Role of Multifunctional Cytoskeletal Filaments in Coronaviridae Infections: Therapeutic Opportunities for COVID-19 in a Nutshell.

A novel coronavirus discovered in 2019 is a new strain of the Coronaviridae family (CoVs) that had not been previously identified in humans. It is known as SARS-CoV-2 for Severe Acute Respiratory Syndrome Coronavirus-2, whilst COVID-19 is the name of the disease associated with the virus. SARS-CoV-2 emerged over one year ago and still haunts the human community throughout the world, causing both healthcare and socioeconomic problems. SARS-CoV-2 is spreading with many uncertainties about treatment and prevention: the data available are limited and there are few randomized controlled trial data on the efficacy of antiviral or immunomodulatory agents. SARS-CoV-2 and its mutants are considered as unique within the Coronaviridae family insofar as they spread rapidly and can have severe effects on health. Although the scientific world has been succeeding in developing vaccines and medicines to combat COVID-19, the appearance and the spread of new, more aggressive mutants are posing extra problems for treatment. Nevertheless, our understanding of pandemics is increasing significantly due to this outbreak and is leading to the development of many different pharmacological, immunological and other treatments. This Review focuses on a subset of COVID-19 research, primarily the cytoskeleton-related physiological and pathological processes in which coronaviruses such as SARS-CoV-2 are intimately involved. The discovery of the exact mechanisms of the subversion of host cells by SARS-CoV-2 is critical to the validation of specific drug targets and effective treatments.

Interferons in the Therapy of Severe Coronavirus Infections: A Critical Analysis and Recollection of a Forgotten Therapeutic Regimen with Interferon Beta.

The pharmacological and immunological properties of interferons, especially those of interferon beta, and the corresponding treatment strategies are described, and the results of studies with different interferons in coronavirus infections are analysed. Furthermore, the data obtained with high-dosed native interferon beta in life-threatening acute viral diseases as well as the results of clinical pilot studies with high-dosed recombinant interferon beta-1a are provided because they serve as the rationale for the proposed therapeutic regimen to be applied in acute viral infections. This regimen differs from those approved for treatment of multiple sclerosis and consists of interferon beta-1a administered as a 24 hour intravenous infusion at a daily dose of up to 90 µg for 3-5 consecutive days. Since under this regimen transient severe side effects can occur, it is analysed which patients are suitable for this kind of treatment in general and if patients with severe coronavirus infections could also be treated accordingly.

Uso de plasma de pacientes recuperados no tratamento de COVID-19: revisão sistemática rápida / Use of plasma from recovered patients in the treatment of COVID-19: rapid systematic review

CONTEXTO: Conforme classificação da OMS, a COVID-19 é uma pandemia de risco muito alto a nível global. Até o momento não existem terapias específicas para a doença, embora diferentes alternativas, incluindo o uso de plasma de pacientes recuperados, estejam em investigação. OBJETIVOS: Identificar, avaliar sistematicamente e sumarizar as melhores evidências científicas disponíveis sobre a eficácia e a segurança do uso de plasma de pacientes recuperados para COVID-19. MÉTODOS: Revisão sistemática rápida (rapid review methodology). RESULTADOS: Após o processo de seleção, 55 estudos foram incluídos: 15 estudos com resultados e 40 estudos clínicos em andamento. Os estudos evidenciaram que o plasma de paciente recuperado para tratamento de SARS-CoV-2 resultou em redução de mortalidade, carga viral e tempo de internação hospitalar. Entretanto, estes estudos têm risco de viés moderado a alto e os estudos clínicos têm amostra pequena. Estes fatores contribuíram para que a certeza na evidência fosse muito baixa. CONCLUSÃO: Esta revisão sistemática rápida identificou 15 estudos com qualidade metodológica baixa a moderada, que avaliaram os efeitos da terapia com plasma de pacientes recuperados para COVID-19. Com base nos achados destes estudos, a eficácia e a segurança do plasma de pacientes recuperados em pacientes com COVID-19 ainda são incertas e seu uso de rotina, para esta situação, não pode ser recomendado, até que resultados de ensaios clínicos em andamento possam ser avaliados.(AU)

Recent discovery and development of inhibitors targeting coronaviruses.

Human coronaviruses (CoVs) are enveloped viruses with a positive-sense single-stranded RNA genome. Currently, six human CoVs have been reported including human coronavirus 229E (HCoV-229E), OC43 (HCoV-OC43), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and MiddleEast respiratory syndrome (MERS) coronavirus (MERS-CoV). They cause moderate to severe respiratory and intestinal infections in humans. In this review, we focus on recent advances in the research and development of small-molecule anti-human coronavirus therapies targeting different stages of the CoV life cycle.

Characterization of amino acid substitutions in feline coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease inhibitor.

Feline infectious peritonitis (FIP) is a highly fatal disease caused by a virulent feline coronavirus in domestic and wild cats. We have previously reported the synthesis of potent coronavirus 3C-like protease (3CLpro) inhibitors and the efficacy of a protease inhibitor, GC376, in client-owned cats with FIP. In this study, we studied the effect of the amino acid changes in 3CLpro of feline coronavirus from a feline patient who received antiviral treatment for prolonged duration. We generated recombinant 3CLpro containing the identified amino acid changes (N25S, A252S or K260 N) and determined their susceptibility to protease inhibitors in the fluorescence resonance energy transfer assay. The assay showed that N25S in 3CLpro confers a small change (up to 1.68-fold increase in the 50% inhibitory concentration) in susceptibility to GC376, but other amino acid changes do not affect susceptibility. Modelling of 3CLpro carrying the amino acid changes was conducted to probe the structural basis for these findings. The results of this study may explain the observed absence of clinical resistance to the long-term antiviral treatment in the patients.

Small-Molecule Antiviral ß-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). ß-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 µM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 µM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, ß-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

Interferon-ß and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays.

The Middle East respiratory syndrome coronavirus (MERS-CoV) presents a novel emerging threat to public health worldwide. Several treatments for infected individuals have been suggested including IFN, ribavirin and passive immunotherapy with convalescent plasma. Administration of IFN-α2b and ribavirin has improved outcomes of MERS-CoV infection in rhesus macaques when administered within 8 h post-challenge. However, detailed and systematic evidence on the activity of other clinically available drugs is limited. Here we compared the susceptibility of MERS-CoV with different IFN products (IFN-α2b, IFN-γ, IFN-universal, IFN-α2a and IFN-ß), as well as with two antivirals, ribavirin and mycophenolic acid (MPA), against MERS-CoV (Hu/Jordan-N3/2012) in vitro. Of all the IFNs tested, IFN-ß showed the strongst inhibition of MERS-CoV in vitro, with an IC50 of 1.37 U ml(-1), 41 times lower than the previously reported IC50 (56.08 U ml(-1)) of IFN-α2b. IFN-ß inhibition was confirmed in the virus yield reduction assay, with an IC90 of 38.8 U ml(-1). Ribavirin did not inhibit viral replication in vitro at a dose that would be applicable to current treatment protocols in humans. In contrast, MPA showed strong inhibition, with an IC50 of 2.87 µM. This drug has not been previously tested against MERS-CoV and may provide an alternative to ribavirin for treatment of MERS-CoV. In conclusion, IFN-ß, MPA or a combination of the two may be beneficial in the treatment of MERS-CoV or as a post-exposure intervention in high-risk patients with known exposures to MERS-CoV.

From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.

This article introduces a series of invited papers in Antiviral Research marking the 10th anniversary of the outbreak of severe acute respiratory syndrome (SARS), caused by a novel coronavirus that emerged in southern China in late 2002. Until that time, coronaviruses had not been recognized as agents causing severe disease in humans, hence, the emergence of the SARS-CoV came as a complete surprise. Research during the past ten years has revealed the existence of a diverse pool of coronaviruses circulating among various bat species and other animals, suggesting that further introductions of highly pathogenic coronaviruses into the human population are not merely probable, but inevitable. The recent emergence of another coronavirus causing severe disease, Middle East respiratory syndrome (MERS), in humans, has made it clear that coronaviruses pose a major threat to human health, and that more research is urgently needed to elucidate their replication mechanisms, identify potential drug targets, and develop effective countermeasures. In this series, experts in many different aspects of coronavirus replication and disease will provide authoritative, up-to-date reviews of the following topics: - clinical management and infection control of SARS; - reservoir hosts of coronaviruses; - receptor recognition and cross-species transmission of SARS-CoV; - SARS-CoV evasion of innate immune responses; - structures and functions of individual coronaviral proteins; - anti-coronavirus drug discovery and development; and - the public health legacy of the SARS outbreak. Each article will be identified in the last line of its abstract as belonging to the series "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses."

Inhibition of Middle East respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody.

We identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also significantly inhibited infection. These findings indicate that both 2F9 and YS110 are potential therapeutic agents for MERS-CoV infection. YS110, in particular, is a good candidate for immediate testing as a therapeutic modality for MERS.

The emerging novel Middle East respiratory syndrome coronavirus: the "knowns" and "unknowns".

A novel lineage C betacoronavirus, originally named human coronavirus EMC/2012 (HCoV-EMC) and recently renamed Middle East respiratory syndrome coronavirus (MERS-CoV), that is phylogenetically closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5, which we discovered in 2007 from bats in Hong Kong, has recently emerged in the Middle East to cause a severe acute respiratory syndrome (SARS)-like infection in humans. The first laboratory-confirmed case, which involved a 60-year-old man from Bisha, the Kingdom of Saudi Arabia (KSA), who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the World Health Organization (WHO) on September 23, 2012. Since then, a total of 70 cases, including 39 fatalities, have been reported in the Middle East and Europe. Recent clusters involving epidemiologically-linked household contacts and hospital contacts in the Middle East, Europe, and Africa strongly suggested possible human-to-human transmission. Clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of MERS-CoV, and the optimal laboratory diagnostic options and potential antiviral targets for MERS-CoV-associated infection.

Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family Coronaviridae.

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

Protective effects of murine recombinant interferon-beta administered by intravenous, intramuscular or subcutaneous route on mouse hepatitis virus infection.

The significance of the route for administration of murine recombinant interferon-beta (IFN-beta) for inducing its therapeutic effects has been studied. BALB/c mice were daily injected intravenously, intramuscularly or subcutaneously with 1.5x10(3), 1. 5x10(4), or 1.5x10(5) IU of IFN-beta, from one day before to 8th day after mouse hepatitis virus (MHV-2) challenge. All mice received IFN-beta survived significantly longer than those without IFN. In the liver of those IFN-treated mice, viral growth and the histopathological damages were extremely alleviated. These results suggest that, irrespective of the differences in the route of administration, IFN-beta markedly suppressed viral activity when its administration was started prior to viral infection. For clinical use, however, further studies are needed on the optimal route for administration if IFN-beta is given after viral infection.

The effect of intranasal nedocromil sodium on viral upper respiratory tract infections in human volunteers.

Two studies involving double-blind group comparative trials in human volunteers compared the effects of intranasal nedocromil sodium (2.6 mg active drug per nostril, q.i.d.) with placebo on clinical symptoms and performance impairment associated with the common cold. In the first study volunteers were challenged with rhinoviruses (RV9 and RV14), and in the second study with respiratory coronavirus. In both studies, active and placebo groups of volunteers were demographically similar. Infection rates in both groups were also similar. There were no withdrawals resulting from unusual symptoms related to either treatment. In the rhinovirus study (19, placebo; 20, nedocromil sodium) daily symptom scores and daily mean nasal secretion weights were significantly lower in the nedocromil sodium-treated group. In the coronavirus study (26, placebo; 27, nedocromil sodium) there was little difference in the severity of colds between the active and placebo-treated groups, but trends favoured nedocromil sodium. In both studies the impairment of performance in volunteers who developed a cold was significantly less in those treated with nedocromil sodium than in those treated with placebo.

A model for persistent murine coronavirus infection involving maintenance via cytopathically infected cell centres.

The relatively cell impermeable hygromycin B was found to inhibit viral but not cellular protein synthesis when added to cultures of murine hepatitis virus (MHV)-infected or mock-infected mouse L-2 fibroblasts. Membrane permeability, as judged by influx of sodium ions, has previously been demonstrated to be an MHV E2 glycoprotein-mediated, cytopathic effect of MHV infection in L-2 cells. It is therefore likely that the selective effect of hygromycin B on viral protein synthesis is a reflection of an increased drug penetration into virus-infected cells. Using hygromycin B as a marker for MHV-induced cell membrane cytopathology, the effects of drug treatment on a persistent MHV infection in mouse LM-K fibroblasts were investigated. MHV persistence in LM-K cells, which normally involves a steady state infection of 0.1 to 1% of the cells in culture, was found to be cured by hygromycin B treatment, as measured by the elimination of infectious virus from the supernatant medium. Hygromycin B also resulted in the eradication of MHV-specific RNA from LM-K cells, arguing against the presence of a non-cytopathically or latently infected subpopulation of cells.