized to be secondary to oxidative membrane stress induced by depletion of adenosine triphosphate inerythrocytes[84]. Hemolytic anemia usually occurs 10 d after therapy but may appear 3-5 d after RBV initiation; it is usually observed with doses of 1 g/d or higher in patients with chronic HCV[85] and HEV viremia[14]. Short-course RBV therapy of medium doses may not cause significant complications[86]. Other side effects include bradycardia[87,88], electrolyte disturbances (hypocalcemia, hypomagnesemia)[89],transaminitis[90], pancreatitis, metallic taste, headache, reduction in bone-mineral density, and central nervous system effects (mood changes, sleep disturbance)[91].

Rbv in measles

Measles is a worldwide and highly contagious (90%) viral illness caused by the measles virus, a single-stranded, negative-sense RNA virus in the genusMorbillivirusof the familyParamixoviridae. Diarrhea is the most common complication, and the majority of deaths are due to pneumonitis or encephalitis[92]. According to 2018 World Health Organization (WHO) reports, 82596 people in 47 of 53 European countries contracted measles, with the 2193 cases reported from Greece[93]. Nearly two-thirds of measles cases were hospitalized, with 72 deaths having occurred in Europe and 140000 globally, mostly among children under the age of 5 years.

RBV inhibits the replication of measles virusin vitro[94,95]. In a cohort of 93 severe measles hospitalized cases during the 2018 outbreak[96], our center treated 13 cases,seven of which were given RBV due to severe pneumonitis. In that cohort, RBV tended to be prescribed to those with numerically lower PaO2/FiO2ratios, whereas others were managed conservatively[96]. RBV was commenced within 5 d from symptom onset at an oral dose of 2.4 g/d for 5-7 d. No patient required intubation, and all recovered completely. We selected high-dose levofloxacin (750 mg intravenously) as an antimicrobial agent based upon a unique animal-study in which levofloxacin was examined for its possible protective effect against Influenza virus-induced lung injury[97]. Levofloxacin exerted a substantial anti-oxidative effect by clearly suppressing the levels of oxidative and nitrative stress metabolites in bronchoalveolar lavage fluid.According to lung histology, levofloxacin significantly suppressed not only the inflammatory infiltration into alveoli and the bronchial pathway but also hemorrhage and necrosis. A double-blind, placebo-controlled trial of oral RBV showed a reduction in the severity duration of measles in children[98].

Given the high risk of measles-associated mortality among immunosuppressed individuals, some authors recommend RBV treatment in measles pneumonia/encephalitis. In a study of severe measles in immunocompromised patients with case fatality rate (CFR) of about 70% for oncology patients and 40% for human immunodeficiency virus-infected patients, the authors observed a rapid defervescence in those treated with RBV[99]. A unique RCT enrolled 100 patients (aged between 6 mo and 47 years)with measles confirmed by positive immunoglobulin M antibody detection to assess the possible beneficial effect of RBV on measles[100]. Fifty patients were treated with oral RBV 200 mg qid (in children, 20 mg/kg/d as syrup) for 7 d. Constitutional symptoms resolved much earlier in the treatment group than in the non-treatment group (3.2 ± 0.6 dvs7.3 ± 0.8 d), and there were no complications or deaths.Importantly, in the comparator group, almost 50% developed pneumonitis, 30%watery diarrhea, and 8% encephalitis, with an overall mortality of 16%.

The reported dosage regimen for RBV varies significantly among different viral infections and studies. According to 2018 European Association for the Study of the Liver recommendations, hepatitis C patients with decompensated cirrhosis without hepatocellular carcinoma can be treated with two antivirals, including RBV 1000 or 1200 mg in patients ﹤ 75 kg or ﹥ 75 kg, respectively, for 12 wk[101]. Immunocompromised patients with chronic HEV viremia were treated with RBV monotherapy at a median dose of 600 mg/d (equivalent to 8.1 mg/kg/d) adjusted to creatinine clearance for 3 mo resulting in 85% sustained viral response (SVR)[14]. In a large cohort, 255 solid organ transplant recipients with chronic HEV infection were treated with RBV monotherapy. After a first course of RBV, the SVR rate was 81.2%, which increased to 89.8% when some patients were offered a second course of RBV. Surprisingly, pretreatment HEV polymerase mutations andde novomutations under RBV did not have a negative impact on HEV clearance. Twenty patients hadde novomutations, 16 of whom were re-treated with RBV, and 12 achieved SVR[15].

One of our measles pneumonitis cases with decompensated liver cirrhosis of autoimmune etiology under maintenance immunosuppressive regimen was started on 2.4 g/d, a dose 2.4-fold to 4.0-fold higher than the regimens recommended in HCV decompensated cirrhosis and chronic HEV viremia, respectively. This high dose was based on findings from Forniet al[102], who treated severe measles pneumonitis cases(not cirrhosis) with intravenous RBV 35 mg/kg/d in three divided doses for the initial 2 d of therapy and 20 mg/kg/d for the remaining 5 d. Intravenous formulations of RBV were unavailable in our country, thus we administered orally 40 mg/kg/d as a loading dose for 3 d and then a maintenance dose of 20 mg/kg/d for the next 4 d,taking into account oral dose bioavailability of approximately 50% and the patient's liver cirrhosis. In one report, high-dose regimens administered for measles pneumonitis comprised a loading dose of 2 g intravenously and then 1 g qid as a maintenance dose in combination with high doses of vitamin A[103]. RBV was discontinued, however, due to transaminitis and acute kidney injury in two patients, both of whom fully recovered[103]. The same dosing regimen was applied to another patient[104], who despite requiring intubation, eventually fully recovered. In a measles outbreak at a pediatric oncologic unit[105], clinicians reported that early RBV treatment of 15 mg/kg orally within 24 h from rash onset resulted in a significantly better outcome (P= 0.009). Several immunocompromised cases have been treated with RBV. A 9-year-old boy with Hodgkin’s disease and a 26-year-old human immunodeficient virus positive patient with measles pneumonitis both fully recovered with early initiation of RBV[106,107]. In contrast, two other immunosuppressed patients with measles pneumonitis and late introduction to RBV succumbed to their illness[108,109].

Taken together, these findings suggest that early administration of treatment(within the first 5-7 d from disease onset) of adequate dosing (40 mg/kg/d p.o.) and duration (2 or 3 wk) in severely immunosuppressed patients is essential for the best therapeutic outcome.

Rbv in SARS

On November 16, 2002, the first known case of atypical pneumonia was reported in Foshan City, Guangdong Province, China, but the cause was not identified until much later. The SARS coronavirus is a positive-sense, single-stranded RNA virus that was the causative pathogen of secondary cases elsewhere in the world. On July 5, 2003, the WHO announced that the epidemic of SARS had been contained worldwide but called for continued vigilance. A total of 8098 people worldwide became sick in 29 countries(mostly in China and other parts of Asia). Of these, 774 died, with a CFR 9.6%[110].Some scattered cases were reported in China until May 2004.

SARS in vitro studies

To examine thein vitroefficacy of several compounds against coronaviruses, Vero and Vero E6 cell lines have been utilized almost exclusively. The easy propagation of coronaviruses in Vero cell lines may be related to the lack of a functional IFN system[111,112]. However, the IFN-dependent pathway function can be activated by exogenously provided IFN. Among the nine retrievedin vitrostudies, only one utilized a cell line other than Vero or Vero E6; while in four studies, Vero cells were examined in combination with other cell lines (Table 3). Three studies concluded that RBV was inactive[112-114], and one demonstrated an inhibitory effect of RBV[115]. In three studies where Vero cell lines were co-examined with other cell lines, researchers concluded that RBV was less active[116] or ineffective (included the Caco2 cell line)[117]; in the third study, the authors concluded that no inosine monophosphatedehydrogenase inhibitor should be used for SARS treatment[118]. They emphasized,however, the need for growth in human cell lines[113]. The combination use of IFNs with RBV was considered the most effective[116,117]. In only one study where fetal rhesus kidney-4 cells were utilized did RBV exhibit an inhibitory effect against SARScoronavirus (CoV)[87].

TabIe 3 Severe acute respiratory syndrome coronavirus, MiddIe East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus-2 in vitro, in vivo (animaI), in silico study resuIts focused on ribavirin treatment

RBV → ﹥ 1000 (Vero), → 9.4 (MA-104), → 2.2 (PK-15), →5.2-8.2 (human cell lines)IFN-β + RBV → 10-fold lower RBV concentration,→ 50-2000-fold,lower IFN concentrations Morgenstern et al[117],2005 FFM1, 6109 Vero, CL14,CaCo2, PK-15, HPEK,MA-104,Synergism → Combination index 0.45 IFN-β + RBV combination inhibits SARS-CoV replication in drastically reduced concentrations The study includes Pitfall 1, the rest are ok RBV → 270, EC90 = 560 RBV → 1253 RBV → EC90 = 225 Barnard et al[118], 2006 SARS Urbani strain Vero 76, Vero E6 MA-104 CaCo2,BALB/c mice RBV → EC90 = 4100 RBV, like other IMPDH inhibitors, may enhance viral replication in lungs. Four days after cessation of therapy, RBV promoted pro-inflammatory cytokine production, although 3 d after RBV administration inhibited pro-inflammatory cytokine production in mice by significantly reducing IL-1α, IL-5, MCP-1 and GMCSF. Authors concluded that their data do not support the use of RBV or other IMPDH inhibitors for SARS treatment The only study with such high RBV’s EC50 in human CaCo2. It is not explained why animals were treated only for 3 d with RBV showing good results and then it was ceased for 4 d, just to confirm the disease worsening with untoward outcomes. Also Pitfall 1 is included in the study Shah et al[119], 2010 VSV, SeV BHK21,BSRT7, HeLa,A549, 4T1,HEp2, Vero RBV Both viruses have the ability to initiate infection in all cell line tested. RBV effectively inhibited VSV in BSRT7, HeLa and HEp2 cells even at the lowest tested drug concentrations. However, RBV had a surprisingly mild effect on VSV in Vero and A549 cells even when used at 1000 μg/mL concentration with a somewhat intermediate effect in 4T1 cells. A similar pattern of RBV-resistance was shown for SeV in BHK21, Vero and A549, suggesting that cellular rather than virus-specific factors determine the dramatic differences in their response to RBV. RBV treatment even at 1000 μg/mL concentration did not produce any statistically significant decrease in cell viability in any of the tested cell lines. The development of cell-based resistance to RBV treatment via decreased RBV uptake can greatly limit RBV activity. RBV uptake was inhibited in most cell lines at both lower and higher NBMPR concentrations, a specific inhibitor of ENT via ENT1, 2, previously shown to be primarily responsible for RBV import into the cells. Dramatic variations were observed in the long-term accumulation of RBV in different cell types. Importantly, it correlated with the antiviral efficacy of RBV in the tested cell lines. All the three RBV-resistant cell lines, BHK21, A549, and especially Vero showed markedly decreased levels of RBV accumulation suggesting that such differences in the intracellular RBV metabolism may be responsible for the natural cell resistance to antiviral RBV treatment. Act-D an inhibitor of DNA-primed RNA synthesis, was able to revert the antiviral effect of RBV against several RNA viruses, with two proposed mechanisms (1) the stabilization of cellular GTP levels, and (2) inhibition of RTP production. ActD had a clear neutralizing effect on RBV in most cell lines. ActD treatment did not inhibit RBV uptake, demonstrating that the observed reversal of RBV antiviral action was not due to interference of ActD with RBV uptake. The observed resistance of VSV and SeV to RBV in Vero, BHK21, and A549 was not due to the generation of RBV-resistant mutants in these cells. Even when the cells were pretreated with RBV starting 24 h before infection, a little effect of RBV on viral replication in RBV-resistant cells was observed, ruling out any possibility of virus adaptation to RBV. In addition, when VSV was passed by 10 to 15 times in HeLa, BSRT7, and BHK21 cells in the presence of sub-inhibitory RBV concentrations, no viral adaptation to RBV was ever observed. RBV uptake in all tested cell lines after 15min treatment showed that no one of the tested cell lines was defective to RBV uptake. In long-term RBV accumulation in cells after 16 h or 24 h treatment four cell lines sensitive to RBV showed significantly higher levels of RBV accumulation compared to RBV-resistant BHK21, A549, and Vero. Vero cells had a particularly low accumulation which explains the highest resistance to RBV. This long-term accumulation is dependent on the cellular metabolism of RBV. Neutral RBV molecule can be transported freely in and out of a cell via ENTs but once it is phosphorylated, negative charged RMP, RDP, or RTP are trapped inside the cells. A good illustration of the difference between the RBV uptake and its long-term accumulation is RBV hyper-accumulation in erythrocytes resulting in hemolytic anemia in some RBV-treated patients. Similarly to nucleated cells, RBV is transported into erythrocytes via ENTs and converted into RMP, RDP, and RTP. However, unlike nucleated cells, erythrocytes lack the phosphatases needed to hydrolyze RMP/RDP/RTP into RBV. Exogenous guanosine had a clear (almost 100%) neutralizing effect on RBV in BHK21, A549 and Vero cells, which are already highly resistant to RBV. However, very little effect was observed on the RBV activities in RBV-sensitive cells, especially HeLa, 4T1, and HEp-2 cells. Unlike guanosine, ActD was able to effectively neutralize RBV in all tested cell lines. Authors hypothesized that RBV antiviral activity in these cell lines depends not only on the depletion of GTP pool (can be restored by guanosine) but also on the successful 5’-phosphorylation of RBV into RMP/RDP/RTP. At the same time they suggested that RBV acts in RBV-resistant cells types primarily via depletion of GTP pool due to insufficient amounts of phosphorylated RBV molecules in these cells, explaining why the effect of RBV can be completely reversed in these cell lines by guanosine CoVs contain the largest known RNA genome and encode an array of 16 viral replicase proteins, including a 3' to 5' exoribonuclease domain, ExoN, within the non-structural protein 14 Nsp14. The exon is the first identified proofreading enzyme for an RNA virus and functions together with other CoV replicases to perform the crucial role of maintaining CoV replication fidelity. In DBT cells, MHV-ExoN+ viruses were resistant to 10 μM of RBV, while MHV-ExoN- virus titers decreased by～200-fold following treatment with same RBV concentrations, a surprising finding because at least 10-fold higher RBV concentrations are required to inhibit poliovirus and chikongunya viruses. Furthermore, they determined the sensitivity of ExoN + and ExoN-at a low multiplicity of infection. Unexpectedly, multi-cycle replication of ExoN-viruses in the presence of RBV was indistinguishable from single-cycle replication. Using qRT-PCR, researchers determined that ExoN-genomic RNA was dose-dependently reduced by RBV, while ExoN + RNA was unaffected. Extracellular addition of guanosine restored ExoN-titers, even in presence of RBV. These data indicate that the antiviral activity of RBV against MHV-ExoN-viruses is occurring, at least in part, through Smith et al[120], 2013 MHV-A59 SARSCoV (Urbani strain)Murine astrocytoma DBT Vero E6

decreasing viral RNA synthesis and inhibition of IMPDH, while the presence of ExoN activity is capable of preventing RBV inhibition of CoV replication. However, the increased sensitivity of MHV-ExoN-to RBV could result from the impairment of undefined functions of ExoN during replication, particularly during RNA synthesis MERS 1280 drugs screened MPA → 0.17 RBV → 9.99-41.45 IFN-α2b → 6709.8 IFN-β1a → 480.5 MDCK IFN-β1b → 17.6 Chan et al[153], 2013 hCo-EMC Vero RBV, MPA → Inactive IFN-β1b and MPA should be considered in the treatment trials of MERS. IMPDH inhibitors inactive in Vero cell line A combination of IFNs with RBV was not tested.Coexistence of Pitfall 1 RBV → 41.45 (Vero), 16.33 (LLC)IFN-α2b → 58.08 (Vero), 13.26 (LLC)Falzarano et al[155], 2013 hCoV-EMC/2012 Vero, LLCMK2 Combination 8-and 16-fold decrease in the inhibitory concentration as either treatment alone Lower sensitivity to RBV for LLC than Vero cells. RBV +IFN-α2b, when combined, the inhibitory concentrations drops to ranges achievable in humans Coexistence of Pitfall 1. Very significant outcomes for IFN + RBV combination Falzarano et al[156], 2013 hCoV-EMC/2012 Rhesus macaque IFN-α2b +RBV Treated animals showed improved clinical parameters, no dyspnea, little evidence on X-ray. They also showed reduced systemic and local proinflammatory markers, significant reduction in viral genome copies in lung tissues and less severe histopathological changes compared to untreated They suggested IFN-α2b + RBV should be considered for early intervention therapy in MERS. The hedgehog signaling pathway was identified as a putative contributor to decreased lung damage Very significant results for the early IFN-α2b + RBV administration MPA → IC50 = 2.87 RBV → ﹥ 250 Hart et al[154], 2014 Hu/Jordan-N3/2012 (Jordan strain)Vero E6 IFN-β→ IC50 = 1.37IFN-β antiviral activity was 16- 41-, 83-,and 117-fold higher than those of IFN-α2b, IFN-γ, IFNtype I and IFN-α2a, respectively INF-β and MPA or a combination should be considered for MERS-CoV infected patients The study involves Pitfall 1 MMF → A single dose did not improve and might have worsened MERS infection Lop/r → Improved clinical, radiological, pathological features, and lowered lung and other tissue viral load Chan et al[157], 2015 EMC/2012 Common marmosets IFN-β1b → Less severe disease and lower tissue viral loads IFN-β and Lop/r are effective against MERS infection in common marmosets. They concluded that potentially effective combinations that should be evaluated could be RBV and IFN- β1b and/or Lop/r. Although high doses of RBV are limited by side-effects, low-dose RBV combined with IFN- β1b and/or Lop/r may be synergistic RBV was not tested, but surprisingly the authors discuss its combination with IFN-β1b and/or Lop/r as potentially effective SARS-CoV-2 Computational studies Kandeel et al The first available crystal structure Molecular modelling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 M-pro were investigated. Phylogenetic analysis 20 drugs

Several years after most of these in vitro studies were published, a very enlightening investigation was conducted to determine the existence of “natural” (without preexposure to drug) resistance to RBV in some cell-types[119]. Seven commonly used cell lines that support replication of both vesicular stomatitis virus and Sendai virus were compared regarding the antiviral activity of RBV. Decreased RBV uptake can greatly limit RBV activity. RBV uptake was inhibited in most cell lines by nitrobenzylthioinosine, a specific inhibitor of ENT1 and ENT2. RBV-resistant cell lines baby hamster kidney 21, A549, and especially Vero showed markedly decreased levels of RBV accumulation. Exogenous guanosine resulted in a neutralizing effect on RBV in already resistant baby hamster kidney 21, A549, and Vero cells but had very small or intermediate effects in RBV-sensitive cells. Actin-D, an inhibitor of DNA-primed RNA synthesis, reverts the antiviral effect of RBVviathe stabilization of cellular GTP levels and the inhibition of RTP production without inhibiting RBV uptake. Data strongly argue that the observed resistance of vesicular stomatitis virus and Sendai virus to RBV was not due to the generation of RBV-resistant mutants in these cells. RBV uptake in all tested cell lines after 15 min treatment, which determines the ability of cells to internalize RBV, showed no defective uptake. However, in long-term RBV accumulation analysis in cells with 16 h or 24 h treatment, four cell lines sensitive to RBV showed significantly higher levels of RBV accumulation compared to RBV-resistant cell lines, thereby explaining the highest resistance to RBV. This long-term accumulation is dependent on the cellular metabolism of RBV[120,121].

Neutral RBV molecules can be transported freely in and out of a cellviaENTs, but once they are phosphorylated, negatively charged RMP, ribavirin diphosphate (RDP),or RTP are trapped inside the cells. Similar to nucleated cells, RBV is transported into erythrocytesviaENTs and converted to RMP, RDP, and RTP. However, unlike nucleated cells, erythrocytes lack the phosphatases needed to hydrolyze RMP/RDP/RTP into RBV[73,76,122]. In a study of radiolabeled RBV after long-term administration, radioactivity was predominantly attributed to RMP and RTP[72].Unlike guanosine, actin-D was able to neutralize effectively RBV in all tested cell lines.Therefore, RBV antiviral activity in RBV-sensitive cell lines depends not only on the depletion of the GTP pool (exogenous guanosine has a small effect on RBV activity)but also on the successful 5’-phosphorylation of RBV into RMP/RDP/RTP. At the same time, in RBV-resistant cells-types RBV acts primarilyviadepletion of the GTP pool due to insufficient amounts of phosphorylated RBV molecules in these cells.

Overall, the activity of RBV is naturally limited in many cell-types. Mostin vitrostudies have inferred RBV inefficiency by testing in cell lines (Vero) that are less likely to phosphorylate the compound[118] (Pitfall 1). When antiviral efficacy and potency are examinedin vitro, multiple cell lines of different origin, including human, should be utilized.

SARS clinical studies

Some international societies have published recommendations or protocols for SARS management and treatment that included RBV[123] (Table 4). During the SARS outbreak, RBV monotherapy or in combination with other drugs was widely administered[124-143] (Table 5). The clinical progression of SARS was mostly uniform,with a tri-phasic pattern[124,131,141,144]. Week 1 was characterized by systemic symptoms that were largely related to the effect of viral replication and cytolysis and generally improved after a few days. In week 2, symptoms reoccur and oxygen desaturation may develop. Taken together, these findings suggest that the lung damage at this phase is related to immunopathological events as a result of an overexuberant host response rather than uncontrolled viral replication. A quarter of patients will progress to Phase III, characterized by acute respiratory distress syndrome (ARDS), necessitating ventilatory support. Examination of the sequential changes in viral load and disease progression suggested that the initial viral replicative phase peaks at around day 10. The key facet of management should include early institution of an effective antiviral agent to decrease the peak viral load and the associated immune-regulatory damage. This therapeutic window should be exploited as early as possible from disease onset and is limited to within the first 5-7 d (Pitfall 2).

An epidemiologic analysis[140] using an integrated database of 1755 cases in Hong Kong concluded that the timing of RBV administration did not seem to influence significantly clinical outcome, despite there being a clear trend in favor of earlier initiation (when RBV commenced on day 1 of symptom onset, CFR = 4.0%; after the first week, CFR = 12.5%; treatment not prescribed, CFR = 29.4%). Non-emergence of statistical significance was attributed to insufficient power to detect a difference because most patients were treated. There were 19 studies (Table 5) that referred to treatment with RBV and/or other drugs for SARS patients, and in four [124,128,137,139], treatment started too late. In the remaining studies, the time-gap between symptom onset and treatment initiation was not reported[89,126,134,142,145] at all or was unclear[12,131,133]. Notably, antivirals were started in Phase II[124] and even post-intubation[137], therefore conferring frustrating results.

TabIe 4 Treatment recommendations for viraI hemorrhagic fevers and coronavirus outbreaks

Besides Pitfall 2, after scrutinizing SARS studies, we noticed very wide fluctuations regarding RBV dosing regimens. In RBV monotherapy, the high-dose intravenous scheme for viral hemorrhagic fevers[123] is the most efficacious[127,128,134,138], but it is associated with the highest adverse-effects rates[89]. In some studies, RBV dosing was equal[126] or even lower[135] than that administered for chronic HCV infection with negative outcomes (Pitfall 3). In patients treated with RBV monotherapy [127,128,132,133] or in combination with low-dose steroids[133,138] during Phases II and III,treatment results were disappointing(Pitfall 4).In contrast,when RBV was combined with pulsed methylprednisolone[127,129,130,135,136,145] or with high-doses of hydrocortisone[132,133] after development of hypoxemia, results were far more promising. Indeed, a review study confirmed the success of RBV combination with pulsed methylprednisolone when dyspnea develops. In addition, they noted that combination with IFNs might be even more helpful[146]. Although another review failed to show statistical difference in several treatment combinations, there was a clear trend in favor of RBV + lopinavir/ritonavir (Lop/r) + steroids, IFN + steroids,and RBV + pulsed methylprednisolone[142].

A large meta-analysis[143] assessed the effectiveness of RBV and corticosteroids as the initial treatment within 2 d of admission for SARS compared with no treatment.Patients without treatment had a CFR of 23.3%, with RBV alone CFR was 8.9%, with steroids alone 29.4%, and with the combination 12.6%. Based on generalized propensity score weighting, the initial findings above were totally reversed as the model predicted that the overall CFR would have been highest (19.2%) if all patientshad been treated within 2 d of admission, whereas it would have been lowest (15.4%)if no treatment applied. As they underlined, the main design analyses were a snapshot of treatment or not within first 2 d of admission. However, they arbitrarily concluded that clinicians should not use RBV and corticosteroids to treat SARS as they provide no benefit in terms of survival (Pitfall 5). This generalization was based on their initial condition of 2 d snapshot findings, and they missed the care during the long intermediate period extending to the final outcome. Initiation of corticosteroids early during Phase I of viral replication may suppress the immune response and allow a higher peak viral load[146] (Pitfall 6). In contrast, two studies[87,140] showed statistically significant superiority in intubation and mortality rates of RBV combination with Lop/r when applied early in the disease course pulsed methylprednisolone was added when necessary. In the second one, CFR was 2.3% in the combination group vs

15 .6% in standard of care (SOC) group (P﹤ 0.05). In the second one, there were no deaths in the combination group, and likelihood of ARDS development was much higher in SOC(28.8%vs2.4%,P﹤0.001).IFN regimens were not widely utilized in the SARS outbreak, except for a prospective study with a limited number of cases that was inconclusive for IFN efficacy[134]. Another preliminary study concluded that the use of IFN alfacon-1 + corticosteroids was associated with reduced desaturation and more rapid resolution of imaging abnormalities[147].

TabIe 5 Severe acute respiratory syndrome coronavirus, MiddIe East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus-2 cIinicaI studies focused on ribavirin treatment

Mp pulses if worsening 0.5 g iv/d for 2-3 doses hospital of whom 13 in ICU (17%) and totally 19 pts intubated radiological findings, and the inverted V viral-load profile suggest that worsening in week 2 is related not to uncontrolled viral replication but rather to immunopathological damage unclear Peiris et al[132], Hong Kong 50 monitored for 12 d 6.7 d 8 mg/kg tid iv 7-10 d Antibiotics, Hc 200 mg tid tailed off 6 pts received treatment before ICU admission all recovered. 31 uncomplicated pts recovered. From 19 complicated pts 1 died No Complicated cases were associated with underlying diseases and delayed use of RBV and steroid treatment.CMR = 2%Ok Booth et al[133],Canada 144/126 First 48 h of hospitalization 2 g ld → 1 g qid × 4 d→ 0.5 g tid × 3 d Antibiotics Ster 40%, Hc 20-50 mg/d × 10 d 103 pts discharged. 8 pts died (6 with DM, 1 with cancer)49% decrease in Hb ﹥ 2 g/dL. 40% transaminitis.14% bradycardia Poor outcome was associated with RBV treated pts but it was not significant Despite unclear time gap between disease onset and RBV initiation, it seems that RBV alone (no Mp pulses, low steroid regimen in only 40% of pts), might not exert a clear benefit (Pitfall 4)Zhao et al[134], China3 190/40. pts allocated to 4 groups Not reported group A: 0.4-0.6 g/d iv Antibiotics 2 pts died. 3 intubated.The rest followed group D→ improved No Early use of high- dose steroids with quinolone + azi gave the best outcome.No advantage from RBV Unclear time-gap, too low RBV dosing (Pitfall 3). RBV treatment alone(Pitfall 4)So et al[135],Hong Kong 31 pts → 1 recovered on antibiotics 5.5 d RBV 400 mg iv tid × 3 d then 1200 mg bid orally× 10-14 d Broad-spectrum antibiotics, Mp 1 mg/kg tid × 5 d then 1 mg/kg bid × 5 d. When worsening pulsed Mp 0.5 g iv. Then Ps 0.5 mg/kg bid × 5 d orally 17 pts showed rapid response. 13 achieved improvement with stepup or pulsed MP. None intubated. No mortality No Protocol provided satisfactory outcomes No mortality reported Lau et al[136], Hong Kong 88 pts → 3 recovered on antibiotics/ 68 5.8 d So et al[135] treatment protocol applied So et al[135] treatment protocol applied 18 pts required ventilation. 30 pts needed Mp pulses. All-cause mortality for pts aged ﹤60 was 0% (0/76) and 3/12 (25%) in aged ﹥ 60.CXRs of all survivors were significantly clearer in discharge No The standard treatment protocol of RBV + steroids and pulsed Mp resulted in satisfactory outcomes Total CMR = 3.4% Ok Dwosh et al[137],Canada 15 pts, treatment data only for 1 case Post-intubation 9 d 2 g ld iv → 1 g qid × 4 d→ 0.5 g tid × 6 d Mp 40 mg × 2 Successfully extubated No No treatment conclusions Late RBV initiation(Pitfall 2)RBV alone or associated with low dose steroids seems insufficient for SARS Phase 2 (Pitfall 4).Sung et al[138], Hong Kong 138/94 3 d (0-11) to admission. RBV started after 48 h 2.4 g ld orally → 1.2 g tid. If dyspnea → 400 mg tid iv Antibiotics Ps 0.5-1 mg/kg. If dyspnea →Hc 100 mg tid. Mp pulses for 3 d (up to 3 g)25/94 pts responded toRBV. Mp in 107 nonresp. → 88.8% success. 15 pts died (mortality 10.9%)Modest degree of anemia in 59%RBV’s role is doubtful in treatment.Pulsed Mp associated with improvement

Possibly RBV is insufficient when applied in respiratory failure Leong et al[139],Singapore 229/97 compared to a group of pts who did not receive RBV on day 6 6.4 d. Duration 5.6 d. Doctordependent RBV use Oral 1.2 g tid iv 400 mg tid Insufficient data Mortality 10.3% vs 12.9%in control. HR of death for RBV 0.78 (P = 0.53). When adjusted for steroids HR =1.03 (P = 0.93)No difference in side effects Use of RBV alone does not seem to confer any benefit Late use of RBV (Pitfall 2), uneven groups,doctor-dependent use of RBV (Pitfall 5). RBV alone seems insufficient(Pitfall 4)Leung et al[140], Hong Kong 1755/1467 met SARS criteria/ 1416 received RBV On symptom onset:25 pts. 1-3 d: 480 pts. 4-6 d: 499 pts. ≥7 d: 412 pts Not reported Not reported 302 died → mortality 17.2%. CFR of 25 pts:4.0%, of 480 pts: 11.1%, of 499 pts: 10.0%, of 412:12.5%, of 51 pts treatment not prescribed: 29.4%No side-effects reported The timing of RBV administration did not seem to statistically significantly influence outcome Authors explain their finding that it possibly results from residual confounding or insufficient power to detect a difference given that most pts were treated (Pitfall 5)Knowles et al[89],Canada 110 pts focused on RBV side-effects Not reported High-dose RBV(total ﹥20 g): 2 g ld → 1 g qid ×4 d → 0.5 g tid × 3 d;Low-dose RBV: 0.4 g iv tid × 4 d → 1.2 g po bid× 7 d Antibiotics 50% steroids 61% hemolytic anemia. 28% transfused with ≥ 1 U of RBCs. A significant decrease (﹥ 2 mg/dL) in Hb was seen at 6.8 d after RBV started, and reached a nadir at 13 d. Anemia associated with higher RBV doses (P =0.005) and prolonged hospital stay (P = 0.001). 35/76 pts developed hypomagnesemia, 32/62 pts developed hypocalcemia. Teratogenic effect: it is recommended that 15 half-lives (6 mo) is required to complete washout after RBV discontinuation In contrast to HK experience where RBV associated side effects have not been detailed, their comparable RBV doses suggest that associated side effects are frequent. The benefits of RBV use may not outweigh the risk of side effects with negative economic consequences on hospitals No outcome results for the 110 pts were reported Chan et al[141], Hong Kong4 75 pts compared with matched cohorts of 643 and 343 pts As soon as SARS diagnosis established. Lop/r 5.5d and 1 d after RBV. Rescue therapy: 18 d 2.4 g oral ld → 1.2 po tid or 8 mg/kg tid × 10-14 d Lop/r 400/100mg bid ×10-14 d. 1 group received it as initial treatment and a 2nd as rescue. In addition,tailing steroids regimen× 21 d and pulsed Mp Lop/r as initial therapy CRF 2.3% vs 15.6%(P ﹤0.05), intubation rate 0%vs 11% (P ﹤ 0.05). As rescue no difference No Early Lop/r initiation in addition to standard treatment protocols (Ho, So)showed significantly beneficial outcomes Combination of early RBV with Lop/r and steroid regimens with pulsed Mp when needed showed statistically significant results in intubation and mortalityrates. Ok Chu et al[87], Hong Kong5 111pts historical controls compared to 41 pts treated with RBV+ Lop/r Once diagnosis was established for RBV.For Lop/r initial treatment group it was started at a median of 3.5 d while in the rescue group at 14 d 4 g oral ld → 1.2 g tid or 8 mg/kg iv tid × 14 d Lop/r 400/100mg bid orally ´ 14 d. Tailing steroid regimen × 21 d and pulsed Mp 21-d adverse outcome(ARDS or death) was 28.8% for the historical control vs 2.4% in the initial treatment group (P﹤ 0.001). No deaths in the treatment group Mild gastrointestinal adverse-effects. Anemia(70%) → 2 pts transfused.26.8% bradycardia Apparent favorable clinical response to combination of Lop/r + RBV +steroids when needed The second study showing statistically significant benefits from the combination of RBV+ Lop/r + ster when early applied in the disease course In Table 2 of the article however, RBV + Lop/r+ ster provided a CFR of 2.3%, IFN + ster 0%,RBV + pulsed Mp 5.9%Cheng et al[142], Hong Kong6 772 No data available No data available Steroids Lop/r No data available No 675 pts received RBV and 44 Lop/r.No obvious difference noted irrespective of treatment combination

and RBV + ster 7.7%compared to a 15.4% of supportive treatment Lau et al[143], Hong Kong,Canada7 Integrated data base containing 1755 HK pts and 191 Toronto cases Within 2 d from hospital admission No data available Data showed for HK pts crude CMR 23.3% in neither treatment, 29.4% in steroids, 8.9% in RBV and 12.6% for combination. For Toronto pts no treatment 20%, RBV 9.3% and RBV + ster 12.8%.Authors adjusted these results for propensity scores and balance was achieved among all pts characteristics. Side-effects not considered in this study Estimated CFRs based on the generalized propensity score weighting, the model predicted that the overall CFR would have been highest if all pts in HK had been treated with RBV + steroids,whereas it would have been the lowest if none treated. Toronto results were consistent. The combination of RBV + ster has no therapeutic benefit The generalized propensity score weighting model prediction reversed the initial finding for CFR 12.7% of the combination to 19.2%and of untreated from 23.3% to 15.4% (!!).Inconclusive study(Pitfall 5)MERS clinical studies Omrani et al[161], Saudi Arabia2 44 with severe pneumonia 20 treated 24 control. Scores APACHE II: 27, and SOFA: 11 3 d from diagnosis 2 g ld → 1.2 g tid 4 d →600 mg tid × 4-6 d.Dosing adjusted to Crcl. Orally RBV Antibiotics, Oseltamivir,PegIFN-α2α sc 180 μg/wk for 2 wk. Hc 200 mg/d in pts with refractory septic shock 41/44 intubated. 14-d mortality: treat 6/20 vs control 17/24 (P = 0.004).28-d: treat: 14/20 vs control 20/24 (P = 0.054)RBV well tolerated. Hb drop in treat ﹥ control (P= 0.002). No differences in transfusions, no treatment discontinuation Significant benefit in 14-d survival.The loss of difference in 28-d might be explained by high initial APACHE II and SOFA scores and several comorbidities Surprisingly,statistically significant results despite that eligible patients had initially severe pneumonia (Phase 2)(Pitfall 4) without high dose steroids applied.Long- lasting IFNs(peg) might not be the best form for acute infections Shalhoub et al[162],Saudi,Arabia2 32 pts were already under MERS pneumonia and some with respiratory failure For IFNs: 1 d after MERS diagnosis.For RBV not reported 2 g ld orally → 600 mg bid Antibiotics, IFN-α2a sc 180 μg/wk × 2 wk. IFNβ1a sc44 μg × 3 times/wk Overall mortality: 22/32(69%). IFN-α2a + RBV:11/13 (85%). IFN-β1a +RBV: 7/11 (64%).Hemodialysis pts: 14/14(100%)No IFN-α2a or IFN-β1a + RBV were ineffective against MERS mortality Unknown time-gap between symptom onset and treatment initiation. Very low RBV dose applied(Pitfall 3). In specific cases with severe pneumonitis high-dose steroids and Mp pulses should have been used for better outcomes(Pitfall 4)Al Ghamdi et al[171],Saudi,Arabia2 51 pts No data reported No data reported Antibiotics, IFN-α, IFNβ, MMF, Hc in 5 pts 31 pts received antivirals(IFNs,RBV) in several combinations, 8 pts MMF all survived. (IFN-β and MMF were given to less severely pts). CMR = 37%No IFN-β and MMF were predictors of increased survival No time gap from symptom onset reported. No dosing reported. Inconclusive study for RBV treatment Choi et al[172], Korea8 186 pts 6 d (1-20 d) 14% of pts within 48 h 81% IFN-α + RBV + Lop/r, 12.7% IFN-α + RBV,5.0% RBV + Lop/r, No dosing regimens reported CMR = 20.4% lower than others ranging 36.5%-65%No Unable to assess the clinical impact of therapies as most pts received antivirals No dosing regimens,not duration reported

Arabi et al[166], Saudi,Arabia9 309/151 pts critically ill received steroids 3 d from ICU admission Antivirals: RBV, IFN, RBV + IFN, oseltamivir. The median of the maximum daily Hc-equivalent was 300 mg with a median duration of 7 d CMR 74.2% vs 57.6% (no steroids). After adjustment for baseline and time-varying confounders the use of steroids was not associated with increased 90-d mortality but with delayed RNA clearance No Steroids were commonly used in critically ill patients with MERS. Pts given steroids were more likely to have 1 or more comorbidities than those who did not (P = 0.001)No Mp pulses were administered.Maximum Hc doses reported (300 mg) are equivalent to only 60 mg of Mp. In addition,authors do not comment about the impact of the coadministered antivirals(Pitfall 5)Habib et al[163], Saudi Arabia2 63/61pts presented with severe illness(pneumonia 87.3% and septicemia 11%)No data reported No data reported No data reported Overall CMR 25.4%.Treated 22.9%. Survivors were more likely to have had received IFN + RBV than those who died (P =0.01)No CMR 25% comparable to that of Omrani 30%, lower than AlMekhlafi(74.2%), Khalid (55%), and Al-Tawgiq(100%). Unable to determine the combination efficacy in the absence of a reference group No dosing regimen, no time-gap from onset.The severity in admission probably implies an advanced disease phase, where antivirals are less effective (Pitfall 4)Arabi et al[166], Saudi,Arabia2 349/144 critically ill all ICU pts 2d from ICU admission but 9 d(6-12) from symptom onset RBV: 2 g ld po → 1.2 g po tid × 4 d → 600 mg tid po × 4-6 d Peg-IFN-α2b → 1.5 mcg/kg sc × 2 wk Per-IFN-α2a → 180 μg/wk× 2 wk Peg-IFN-β1a →44 mg sc × 3/wk Crude CMR was higher in antiviral treated group 73.6% vs 61.5% (P = 0.02). However, with a marginal structural model there was no significant difference in 90-d mortality (aOR: 1.03; 95%CI: 0.73-1.44, P = 0.87).Also, no significant difference in RNA clearance (aOR:0.65; 95%CI: 0.3-1.44, P = 0.29)During ICU stay RBV/IFN treated pts were more likely to receive steroids(59.7% vs 44.9%P = 0.006). Future studies should test the efficacy of newer antiviral interventions Very late antiviral initiation. Possible higher needs for steroids in antiviral -treated group could imply more severely ill pts (Pitfalls 2, 5)AlMekhlafi et al[167],Saudi,Arabia2 31 pts in ICU. 13 pts received RBV+ IFNα2α ICU pts Not reported Not reported CMR 74.2%. Among 13 pts who were given antivirals, 9 died No All pts who received either oseltamivir or RBV + IFN-α2a had no favorable outcomes Antivirals may have no efficacy in Phase II-III of MERS (Pitfall 4)Khalid et al[168], Saudi,Arabia2 14 pts intubated 11 pts received RBV 6 d Not reported Antibiotics RBV + Peg-IFN-α2a, Mp 1 mg/kg/d × 7 d 9 pts died in the ICU, 5 discharged No MERS with ARDS has high mortality rates. The role of RBV + IFN warrants further evaluation Antivirals may have no effect in Phase II-III of MERS-infected pts under mechanical ventilation (Pitfall 4)Khalid et al[164], Saudi,Arabia2 6 pts, 3 cases 74-84 yr, 3 cases 17-54 yr 1st group 12-19 d; 2 nd group 1-2 d 2 g ld → 1.2 g tid × 4 d→ 0.6 g tid × 4-6 d IFN-α2b sc 180 μg/wk ×2 wk. 1 case received pulsed Mp and recovered 1st group pts all died. 2nd group all recovered No Combination of RBV and IFN-α2b have a role in treatment of MERS if started early in disease course Very late (12-19 d)antiviral initiation in 1st group when disease is already in the ARDS phase (Pitfall 4). 1 case was helped by Mp pulses Al-Tawfiq et al[169],Saudi,Arabia 5/5 11-21 d (after admission)2 g ld → 400 mg po tid Antibiotics Oseltamivir IFN-α2b Mp 40 mg tid or Ps 40 mg/d All died No All pts were already intubated when treatment started Antivirals in Phase 2,very low RBV dosing,low ster dosing for Phase 2-3 (Pitfalls 2, 3,4)

Park et al[159], Korea2 43 HCW with high-risk exposure to MERS pneumonia pts. 21 HCW with more severe exposure received PEP.22 HCW no PEP Within 36 h after unprotected exposure RBV 2.0 g ld orally →1.2 g tid × 4 d → 600 mg tid × 6-8 d Lop/r 400/100 mg bid× 11-13 d 6/43 HCW exposed developed MERS infection. The attack rate was lower in the PEP vs no-PEP (0% vs 28.6% OR:0.405 P = 0.009). No MERS infection in PEP group.Only PEP therapy reduced significantly the risk of MERS infection(OR: 0.714; P = 0.009)Mild: diarrhea, nausea,anemia, stomatitis,leucopenia,hyperbilirubinemia. No PEP discontinuation. All normalized after completion of PEP PEP therapy was associated with a 40% decrease in the risk of infection The only study reporting results of PEP prophylaxis with the combination of Lop/r +RBV. Ok COVID-19 clinical studies Tong et al[189], China2 115/44 pts Severe disease. 9 pts intubated 28 pts NINV 8 d from onset 4 d from diagnosis 500 mg iv bid Antibiotics Negative conversion time of SARS-CoV-2 test in RBV vs control (12.8 d vs 14.1 d, P = 0.314) CFR 17.1% vs 24.6% (P = 0.475)No side effects. No difference in anemia RBV administration was doctordependent and sometimes RBV was out of stock. In severe COVID-19 RBV is not associated with improved negative conversion time for SARSCoV-2 test or improved mortality Pitfall 2. Relatively moderate RBV dosing(Pitfall 3). Possibly not regular RBV administration (Pitfall 5)Li et al[190],China2 151 pts, Number of pts treated with RBV was not specified. Moderate to critical disease Not reported 500 mg iv bid or tid ×10 d Umifenovir Lop/r,Traditional medicine,Peramivir, Oseltamivir,Penciclovir Ganciclovir 25 pts discharged 25 pts hospitalized 79 pts clinical improvement7 died (CFR= 4.6%)The use of two-step clustering and subgroup analysis enabled an in-depth analysis of the effects of single or combined antiviral therapy. Following the antiviral therapy, there was indeed an improvement of severe patients' condition. Combination was superior to single or dual agents. A quadruple combinationof Umifenovir + RBV + Lop/r+ Lianhua Qingwen has been recommended for critically ill COVID-19 pts Incomplete data (timegap from symptom onset to treatment initiation) (Pitfall 5)Yuan et al[191], China2 94 pts, 46 pts IFN-α +Lop/r.21 pts IFN-α +Lop/r + RBV. Median age 40 yr. 15 pts, 1 or 2 comorbiditie. Mild disease: 8 pts.Moderate: 75 pts.Critical: 11 pts Hospitalized 7d after symptom onset No data reported No data reported Significant correlation between the length of hospital stay and PCR negative conversion time in pts treated with IFN +Lop/r (P = 0.012) and with IFN + Lop/r + RBV (P = 0.0215). No death, no intubation, all recovered No These two regimens might be beneficial for COVID-19 treatment Pitfall 2. No dosing regimens reported. Ok Wu et al[192], China9 80/80 pts, 41 females,46.1 yr. 77 pts mild to moderate symptoms. 3 pts severe. 38 pts chronic diseases Not reported Not reported. Duration 7 d Moxifloxacin duration 7 d12 pts Mp to alleviate the shortness of breath No death, no INV. 35 pts NINV. 55 pts abnormal chest CT. 3 pts transaminitis. 1 pt hemodialysis. As of writing, 21 pts discharged(stay 8 d)No Notably, infected patients may be falsely excluded based on 2 consecutively negative respiratory pathogenic PCR tests Surprisingly, authors do not discuss at all the role of treatment administered (RBV +Mp + Moxi) (Pitfall 5)Impressive findings for both antivirals in reducing mortality in severe cases. The beneficial effect of RBV in cardiac injury is supported by another Chen et al[193], China2 681 pts with severe disease/279 received RBV. 375 pts had comorbidities. Median 65 yr. 40-65 yr 46.1% of pts, ﹥ 65 yr 47.1% of pts No time-gap between symptom onset and initiation of treatment, no dosing regimens reported, or drug combinations. 666 pts received antivirals, antibiotics (83.8%), IVIG (54.6%), and steroids (48.8%)In a report from China overall mortality from COVID-19 was 2.3% while in critical cases 49%.In another from Italy CFR was 26% in ICU pts. Another study indicated a mortality of 15%while in ICU cases 38%. In this study CFR was 15.3%. 45.8% of the pts had preexisting cardiovascular disease, of which 23.4% died. In multivariate analysis, RBV and arbidol were positively associated with death, OR: 0.208 (95%CI: 0.07-0.618; P = 0.005). Of notice, RBV might have a beneficial effect in severe COVID-19 pts with cardiovascular diseases and cardiac injury by disease. Therefore, every drug regimen should include arbidol or RBV for severe cases

1Prospective.2Retrospective.3Prospective randomized.4Multicenter retrospective matched cohort.5Open non-randomized prospective.6Review.7Systematic review.8Retrospective observational.9Multicenter retrospective.10Randomized, open-label, prospective trial.11Multicenter randomized prospective.12Open label parallel trial.13Randomized controlled trial.14Meta-analysis.aOR: Adjusted odds ratio; ARDS: Acute respiratory distress syndrome; bid: Bis in die; bw: Body weight; CI: Confidence interval; COVID-19: Coronavirus disease 2019; CFR: Case fatality rate; Crcl: Creatinine clearance; GI: Gastrointestinal;Hb: Hemoglobin; HCQ: Hydroxychloroquine; HR: Hazard ratio; ICU: Intensive care unit; IFN: Interferon; IVIG: Intravenous immunoglobulin; neb: Nebulizer; Lop/r: Lopinavir/ritonavir; MERS: Middle East respiratory syndrome; MMF:Mycophenolate mofetil; OR: Odds ratio; pts: Patients; RR: Relative risk; SARS: Severe acute respiratory syndrome; RBV: Ribavirin; Sof: Sofosbuvir; ster: Steroids; tid: Ter in die.

In conclusion, Severe acute respiratory syndrome-associated coronavirus (SARSCoV) treatment with RBV alone or in combination with low-dose steroids in Phase II of the disease is probably ineffective (Pitfall 4), while early initiation of RBV + Lop/r combination decreases the viral load and significantly lowers the need for steroid use,intubation, and finally mortality. When ARDS develops (Phase III), pulsed methylprednisolone should be administered in addition to adequate ventilation.

MERS outbreak

Since September 2012, WHO has been notified of 2519 Laboratory-confirmed cases of MERS in 27 countries, including 866 associated deaths (CFR = 34.3%) globally[148]. A majority of cases were reported from Saudi Arabia. By the end of January 2020,confirmed Middle East respiratory syndrome coronavirus (MERS-CoV) cases have occurred every year, mostly in the Middle East. MERS β-coronavirus is closely related to two Asian bat β-coronavirus (HKU4 and HKU5) in lineage C. In contrast to SARSCoV, which uses angiotensin-converting enzyme 2 to gain entry into cells, MERS uses dipeptidyl peptidase 4 as a functional receptor[149]. MERS-CoVin vivotargets type II alveolar cells, Clara cells, and endothelial cells but not ACE-2-expressing ciliated epithelial cells infected by SARS-CoV. MERS-CoV, unlike SARS-CoV, can also infect and replicate in human monocyte-derived macrophages[150]. This increases the expression of major histocompatibility complex class I and co-stimulatory molecules,leading to a more exaggerated activation of the immune response. These differences in receptor usage and susceptibility to type I and type III IFN may account for the differences in disease patterns, organ tropism, and virus shedding[150-152].

MERS in vitro studies

A chemical library of 1280 known drugs against influenza A was assessed for possible anti-MERS-CoV activity[153]. In the Madin-Darby canine kidney cell line,mycophenolic acid (MPA), RBV, and IFNs were active against MERS-CoV, while in Vero cells, RBV and MPA were inactive (Pitfall 1). Scientists concluded that IFN-β1b and MPA should be considered for treatment trials. Similar were the findings in anotherin vitrostudy[154] that also fell into Pitfall 1. When both Vero and LLC-MK2 cell lines were utilized, authors concluded that the latter was more sensitive to RBV,and when combined with IFN-α2b, inhibitory RBV concentrations were achievable in humans[155]. Additionally, two animal studies with MERS-infected rhesus macaques[156] and common marmosets[157]were performed. In the former, scientists concluded that IFN-α2b + RBV should be considered for early intervention therapy in MERS and in the latter that low dose RBV combined with IFN-β1b and/or Lop/r may have synergistic effects.

MERS clinical studies

Physicians have published recommendations for the antiviral treatment of MERS-CoV infection and propose the combination of IFN-α2a + RBV + Lop/r[158], and the combination of RBV + Lop/r for post-exposure prophylaxis[159]. Early drug administration is essential in MERS as there is a more rapid progression to death than SARS[160]. In MERS-CoV published studies (Table 5), antiviral treatment was commenced very late in the disease course in patients with severe pneumonia and respiratory failure[161-164] (Pitfall 2), or in patients already in the intensive care unit (ICU) or intubated[165-170] (Pitfalls 2, 4). Characteristically, in a case-series of five patients who were intubated when antivirals (IFN-α2b + RBV) and low-dose steroids were initiated,mortality was 100%[169]. The majority of MERS clinical studies fell under Pitfall 2,probably because the disease progression is much more rapid than SARS-CoV.However, in a study by Omraniet al[161], when antivirals were initiated 3 d after symptom onset, some statistically significant findings and trends were provided.Forty-four MERS patients (73% men, mean 65.5-year-old) with a median of three comorbidities and with severe pneumonia [APACHE II score of 27, Sequential Organ Failure Assessment (SOFA) score of 11] were treated with the antiviral combination of Peg-IFN-α2a + RBV. Fourteen-day mortality was 6/20vs17/24 in the comparator group (P= 0.004). Nevertheless, 28-d mortality did not show any significant difference(CRF 70%vs83.3%,P= 0.054), probably due to the small number of cases, the high initial APACHE II and SOFA scores, and the comorbidities but mostly due to the fact that antiviral treatment is ineffective in Phases II-III of the disease.Moreover,they did not administer immunosuppressive/immunomodulatory therapy for ARDS (such as pulsed methylprednisolone) (Pitfall 4). A retrospective study[162] aimed to find potent efficacy of IFN-a2a or IFN-b1a in combination with RBV in MERS pneumonia but concluded that there was lack of efficacy (Pitfalls 2, 3, 4 present in this study). In a retrospective study[171] in which 31 patients received a number of different antiviral combinations (overall CFR of 37%), authors concluded that only any IFN (mainly IFNβ,P= 0.009) and mycophenolate mofetil treatment (P= 0.019) were predictors of increased survival in the univariate analysis. In a Korean[172]retrospective observational study, the lowest ever CFR of 20.4% was reported. The triple combination of IFN+ RBV + Lop/r was administered in 112 pts and accounted for a CFR of 17.9%, while combination therapy IFN + RBV provided only 5.6% (1/18). Researchers attributed this low CFR to the application of aggressive treatment measures, including antiviral agents early from disease onset (median 6 d). Another study confirmed these findings[163] in patients with pneumonia and/or sepsis. The combination of IFN-α + RBV resulted in a relatively satisfactory CFR (22.9%), whereas patients who survived were more likely to have had received the combination therapy than patients who died (P=0.01). No steroid use was reported in this study too (Pitfall 4). Researchers in a multicenter study[165] with ICU patients deduced that the use of steroids was not associated with increased 90 d mortality but with delayed RNA clearance.Nevertheless, the patients who received steroids had one or more comorbidities (e.g.,diabetes, chronic pulmonary, and cardiac diseases) compared to those who did not receive steroids (P= 0.001). Furthermore, they utilized a maximum hydrocortisone equivalent of 300 mg/d, which corresponds to 60 mg of methylprednisolone, far from pulsed methylprednisolone dosing (Pitfall 3).

In a retrospective study of critically ill patients, the association of RBV + IFN was evaluated[166]. Using a marginal structural model, RBV + IFN was not associated with changes in 90 d mortality or with more rapid MERS-CoV RNA clearance. This large study, however, also fell under specific pitfalls. The time-gap from symptom onset to treatment initiation was 9 d (Pitfall 2). Furthermore, patients under antiviral treatment received statistically significant (P= 0.006) more steroids compared than the rest, an intervention implying that those were more severe patients than the former group.Steroid dosing regimen and time of steroid administration was not reported (Pitfalls 3,4). In a number of ICU and intubated patients suffering MERS infection with very high CFRs (74.2%[167] and 64.3%[168]), the authors concluded that RBV + IFN combination had no favorable outcome in patients with ARDS, a finding consistent with Pitfall 4. In a small case-series, RBV + IFN-α2b was administered within 1-2 d of admission to three patients, all of whom survived. In contrast, three other patients who received therapy 12-19 d after admission did not survive[164]. Clinicians inferred that combination treatment has a role in MERS infection only when given early in the disease course.

Coronaviruses have been shown to suppress IFN response in hosts[149]. A subdued IFN response diminishes antigen presentation and reduces antiviral adaptive Th-1 immune response[173,174]. Therefore, recombinant IFNs have been identified as a treatment modality for MERS for their ability to augment host response. Type I (α, β)[112,121,175,176], type II (γ)[177,178], and type III (λ) IFNs[177] exhibit activity against SARS-CoV. IFN-β is the most potent[112,117,179]when compared with IFN-α and -γ.MERS-CoV is 50-100 times more sensitive to IFN-α than SARS-CoV in Vero cells[180].As viruses that cause lysis of their target cells are most effectively inhibited by IFNs in uninfected cells, IFNs have their highest utility in prophylaxis or early post-exposure.IFNs display synergistic characteristics. When administered together, IFN-β and IFN-γ inhibited SARS-CoV plaque formation 30-fold and inhibited replication by 3000-fold[176,178]. The combination of IFN-α2b and RBV was effective in reducing MERS-CoV replication in Vero and LLC-MK2 cells[155]. The biological plausibility of the combination was studiedviamicroarray, which showed that RBV and IFN-α targeted MERS-CoV genes were involved in pathogen recognition, cytokine release, and immune responses[181]. On the other hand, lopinavir was found to inhibit MERS-CoVin vitroin Vero E6 and in Huh7 cells, at a mean half maximal effective concentration of 8.0 mmol/L in a screen of 348 Food and Drug Administration-approved drugs for anti-MERS-CoV activity[182]. Taking into account all these data and considering the MERS clinical studies with the least pitfalls and the lowest CFRs, we conclude that the combination of RBV with IFNs (mainly PEG-IFN-α2α)[161,163,164,172] or triple combination with Lop/r[172] is the best antiviral early treatment in MERS-CoV infection. The role of steroids as immunomodu-latory/immunosuppressive therapy in Phases II-III (ARDS) was not thoroughly studied in MERS, and patients were managed at this Phase solely by mechanical ventilation or extracorporeal membrane oxygenation.

COVID-19 outbreak

A novel, very contagious coronavirus (nCoV-19) was first identified in humans in December 2019 in Wuhan, China, and it quickly spread globally. Data provided by the WHO Health Emergency Dashboard report almost 50 million confirmed cases of COVID-19, including 1.24 million deaths, with confirmed cases in 219 countries(accessed November 08, 2020) https://www.who.int/emergencies/diseases/novelcoronavirus 2019?gclid=Cj0KCQjw2or8BRCNARIsAC_ppyblMJawKCnLtU9F6oYu WCpdraGvdC7QvkrQKqF5 LpbNX5G7kBUefqYaAkB3EALw_wcB. nCoV-19 virus was subsequently termed the SARS-CoV-2 virus, as it is very similar to SARS-CoV.Indeed, in phylogenetic analysis there is 96.08% identity between COVID-19 and SARS-CoV main protease, called 3-C-like protease (M-pro), while identity is only 51.61% between COVID-19 and MERS-CoV M-pros.

In computational (in silico) studies (Table 3), molecular modelling, virtual screening,docking, and sequence comparison statistics of the COVID-19 M-pro were investigated. In the Schrodinger glide docking module, RBV and telbivudine were ranked at the 2ndand 3rdpositions in SARS-CoV-2 M-pro inhibition, respectively where RBV was shown to form two hydrogen bonds with M-pro[183]. In another in silico study,scientists used the optimized COVID-19 and SARS RdRps, and found that sofosbuvir and RBV compete with physiological nucleotides for the RdRp active site and form seven and 13 H-bonds, respectively, suggesting that they can be used against COVID-19 with promising results[184].In a transcriptosome-based drug repositioning study,scientists using bronchoalveolar lavage fluid transcriptome data of eight COVID-19 patients and 20 healthy controls and found that the endocytosis and lysosome pathways are highly involved in the disease and that the regulation of genes involved in neutrophil degranulation was disrupted[185]. The principle of transcriptome-based drug screening identifies drugs that are capable of restoring virus-induced gene expression dysregulation rather than directly targeting viral or human proteins. They identified a total of 1569 differentially expressed genes, consisting of 872 genes with upregulated expression and 697 genes with downregulated expression. Two Food and Drug Administration-approved antiviral drugs (saquinavir and RBV) were identified in the coexpression-based drug enrichment analysis for the prevention and treatment of COVID-19 pneumonia. In contrast, in twoin vitrostudies utilizing nCoV-19 isolates,RBV did not exert any inhibitory effects (half maximal effective concentration 109.5-500 μmol/L). However, in both studies[186,187] Vero cell lines were utilized (Pitfall 1),which are naturally RBV-resistant, as they are inefficient at converting RBV into its mono- and triphosphate forms[119].

A literature search retrieved 137 articles, 12 of which reported on RBV monotherapy or in combinations in COVID-19 and were considered eligible (Table 5) (accessed November 14, 2020). RBV was also included in the Chinese treatment guide[188].There were four randomized open-labeled prospective studies, six retrospective studies, and two meta-analyses. In one study[189], researchers compared retrospectively 71 patients with severe COVID-19 treated with SOC and 44 patients treated with RBV. SOC applied in both groups included ventilation, corticosteroids,and intravenous immunoglobulin. Despite the obvious Pitfall 2 (median time from symptom onset was 8 d), the relatively low (500 mg bis in die) RBV monotherapy dosing (Pitfall 3), and the unclear corticosteroid background treatment, there was a trend in favor of RBV in the negative conversion time of reverse transcription polymerase chain reaction (12.8 dvs14.1 d), in intubation rates (4.5%vs9.9%), and in CFR (17.1%vs24.6%). Because those findings were statistically insignificant, authors extrapolated their results to conclude RBV insufficiency for severe COVID-19, despite the serious study design issues (small number of patients, doctor-dependent RBV administration, and irregular administration-sometimes RBV was out of stock) (Pitfall 5).

In another retrospective study[190], 151 moderately to critically ill patients were recruited, and combination treatment was superior to single or dual antiviral agents.Use of a quadruple combination therapy (RBV + Lop/r + umifenovir + Lianhua Qingwen) significantly improved severe COVID-19 patients. Yuanet al[191] aimed to evaluate the correlation between viral clearance and blood biochemical index of 94 patients suffering predominantly moderate COVID-19 who were hospitalized 7 d after symptom onset (Pitfall 2). Correlation analysis indicated that the duration of hospital stay was significantly correlated with polymerase chain reaction negative conversion times in IFN-α + RBV + Lop/r group (P= 0.0215) as well as in IFN-α + Lop/r group (P= 0.012). In addition, there were no intubations or deaths, and all were discharged.Authors inferred that these two therapeutic regimens are beneficial for the treatment of COVID-19 infected patients. Wuet al[192] described 80 imported cases in Jiangsu Province (China) with mild to moderate symptoms and compared them with those from Wuhan. They presumed that the cases in Jiangsu exhibited mild or moderate symptoms and no obvious gender susceptibility. As of their writing, no one was intubated or died, and 21 patients were discharged. However, they did not mention at all the possible therapeutic role of treatments applied (RBV to all patients, methylprednisolone in 12 patients at an appropriate dose to alleviate the shortness of breath, and moxifloxacin). The largest retrospective study enrolled 681 patients with severe COVID-19[193] in order to clarify their epidemiological, clinical, and therapeutic features. Their median age was 65 years, and 55.1% had comorbidities, with incomplete data regarding the time-gap from symptom onset to treatment initiation,the dosing regimens, and possible drug-combinations administered. Overall mortality of this cohort was 15.3%; this was similar to CFR in another study in which higher CFR(38%) was calculated for ICU patients[194] and comparable (26%) with another report from Italy[195]. Out of 681 patients, 45.8% had cardiovascular disease, and 23.4% of them succumbed. In multivariate analysis, RBV was independently associated with predicting the risk of death in COVID-19 patients [odds ratio: 0.477 (0.232-0.982);P=0.044] as well as arbidol. Multivariable logistic regression in patients combined with COVID-19 and cardiovascular disease performed to evaluate the efficiency of the intervention showed that RBV was also significantly effective in these patients in two different models applied [odds ratio: 0.208 (0.070-0.618);P= 0.005], in accordance with the findings of the total cohort. The authors inferred that RBV and arbidol were effective in patients with severe COVID-19, especially in the subgroup of those with cardiovascular comorbidities and cardiovascular injury by SARS-CoV-2. The latter would not be surprising, taking into account another report[196] in which scientists compared the pharmacokinetic profiles and tissue distribution of antiviral drugs and concluded that RBV is highly concentrated in the heart and the intestines. Other clinicians treated six critical and two severe out of 75 pediatric patients[197] with IFNα nebulizer and intravenous RBV with favorable outcomes.

In one meta-analysis[198], it was concluded that, except for Lop/r for which they found low quality evidence in decreasing ICU stay, there was no persuasive evidence demonstrating important benefit for any antiviral in COVID-19. Notwithstanding,they came to the contradictory conclusion that evidence had not excluded the important benefit of each treatment. On the contrary, another meta-analysis[88] that evaluated the efficacy and safety of antiviral therapeutic options in coronaviruses infections, scientists inferred that therapeutic interventions notably reduce mortality[relative risk (RR): 0.65, 95% confidence interval (CI): 0.44-0.96,I2= 81.3%]. In a subgroup analysis, the combination of RBV and corticosteroids remarkably decreased mortality (RR: 0.43, 95%CI: 0.27-0.68). Besides, Lop/r IFN + RBV, RBV, and combination of Lop/r + RBV + corticosteroids showed a tendency to lower mortality,remarkably improve clinical manifestations (RR: 1.52, 95%CI: 1.05-2.19), and improve radiographical findings (RR: 1.62, 95%CI: 1.11-2.36,I2= 11.0%), even without manifesting clear effects on virological eradication, incidence of ARDS, intubation, and adverse events. The Lop/r-based combination showed superior virological eradication and radiographical improvement with reduction in the rate of ARDS. Conversely, RBV might cause more safety concerns, especially bradycardia.

Of the four RCTs of COVID-19 therapy that included RBV, two of them were based on sofosbuvir/daclatasvir (Sof/d) combination. The first one[199] compared Sof/d with RBV orally, with both arms being treated with SOC (Lop/r and hydroxychloroquine). Although timing of treatment initiation from symptom onset was unknown and there was biased low RBV oral dosing (1.200 mg) and confounding“background” antiviral SOC, researchers deduced that the duration of stay and mortality were lower in the Sof/d group. The relative risk of death for patients treated with Sof/d was 0.17 (95%CI: 0.04-0.73;P= 0.02). In the other RCT[200], researchers evaluated the efficacy of Sof/d in combination with RBV for hospitalized COVID-19 patients with moderate disease compared with SOC. Although it fell under Pitfalls 2,3, same as the previous one, it was even more confounding as the SOC group could have also received RBV according to the given recommendations at the time of the study. The authors deduced only trends for recovery and lower deaths in the Sof/d +RBV arm. Huanget al[201] conducted an open-label RCT to evaluate the efficacy of three antiviral combination regimens. The results indicated that there were no significant differences among the three regimens in terms of antiviral effectiveness.Furthermore, the combination of RBV and Lop/r was associated with a significant increase in gastrointestinal adverse events, suggesting that RBV and LPV/r should not be co-administered to COVID-19 patients simultaneously. No mortality was recorded in that study. Finally, Hunget al[202] published a multicenter, open-label, phase 2 RCT trial to assess the efficacy and safety of combined IFN-β1b+Lop/r+RBV for treating patients with COVID-19. In total, 127 patients were recruited; 81 were assigned in the combined treatment group and 41 were in the control group and received Lop/r monotherapy. For the primary endpoint, which was time from start of treatment to negative nasopharyngeal swab, the combination group had a significantly shorter median time [7 dvs12 d, hazard ratio (HR): 4.37 (1.86-10.24);P= 0.0010]; clinical improvement was better in the combination group, with a significantly shorter time to complete alleviation of symptoms, defined as a National Early Warning Score 2 of 0 [4 dvs8 d, HR: 3.92 (1.66-9.23);P= 0.0001] and SOFA score of 0 [3.0 dvs8.0 d, HR: 1.89(1.03-3.49);P= 0.041]. The significantly better clinical and virological response was also reflected in the shorter median hospital stay in the combination group [9.0 dvs14.5 d, HR: 2.72 (1.2-6.13);P= 0.016]. Eight patients were given stress doses of corticosteroids in the second week from symptom onset. Of the 127 patients, 17 developed oxygen desaturation and required oxygen treatment; six were admitted to the ICU, of whom five required noninvasive ventilation, and a 96-year-old female patient required intubation but was successfully extubated. There were no reported deaths during this study. Interestingly, post-hoc subgroup comparison of the 76 patients who started early treatment less than 7 d after symptoms onset showed better clinical outcomes(time to National Early Warning Score 2 of 0: 4 dvs8 d;P= 0.0001; time to SOFA score of 0: 3 dvs7 d;P= 0.001), shorter duration of hospital stay (8 dvs15 d;P= 0.003), and better virological outcomes (time to negative viral loads all specimens 7 dvs13 d;P=0.0001) in the combination group (52 patients) than in the control group across all measured variables except stool samples. Scientists concluded that the early triple antiviral therapy was safe and superior to Lop/r alone in alleviating symptoms and shortened the duration of viral shedding and hospital stay in patients with mild to moderate COVID-19.

DISCUSSION

Under the aggravating pressure of an emerging pandemic it is burdensome to design and conduct well-organized, double-blind, RCTs. These difficulties may explain why the majority of published studies in SARS, MERS, and COVID-19 outbreaks were uniformly inconclusive. We have identified five specific pitfalls to which many studies fall victim. These include use of inappropriate cell lines (Pitfall 1), not fully understanding the clinical course of the disease (Pitfall 2), and incorrect pharmacology of applied treatments (Pitfalls 3, 4). Another pitfall is the misinterpretation of study results with generalizations that are disrespectful to study design conditions and the primary end-point (Pitfall 5). Other important confounding factors were the concurrent administration of multiple therapies and the absence of a control group in some of them. In Table 6, studies of all three coronaviruses that produced statistically significant results are summarized.

Table 6 Studies of all coronavirus outbreaks with statistically significant findings

ARDS: Acute respiratory distress syndrome; CFR: Case fatality rate; HCQ: Hydroxychloroquine; HR: Hazard ratio; ICU: Intensive care unit; IFN:Interferon; Lop/r: Lopinavir/ritonavir; MERS: Middle East respiratory syndrome; MMF: Mycophenolate mofetil; OR: Odds ratio; RBV: Ribavirin; RR:Relative risk.

Siddiqiet al[203] proposed a clinical-therapeutic staging classification of COVID-19.In stage I, infection occurs at the time of inoculation, and there is early establishment of disease. During this period, SARS-CoV-2 multiplies and establishes residence in the host, primarily focusing on the respiratory system. In stage II of established pulmonary disease, viral multiplication and localized inflammation in the lung are the norm, while patients develop a viral pneumonia and possibly hypoxia. Imaging reveals bilateral infiltrates or ground glass opacities. In early stage IIa (without significant hypoxia), the use of corticosteroids may be avoided. However, if hypoxia ensues (stage IIb), it is likely that patients will progress to requiring mechanical ventilation, and in that situation, the use of anti-inflammatory therapy, such as corticosteroids, may be useful and can be judiciously employed[203]. A minority of COVID-19 patients will transition into the most severe stage of the illness (stage III),which manifests as an extrapulmonary systemic hyperinflammation syndrome with elevated markers of systemic inflammation. COVID-19 infection results in a decrease in helper, suppressor, and regulatory T cell counts[204]. Inflammatory cytokines and biomarkers such as IL-2, IL-6, IL-7, granulocyte colony-stimulating factor, macrophage inflammatory protein 1a, tumor necrosis factor alpha, C-reactive protein, ferritin, and D-dimer are significantly elevated in patients with more severe disease[205]. A form akin to secondary hemophagocytic lymphohistiocytosis and systemic organ involvement may occur in this advanced stage[206]. Tailored therapy in stage III hinges on the use of immunomodulatory agents to reduce systemic inflammationbefore it overwhelmingly results in multiorgan dysfunction. The use of corticosteroids may be justified in concert with the use of cytokine inhibitors such as tocilizumab (IL-6 inhibitor) or anakinra (IL-1 receptor antagonist)[206].

The clinical progression of SARS was mostly uniform[131] and very similar to that of COVID-19, with a tri-phasic pattern. Week 1 was characterized by systemic symptoms that generally improve after a few days. The increasing viral load during this phase suggests that the symptoms are largely related to the effect of viral replication and cytolysis. As the disease progresses into week 2, the patients frequently experience recurrence of fever, onset of diarrhea, and oxygen desaturation with shifting radiographic shadows. If viral induced damage was the primary pathological mechanism, such a flitting pattern of radiological change is difficult to explain. The timing of the immunoglobulin G seroconversion, which starts on day 10, seems to correlate with falls in viral load, which occur between days 10 and 15. There is a progressive decrease in rates of viral shedding from nasopharynx, stool, and urine from days 10 to 21 after symptom onset.The severe clinical worsening that may occur at this time cannot be explained by uncontrolled viral replication. The lung damage is related to immunopathological events as a result of an overexuberant host response,rather than uncontrolled viral replication[129,131]. Twenty percent of patients may progress to Phase III, characterized by ARDS, necessitating ventilatory support.Inevitably, several patients will develop nosocomial sepsis during this phase of endorgan damage and severe lymphopenia. Interestingly, in terms of pathogenesis, in pulmonary reovirus infection in athymic mice, a lower plaque-forming value of 106is associated with pathological changes of bronchiolitis obliterans organizing pneumonia(BOOP), whereas a higher inoculum of 107is associated with ARDS[207]. Thus, rapid reduction in viral load is critical for the development of a more severe disease stage.

However, there is a critical difference between COVID-19 and the other two coronaviruses. The viral loads of SARS and MERS peak at around days 7-10 after symptom onset[131,142], implying that there is a therapeutic window that could be exploited[87]. In contrast, the viral load of COVID-19, as detected in posterior oropharyngeal saliva samples, is highest at presentation with higher viral loads in the nose than in the throat[208,209], or it peaks at around 5-6 d after symptom onset in throat swab and sputum samples[40]. After the first week, COVID-19 viral load gradually declines[210]. This suggests there is very short time-interval after symptom onset during which a viable antiviral therapy will be beneficial. Targeting selected patients during stage I not only will minimize contagiousness but may prevent progression to higher disease severity. SARS-CoV-2 is highly contagious, as the basic reproduction number (R0) is approximately 2-3.5[211,212]. Thus, except for protecting from disease progression, the reduced viral load would also translate to reduced virus shedding, thereby reducing the risk of secondary transmission and thus acting as a prophylaxis.

For SARS,scientists concluded that in order to lessen the risk of progression to the ARDS phase, an effective antiviral was necessary to reduce the viral load and decrease the initial cytolytic damage in Phase I, which in turn may result in decreased immunopathological damage during Phase II[131]. Therefore, an antiviral therapy in stage I of COVID-19 may have the greatest benefit. The doses of RBV used in reports from Hong Kong[129,131] were associated with a reversed V-shaped curve of viral load, excluding the absence of antiviral activity. The characteristic finding on computed tomography mimicked that in BOOP. In addition, the similarity of the histologic features to those of early ARDS in post-mortem studies prompted physicians to use corticosteroids in combination with RBV for the treatment of SARS[129], as corticosteroid therapy had been used with some success in BOOP[144]. Corticosteroids may decrease the release of macrophage-derived inflammatory cytokines[213]. In a Canadian SARS outbreak,RBV was administered early with steroids, and no conclusive results of efficacy could be established despite viral and symptom flare-up in a portion of patients after treatment cessation[126,133]. High dose methylprednisolone should be avoided in the early phase of SARS and SARS-CoV-2 as viral clearance by host immunity might be hampered.

It is more than clear since the SARS outbreak that an efficient antiviral treatment,administered as early as possible following COVID-19 onset, is the critical step to reduce viral load and restrain disease progression to stages II and III. In contrast to SARS, in which the time from symptom onset to highest viral loads may exceed 1 wk,the viral load in SARS-CoV-2 peaks with presentation or within the first few days.That is the reason why prompt antiviral initiation is crucial. If disease progresses to stage IIb and stage III, antivirals probably have no beneficial effect. At the stage of aberrant immunopathological damage in lungs and extrapulmonary systemic hyperinflammation syndrome, with cytokine storm and possible occurrence of secondary hemophagocytic lymphohistiocytosis, an effective immunosuppressive/immunomodulator treatment is needed.

Several studies in coronavirus diseases have shown that the combination of RBV with steroids and especially pulsed methylprednisolone when hypoxia develops or in worsening pulmonary infiltrates can yield improvement in imaging findings and final outcome. It should be kept in mind that corticosteroids may delay viral clearance,prolonging infections while reducing the symptomatic inflammatory cytokines [214-216]. Although initiating steroids early can prevent the cytokine storm and lung damage, starting too early might inhibit antibody production, thus prolonging the natural course of the disease[125]. Hemophagocytosis has been attributed to cytokine dysregulation, and intervention with steroids might modulate this cytokine response and prevent a fatal outcome, as it has been proposed for other causes of ARDS[217]. In a retrospective cohort[207] of patients with severe pneumonia COVID-19 and subsequent ARDS, the administration of methylprednisolone appeared to reduce the risk of death in patients with ARDS (HR: 0.38; 95%CI: 0.20-0.72;P= 0.003). Those who received methylprednisolone treatment were much more likely to develop ARDS,likely owing to confounding by indication (e.g., sicker patients were more likely to be given methylprednisolone). These concerns may be avoided if corticosteroids are applied during the proper time window of the disease; not during the early phase, but when hypoxia and apparent imaging findings are established. Finally, specific immunomodulatory compounds (tocilizumab and anakinra) are beneficial in stages IIb and III. In COVID-19 pneumonia with ARDS characterized by hemophagocytic lymphohistiocytosis, the response to tocilizumab was rapid, sustained, and associated with significant clinical improvement[218].

Accumulated data from all coronavirus outbreaks converge to the conclusion that antiviral combination therapy is more beneficial than antiviral monotherapy. The checkerboard assay demonstrated synergism between lopinavir and RBV at a low viral inoculum[87]. RBV combined with Lop/r seems to improve the clinical efficacy of SARS[161]. Use of IFNs was not considered by most clinicians during the SARS epidemic because of their known proinflammatory activity, which may potentiate the inflammatory damage initiated by the viral infection[219]. Early administration of IFN protected mice from lethal MERS-CoV infection, while late administration of exogenous IFN promoted the proinflammatory cytokine response and inhibited the optimal virus-specific T cell response[220,221]. IFN-β1a or leukocytic IFN-α with RBV appeared to be the most effective combination[116]. RBV concentrations inhibiting virus production in combination with IFN-β were at least 10-fold lower compared to monotherapy, with highly synergistic antiviral effects of combination treatment[117].In animal studies, researchers concluded that potentially effective combinations were RBV with IFN-β1b and/or Lop/r[157]. Moreover, when both IFN-α2b and RBV were applied as a combination, significant synergism was observed, with 8-fold reduction in half maximal inhibitory concentration of IFN-α2b and 16-fold reduction in that of RBV[108], while the combination improved MERS infection in rhesus macaque[156]. IFN-β combined with Lop/r had better efficacy to treat MERS-CoV[140].

RBV is a broad-spectrum nucleoside that is phosphorylated in virus-infected cells,and its product acts as a competitive inhibitor of virus synthetase, interfering with early viral transcription events and hindering the synthesis of ribonucleoproteins and virus replication and spread[222]. RBV's multiple mechanisms of action likely support its longevity and quality as a clinical resource. The risk of RBV-associated anemia―although substantial and in need of careful monitoring―might not hinder the use of RBV for patients with severe coronavirus infections, especially if a survival benefit can be confirmed.

CONCLUSION

COVID-19 behaves like a bipolar disease. On the one hand it is a mild, self-limiting viral respiratory tract infection, and the majority of patients recover with no sequelae.On the other hand, it is a severe pneumonitis with a deadly systematic auto-inflammatory disease component. With respect to the most significant studies and to those that fell under the least serious pitfalls analyzed in this article, we conclude by presenting below a scheme of treatment modalities tailored to COVID-19 disease stages, which could, if timely applied, be beneficial.

Suggestion 1 (Stage I treatment)

IFN-β + RBV + Lop/r should be commenced early after disease onset at doses as reported by Hunget al[202]. Concerns: IFN-β should not exceed the first week of the disease due to its proinflammatory activity; the other antivirals should not be administered for more than 7-10 d and unequivocally when severe hypoxia develops due to the loss of their benefit and subsequent prevalence of their side-effects.Rationale: The effective reduction of viral load and subsequent deterrence of disease progress; the reduction of virus shedding, thus reducing the risk of secondary transmission, therefore acting as a prophylaxis. Implementation: Certainly, this antiviral combination would not be initiated in most COVID-19 cases, as the majority of patients have a benign, self-limited illness. This approach targets selected patients,as specific age groups present severe morbidity and mortality. According to Centers for Disease Control and Prevention COVID-19 data tracker (https://www.cdc.gov/covid-data-tracker/index.html#demographics), CFR by age groups are: 18-49 (0.5%-3.1%); 50-64, 15.2%; 65-74, 21%; 75-84, 26.6%; and 85+, 32.2%. Therefore, all patients older than 50 years should be treated. For younger patients (18-years-old to 49-years-old), stage I treatment should be applied only to adults with certain underlying medical conditions (e.g., cardiovascular diseases, diabetes, chronic obstructive pulmonary disease, obesity, smoking, chronic kidney disease, cancer, and immunosuppression in solid organ transplant recipients, https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/people-at-increased-risk.html)that are independent risk factors for severe illness and negative COVID-19 outcome.The remarkable advantage of this therapeutic regimen is that it can be applied on an outpatient basis with the patient at home in quarantine.

Suggestion 2

Corticosteroids and tocilizumab or anakinra should be used for severe pneumonitis and for stage III of systemic hyperinflammation syndrome with cytokine storm and possible occurrence of secondary hemophagocytic lymphohistiocytosis. If not initiated early, corticosteroids have been shown to be beneficial in reducing intubation and mortality rates in all coronaviruses infections. The combination of corticosteroids with tocilizumab showed superior survival outcome when compared with SOC treatment and treatment with corticosteroids alone or in combination with anakinra.Furthermore, corticosteroid use either alone or in combination with tocilizumab or anakinra was associated with reduced hospital mortality for patients with cytokine storm compared with patients receiving SOC treatment[223]. Proposal A:Dexamethasone 6 mg/d for 10 d in those with respiratory failure or those intubated[224]. Proposal B: High-dose steroids with pulsed methylprednisolone being the most tested and effective in SARS and MERS studies at a dose of 0.5-1.0 g/d for 2-3 d or in a total of 3 g. It has been shown that pulsed methylprednisolone not only restricts radiological abnormalities and restores respiratory failure but may also restrain the cytokine storm. Proposal C: Immediate treatment with methylprednisolone 250 mg intravenously on day 1,followed by methylprednisolone 80 mg intravenously on days 2-5. In cases with lack of clinical improvement or worsening in respiratory status,escalation of immunosuppressive treatment with a monoclonal antibody directed against the IL-6 receptor and tocilizumab should follow between day 2 and day 5(single-dose tocilizumab, 8 mg/kg intravenous, max 800 mg)[225,226].

Suggestion 3

Regarding measles pneumonitis, RBV seems an adequate treatment at a dose of 600 mg qid for 5-7 d. In case of malignancies, especially hematological and severely immunosuppressed patients, a longer regimen of 2-3 wk may be required.

ARTICLE HIGHLIGHTS

Research background

Ribavirin is a broad-spectrum nucleoside antiviral drug that despite it has been widely used clinically for almost five decades, evidence regarding its efficacy in viral infections remains conflicting. Ribavirin use has only been established in chronic hepatitis C virus infection, chronic hepatitis E virus infection in transplant recipients,respiratory syncytial virusin children, and some of the viral hemorrhagic fever viruses.Ribavirin was widely utilized alone, or in combination with other compounds in severe acute respiratory syndrome (SARS), Middle-East respiratory syndrome (MERS),and coronavirus disease 2019 (COVID-19) outbreaks. Despite the large amount of data however, the conclusions of all three coronaviruses studies concerning ribavirin efficacy have been contradictory. The present review article aims to clarify the underlying reasons for these discrepancies including possible study design inaccuracies and failures, misinterpretations of data, and to address these potential confounds. Moreover, the possible role of ribavirin in COVID-19 therapeutic schemes is thoroughly studied.

Research motivation

COVID-19 pandemic has emerged as a global health issue with the highest significance and is currently the number one priority for scientists worldwide. During the 2018 measles outbreak, we hospitalized a number of adult cases suffering measles pneumonitis and treated them with Ribavirin (RBV). Because of the lack of specific guidelines on severe measles disease treatment in adults, we reviewed the literature on RBV dosing regimens and outcomes in any infectious disease. The most amount of clinical data available was for SARS and MERS, where RBV was widely utilized. While preparing the measles/RBV study for publication, the new COVID-19 outbreak emerged, prompting us to focus heavily on COVID-19 treatment with RBV alone or in combination with other compounds.

Research objectives

To shed light in and clarify the confounding factors of ribavirin treatment studies regarding SARS, MERS, and COVID 19 and to propose a therapeutic scheme for COVID-19 that would be tailored to its distinct disease stages.

Research methods

A meticulous electronic search of PubMed database was performed covering a period of over five decades up to October 15, 2020 using the terms “ribavirin”, “treatment” in combination with “measles”, “SARS”, “MERS”, and “COVID-19”. All review articles referring to COVID-19 treatment were searched and studied, regardless of whether“ribavirin” was included in key words.In vitro, animal and clinical studies, reviews,and meta-analyses in English language only were considered for data extraction. The citations in each article were reviewed to locate additional references that were not retrieved during the initial search. Eligible to be included in the review were those studies referring to RBV treatment alone or in combination and/or those reporting on its dose regimens, adverse effects, or outcomes. The literature search was performed and described with respect to PRISMA guidelines.

Research results

A total of 32 severe acute respiratory syndrome-associated coronavirus studies, 18 Middle East respiratory syndrome coronavirus studies, and 17 severe acute respiratory syndrome-2 associated coronavirus studies were considered eligible to be included in this review. The burden of designing and conducting well-organized, double-blind,randomized controlled trials under the difficulties and pressures of an emerging pandemic is obvious. Hence, many of those studies succumbed to specific pitfalls that resulted in conflicting evidence regarding the clinical efficacy of ribavirin for coronaviruses infections. We detected six pitfalls that were carefully identified and described in this review and comprise: utilization of naturally ribavirin-resistant Vero cell lines inin vitrostudies; study design inconsistent with the well-established clinical course of disease (i.e., antiviral administration late in the disease course or early use of corticosteroids); inappropriate pharmacology of applied treatments (i.e.dosing regimens, treatment duration); and misinterpretation of study results with misconceived generalizations. Considering all those studies with their pitfalls and mostly taking into account those with statistically significant outcomes, we concluded to a comprehensive treatment for COVID-19 documented by thorough, long-term investigation of ribavirin regimens in coronavirus infections which is strictly tailored to distinct disease stages.

Research conclusions

COVID-19 behaves like a bipolar disease being an asymptomatic or mild, self-limiting viral respiratory tract infection with the majority of patients recovering without sequelae on the one hand, and on the other it may progress to a severe pneumonitis with a deadly systematic auto-inflammatory disease component. Documented by accumulated data from the three coronaviruses studies and considering the six identified pitfalls to which most of the studies fall victim, the early antiviral treatment is crucial for reducing viral load, transmission, and preventing disease progression to severity. Interferon-β + Ribavirin + Lopinavir/ritonavir should be commenced as early as possible after disease onset resulting in an effective reduction of viral load and subsequent deterrence of disease progress, reduction of virus shedding, and thus reducing the risk of secondary transmission, therefore acting as a prophylaxis. This approach could target selected patients, as specific age groups (older than 50 years)present severe morbidity and mortality, as well as younger patients with wellrecognized independent risk factors for severe illness and increased mortality. The remarkable advantage of this therapeutic regimen is that it can be applied on an outpatient basis with the patient at home in quarantine. On the other hand, corticosteroids and anti-interleukin monoclonal antibodies (tocilizumab or anakinra) should be used for severe pneumonitis and for systemic hyperinflammation syndrome with cytokine storm and possible occurrence of secondary hemophagocytic lymphohistiocytosis. Corticosteroids in COVID-19 comprise dexamethasone 6 mg/d as previously shown to reduce mortality; methylprednisolone 250 mg intravenously on day 1, 80 mg on days 2-5 and escalation of immunosuppressive treatment with a monoclonal antibody directed against the interleukin-6 receptor (tocilizumab) when respiratory status worsens; a third corticosteroid treatment proposal being the most tested and effective in SARS and MERS studies includes high-dose steroids with pulsed methylprednisolone at a dose of 0.5-1. 0 g/d for 2-3 d or for a total of 3 g. Finally, regarding measles pneumonitis, ribavirin seems an adequate treatment at a dose of 600 mg for 5-7 d, but in cases of hematological malignancies, or severely immunosuppressed patients, a longer regimen of 2-3 wk may be required.

Research perspectives

It is of paramount importance to confirm the efficacy of the early triple antiviral combination in reducing viral load, transmission, and preventing disease progress to severity by conducting Phase III randomized controlled trials, as this early triple antiviral combination efficacy has already been determined for COVID 19 in a Phase II clinical trial with statistically significant outcomes. It would be of great interest also to perform clinical studies to determine the impact of pulsed methylprednisolone on COVID 19 pneumonitis and/or on cytokine storm compared with the already approved approaches such as administration of dexamethasone 6 mg/ d. COVID 19 is the pinpoint of interest of all scientists worldwide.