Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Valoración y seguimiento de los pacientes con sospecha de COVID-19 en la primera ola pandémica en una zona urbana de Andalucía / Assessment and follow-up of patients with suspected COVID-19 in the first pandemic wave in an urban area of Andalusia (Spain)

Objetivos: Conocer las características de la atención inicial y el seguimiento telefónico de pacientes con sospecha de COVID-19 en la primera ola de la pandemia. Diseño: Observacional retrospectivo (auditoría de historias clínicas).Emplazamiento: Centro de salud urbano. Participantes: Casos probables de SARS-CoV-2 (15/03/2020 a 15/06/2020). Mediciones principales Ámbito de atención inicial y seguimiento telefónico (número de llamadas y duración). Variables sociodemográficas (incluyendo estructura familiar). Curso clínico (sintomatología, vulnerabilidad, pruebas, ingreso hospitalario y desenlace). Resultados Trescientos uno pacientes (51,5 [±17,8] años, 23% vulnerables, 17% estructura familiar no nuclear). Valoración inicial en el centro de salud (59,8%: telefónica; 25,2%: presencial). En urgencias hospitalarias (11%) presentaron síntomas similares que en atención primaria, predominan estructuras familiares no nucleares (p<0,05; test χ2), realizando más pruebas (p<0,05; test χ2). En domicilio (3,9%) son pacientes ancianos vulnerables (p<0,01, test ANOVA). El seguimiento telefónico duró 17,1 [±10,3] días con 8,2 [±4,4] llamadas, superior si provenían de urgencias o domicilio (p<0,03; test ANOVA). Se incrementa tras ≥2 consultas presenciales (OR: 4,8), la presencia de síntomas de alarma (OR: 2,3) y la edad ≥45 años (OR: 2,0). Se realizaron pocas pruebas de confirmación (19,3% antigénicas, 13% serologías). El 15,3% ingresos hospitalarios (todos valorados en centros de salud), con 6,3% casos severos y 2,3% exitus. Conclusión Durante la primera ola de la pandemia, la población optó por ser atendida de forma telemática en su centro de salud. Las valoraciones iniciales en urgencias del hospital se relacionan con la falta de apoyo social, pero no con mayor gravedad clínica. El seguimiento telefónico fue aceptado por la población y permitió seleccionar a los pacientes con peor curso clínico.

ObjectiveTo know the characteristics of the initial care and telephone follow-up of patients with suspected COVID-19 in the first wave of the pandemic. Design: Observational, retrospective (audit of medical records).Location: Urban Primary Care Center of Andalusia (Spain). Participants: Probable cases of SARS-CoV-2 (from 20/03/15 to 20/06/15). Principal measurements Initial medical assessment (place and modality) and telephone follow-up (number of calls and duration). Sociodemographic variables (including family structure). Clinical course (symptoms, vulnerability, tests, hospital admission and outcome). Results Three hundred one patients (51.5±17.8 years; 23% vulnerable people; 17% non-nuclear family structure). First assessment in Primary Care by phone (59.8%) and face-to-face (25.2%). At the hospital emergency department (11%), patients were more frequently from non-nuclear families (P<.05 χ2) and more tests were carried out (P<.05 χ2) despite having similar symptoms. Vulnerable elderly patients needed home health care (P<.01 ANOVA). 8.2±4.4 follow-up phone calls were made per patient, for 17.1±10.3 days. It increases after ≥2 face-to-face consultations (OR 4.8), the presence of alarm symptoms (OR 2.3) and age ≥45 years (OR 2.0). Few confirmatory tests were performed (19.3% antigenic, 13% serology). The 15.3% hospital admissions (all assessed previously in Primary Care), with 6.3% severe cases and 2.3% death. Conclusion Population chose to be attended in Primary Care during the pandemic first wave, above all by phone. Telephone follow-up was well accepted and useful to select patients with serious complications. Initial medical assessment in the hospital emergency department was related to a lack of social support but not with greater clinical severity.

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites.

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

PABPC4 Broadly Inhibits Coronavirus Replication by Degrading Nucleocapsid Protein through Selective Autophagy.

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.

Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Programmed -1 Ribosomal Frameshifting in coronaviruses: A therapeutic target.

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.

Lower respiratory tract infection in the community: associations between viral aetiology and illness course.

OBJECTIVES: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. METHODS: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. RESULTS: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. CONCLUSIONS: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.

Afecção Pulmonar pelo Vírus SARS-CoV-2: Clínica e Diagnóstico / SARS-CoV-2 Lung Infection: Clinic and Diagnosis

O pulmão é o órgão que diariamente está exposto e é agredido por diferentes infecções e poluentes do meio ambiente e do local de trabalho. O coronavírus SARS-CoV-2 é o agressor mais recente que chega ao pulmão através das células respiratórias das vias aéreas superiores e com enorme capacidade de desenvolver novas infecções. Este vírus liga-se ao receptor da Enzima Conversora de Angiotensina humana (hACE-2), causa sintomas constitucionais e respiratórios. A média de novos casos gerados por um caso com a infecção provocada pelo novo Coronavírus (R0) oscila entre 2,24 (IC 95%: 1,96-2,55) e 3,58 (IC 95%: 2,89-4,39)21, ou seja um indivíduo com COVID-19 pode infectar cerca de 2 a 4 pessoas, o que caracteriza a sua elevada contagiosidade. A doença disseminou-se por diferentes países e continentes. Em Março de 2020 foi caracterizada pela Organização Mundial da Saúde (OMS) como uma pandemia, tornando-se uma preocupação séria e um desafio extremo para a sua contenção. O tempo de incubação após o contágio pode variar de dois a 14 dias. Durante este período, também conhecido como período "pré-sintomático", algumas pessoas infectadas podem ser contagiosas de um a três dias antes do início dos sintomas. Neste contexto, o sistema respiratório não é só o principal órgão a ser agredido, mas também o principal responsável pela sua transmissibilidade.

The lung is the organ that is daily exposed and assaulted by different infections and pollutants from the environment and workplace. The SARS-CoV-2 coronavirus is the most recent aggressor that reaches the lung through the respiratory cells of the upper airways and with enormous capacity to develop new infections. This virus binds to the human angiotensin-converting enzyme receptor (hACE-2), causes constitutional and respiratory symptoms. The average number of new cases generated by a case with the infection caused by the new coronavirus (R0) ranges from 2.24 (95% CI: 1.96-2.55) to 3.58 (95% CI: 2.89-4.39)21, i.e., an individual with COVID-19 can infect about 2 to 4 people, which characterizes its high contagiousness. The disease has spread to different countries and continents. In March 2020 it was characterized by the World Health Organization (WHO) as a pandemic, making it a serious concern and an extreme challenge to contain. The incubation time after contagion can range from two to 14 days. During this period, also known as the "pre-symptomatic" period, some infected people may be contagious from one to three days before the onset of symptoms. In this context, the respiratory system is not only the main organ to be attacked, but also the main organ responsible for its transmissibility.

The past, present and future of RNA respiratory viruses: influenza and coronaviruses.

Influenza virus and coronaviruses continue to cause pandemics across the globe. We now have a greater understanding of their functions. Unfortunately, the number of drugs in our armory to defend us against them is inadequate. This may require us to think about what mechanisms to address. Here, we review the biological properties of these viruses, their genetic evolution and antiviral therapies that can be used or have been attempted. We will describe several classes of drugs such as serine protease inhibitors, heparin, heparan sulfate receptor inhibitors, chelating agents, immunomodulators and many others. We also briefly describe some of the drug repurposing efforts that have taken place in an effort to rapidly identify molecules to treat patients with COVID-19. While we put a heavy emphasis on the past and present efforts, we also provide some thoughts about what we need to do to prepare for respiratory viral threats in the future.

Quantification of Coronaviruses by Titration In Vitro and Ex Vivo.

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).

Determining How Coronaviruses Overcome the Interferon and Innate Immune Response.

All viruses have to overcome the innate immune response in order to establish infection. Methods have been developed to assay if, and how, viruses overcome these responses, and many can be directly applied to coronaviruses. Here, in vitro methods to determine how coronaviruses overcome this response are described.

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

[Role of cyclophilin A during coronavirus replication and the antiviral activities of its inhibitors].

Cyclophilin A (CypA) is a widely distributed and highly conserved protein in organisms. It has peptidyl-prolyl cis/trans isomerase activity and is a receptor for cyclosporin A (CsA). Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. Seven types of coronaviruses are currently known to infect humans, among which SARS-CoV, MERS-CoV, and SARS-CoV-2 are fatal for humans. It is well established that CypA is essential for the replication of various coronaviruses such as SARS-CoV, CoV-229E, CoV-NL63, and FCoV. Additionally, CsA and its derivatives (ALV, NIM811, etc.) have obvious inhibitory effects on a variety of coronaviruses. These results suggest that CypA is a potential antiviral target and the existing drug CsA might be used as an anti-coronavirus drug. At the end of 2019, SARS-CoV-2 raged in China, which seriously theatern human health and causes huge economic lases. In view of this, we describe the effects of CypA on the replication of coronaviruses and the antiviral activities of its inhibitors, which will provide the scientific basis and ideas for the development of antiviral drugs for SARS-CoV-2.

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.

Mouse Ifit1b is a cap1-RNA-binding protein that inhibits mouse coronavirus translation and is regulated by complexing with Ifit1c.

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.