Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.

SARS Unique Domain (SUD) of Severe Acute Respiratory Syndrome Coronavirus Induces NLRP3 Inflammasome-Dependent CXCL10-Mediated Pulmonary Inflammation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) initiates the cytokine/chemokine storm-mediated lung injury. The SARS-CoV unique domain (SUD) with three macrodomains (N, M, and C), showing the G-quadruplex binding activity, was examined the possible role in SARS pathogenesis in this study. The chemokine profile analysis indicated that SARS-CoV SUD significantly up-regulated the expression of CXCL10, CCL5 and interleukin (IL)-1ß in human lung epithelial cells and in the lung tissues of the mice intratracheally instilled with the recombinant plasmids. Among the SUD subdomains, SUD-MC substantially activated AP-1-mediated CXCL10 expression in vitro. In the wild type mice, SARS-CoV SUD-MC triggered the pulmonary infiltration of macrophages and monocytes, inducing CXCL10-mediated inflammatory responses and severe diffuse alveolar damage symptoms. Moreover, SUD-MC actuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-dependent pulmonary inflammation, as confirmed by the NLRP3 inflammasome inhibitor and the NLRP3-/- mouse model. This study demonstrated that SARS-CoV SUD modulated NLRP3 inflammasome-dependent CXCL10-mediated pulmonary inflammation, providing the potential therapeutic targets for developing the antiviral agents.

Prefusion spike protein conformational changes are slower in SARS-CoV-2 than in SARS-CoV-1.

Within the last 2 decades, severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) have caused two major outbreaks; yet, for reasons not fully understood, the coronavirus disease 2019 pandemic caused by SARS-CoV-2 has been significantly more widespread than the 2003 SARS epidemic caused by SARS-CoV-1, despite striking similarities between these two viruses. The SARS-CoV-1 and SARS-CoV-2 spike proteins, both of which bind to host cell angiotensin-converting enzyme 2, have been implied to be a potential source of their differential transmissibility. However, the mechanistic details of prefusion spike protein binding to angiotensin-converting enzyme 2 remain elusive at the molecular level. Here, we performed an extensive set of equilibrium and nonequilibrium microsecond-level all-atom molecular dynamics simulations of SARS-CoV-1 and SARS-CoV-2 prefusion spike proteins to determine their differential dynamic behavior. Our results indicate that the active form of the SARS-CoV-2 spike protein is more stable than that of SARS-CoV-1 and the energy barrier associated with the activation is higher in SARS-CoV-2. These results suggest that not only the receptor-binding domain but also other domains such as the N-terminal domain could play a crucial role in the differential binding behavior of SARS-CoV-1 and SARS-CoV-2 spike proteins.

SARS-CoV-2 nsp5 Exhibits Stronger Catalytic Activity and Interferon Antagonism than Its SARS-CoV Ortholog.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an enormous threat to economic activity and public health worldwide. Previous studies have shown that the nonstructural protein 5 (nsp5, also called 3C-like protease) of alpha- and deltacoronaviruses cleaves Q231 of the NF-κB essential modulator (NEMO), a key kinase in the RIG-I-like receptor pathway, to inhibit type I interferon (IFN) production. In this study, we found that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at multiple sites (E152, Q205, and Q231). Notably, SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5. Sequence and structural alignments suggested that an S/A polymorphism at position 46 of nsp5 in SARS-CoV versus SARS-CoV-2 may be responsible for this difference. Mutagenesis experiments showed that SARS-CoV-2 nsp5 (S46A) exhibited poorer cleavage of NEMO than SARS-CoV-2 nsp5 wild type (WT), while SARS-CoV nsp5 (A46S) showed enhanced NEMO cleavage compared with the WT protein. Purified recombinant SARS-CoV-2 nsp5 WT and SARS-CoV nsp5 (A46S) proteins exhibited higher hydrolysis efficiencies than SARS-CoV-2 nsp5 (S46A) and SARS-CoV nsp5 WT proteins in vitro. Furthermore, SARS-CoV-2 nsp5 exhibited stronger inhibition of Sendai virus (SEV)-induced interferon beta (IFN-ß) production than SARS-CoV-2 nsp5 (S46A), while introduction of the A46S substitution in SARS-CoV nsp5 enhanced suppression of SEV-induced IFN-ß production. Taken together, these data show that S46 is associated with the catalytic activity and IFN antagonism by SARS-CoV-2 nsp5. IMPORTANCE The nsp5-encoded 3C-like protease is the main coronavirus protease, playing a vital role in viral replication and immune evasion by cleaving viral polyproteins and host immune-related molecules. We showed that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleave the NEMO at multiple sites (E152, Q205, and Q231). This specificity differs from NEMO cleavage by alpha- and deltacoronaviruses, demonstrating the distinct substrate recognition of SARS-CoV-2 and SARS-CoV nsp5. Compared with SARS-CoV nsp5, SARS-CoV-2 nsp5 encodes S instead of A at position 46. This substitution is associated with stronger catalytic activity, enhanced cleavage of NEMO, and increased interferon antagonism of SARS-CoV-2 nsp5. These data provide new insights into the pathogenesis and transmission of SARS-CoV-2.

Surviving SARS and living through COVID-19: Healthcare worker mental health outcomes and insights for coping.

OBJECTIVE: Explore how previous work during the 2003 Severe Acute Respiratory Syndrome (SARS) outbreak affects the psychological response of clinical and non-clinical healthcare workers (HCWs) to the current COVID-19 pandemic. METHODS: A cross-sectional, multi-centered hospital online survey of HCWs in the Greater Toronto Area, Canada. Mental health outcomes of HCWs who worked during the COVID-19 pandemic and the SARS outbreak were assessed using Impact of Events-Revised scale (IES-R), Generalized Anxiety Disorder scale (GAD-7), and Patient Health Questionnaire (PHQ-9). RESULTS: Among 3852 participants, moderate/severe scores for symptoms of post- traumatic stress disorder (PTSD) (50.2%), anxiety (24.6%), and depression (31.5%) were observed among HCWs. Work during the 2003 SARS outbreak was reported by 1116 respondents (29.1%), who had lower scores for symptoms of PTSD (P = .002), anxiety (P < .001), and depression (P < .001) compared to those who had not worked during the SARS outbreak. Multivariable logistic regression analysis showed non-clinical HCWs during this pandemic were at higher risk of anxiety (OR, 1.68; 95% CI, 1.19-2.15, P = .01) and depressive symptoms (OR, 2.03; 95% CI, 1.34-3.07, P < .001). HCWs using sedatives (OR, 2.55; 95% CI, 1.61-4.03, P < .001), those who cared for only 2-5 patients with COVID-19 (OR, 1.59; 95% CI, 1.06-2.38, P = .01), and those who had been in isolation for COVID-19 (OR, 1.36; 95% CI, 0.96-1.93, P = .05), were at higher risk of moderate/severe symptoms of PTSD. In addition, deterioration in sleep was associated with symptoms of PTSD (OR, 4.68, 95% CI, 3.74-6.30, P < .001), anxiety (OR, 3.09, 95% CI, 2.11-4.53, P < .001), and depression (OR 5.07, 95% CI, 3.48-7.39, P < .001). CONCLUSION: Psychological distress was observed in both clinical and non-clinical HCWs, with no impact from previous SARS work experience. As the pandemic continues, increasing psychological and team support may decrease the mental health impacts.

Interindividual immunogenic variants: Susceptibility to coronavirus, respiratory syncytial virus and influenza virus.

The coronavirus disease (Covid-19) pandemic is the most serious event of the year 2020, causing considerable global morbidity and mortality. The goal of this review is to provide a comprehensive summary of reported associations between inter-individual immunogenic variants and disease susceptibility or symptoms caused by the coronavirus strains severe acute respiratory syndrome-associated coronavirus, severe acute respiratory syndrome-associated coronavirus-2, and two of the main respiratory viruses, respiratory syncytial virus and influenza virus. The results suggest that the genetic background of the host could affect the levels of proinflammatory and anti-inflammatory cytokines and might modulate the progression of Covid-19 in affected patients. Notably, genetic variations in innate immune components such as toll-like receptors and mannose-binding lectin 2 play critical roles in the ability of the immune system to recognize coronavirus and initiate an early immune response to clear the virus and prevent the development of severe symptoms. This review provides promising clues related to the potential benefits of using immunotherapy and immune modulation for respiratory infectious disease treatment in a personalized manner.

No evidence of SARS-CoV-2 in hospitalized patients with severe acute respiratory syndrome in five Italian hospitals from 1st November 2019 to 29th February 2020.

BACKGROUND: On 9th January 2020, China CDC reported a novel coronavirus (later named SARS-CoV-2) as the causative agent of the coronavirus disease 2019 (COVID-19). Identifying the first appearance of virus is of epidemiological importance to tracking and mapping the spread of SARS-CoV-2 in a country. We therefore conducted a retrospective observational study to detect SARS-CoV-2 in oropharyngeal samples collected from hospitalized patients with a Severe Acute Respiratory Infection (SARI) enrolled in the DRIVE (Development of Robust and Innovative Vaccine Effectiveness) study in five Italian hospitals (CIRI-IT BIVE hospitals network) (1st November 2019 - 29th February 2020). OBJECTIVES: To acquire new information on the real trend in SARS-CoV-2 infection during pandemic phase I and to determine the possible early appearance of the virus in Italy. MATERIALS AND METHODS: Samples were tested for influenza [RT-PCR assay (A/H1N1, A/H3N2, B/Yam, B/Vic)] in accordance with the DRIVE study protocol. Subsequently, swabs underwent molecular testing for SARS-COV-2. [one-step real-time multiplex retro-transcription (RT) PCR]. RESULTS: In the 1683 samples collected, no evidence of SARS-CoV-2 was found. Moreover, 28.3% (477/1683) of swabs were positive for influenza viruses, the majority being type A (358 vs 119 type B). A/H3N2 was predominant among influenza A viruses (55%); among influenza B viruses, B/Victoria was prevalent. The highest influenza incidence rate was reported in patients aged 0-17 years (40.3%) followed by those aged 18-64 years (24.4%) and ≥65 years (14.8%). CONCLUSIONS: In Italy, some studies have shown the early circulation of SARS-CoV-2 in northern regions, those most severely affected during phase I of the pandemic. In central and southern regions, by contrast no early circulation of the virus was registered. These results are in line with ours. These findings highlight the need to continue to carry out retrospective studies, in order to understand the epidemiology of the novel coronavirus, to better identify the clinical characteristics of COVID-19 in comparison with other acute respiratory illnesses (ARI), and to evaluate the real burden of COVID-19 on the healthcare system.

COVID-19 associated symmetrical peripheral gangrene: A case series.

BACKGROUND AND AIMS: The novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has turned the world topsy-turvy since its onset in 2019. The thromboinflammatory complications of this disease are common in critically ill patients and associated with poor prognosis. Symmetrical peripheral gangrene (SPG) is characterized by symmetrical distal gangrene in absence of any large vessel occlusion or vasculitis and it is usually associated with critical illness. Our aim was to report the clinical profile and outcome of patients diagnosed with SPG associated with COVID-19. To the best of our knowledge, no such similar cases have been reported till date. METHODS: In this case series, we have discussed the clinical presentation, laboratory parameters and outcome in a series of two patients of SPG associated with COVID-19 and also compared those findings. Due to paucity of data, we also reviewed the literature on this under-diagnosed and rarely reported condition and association. RESULTS: Two consecutive patients (both males, age range: 37-42 years, mean: 39.5 years) were admitted with the diagnosis of COVID-19 associated SPG. Both patients had clinical and laboratory evidence of disseminated intravascular coagulation (DIC). Leucopenia was noted in both patients. Despite vigorous therapy, both patients succumbed to their illness within a fortnight of admission. CONCLUSION: SPG in the background of COVID-19 portends a fatal outcome. Physicians should be aware of its grim prognosis.

Impact of COVID-19 on Pregnancy.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is an emerging disease. There has been a rapid increase in cases and deaths since it was identified in Wuhan, China, in early December 2019, with over 4,000,000 cases of COVID-19 including at least 250,000 deaths worldwide as of May 2020. However, limited data about the clinical characteristics of pregnant women with COVID-19 have been reported. Given the maternal physiologic and immune function changes during pregnancy, pregnant women may be at a higher risk of being infected with SARS-CoV-2 and developing more complicated clinical events. Information on severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) may provide insights into the effects of COVID-19's during pregnancy. Even though SARS and MERS have been associated with miscarriage, intrauterine death, fetal growth restriction and high case fatality rates, the clinical course of COVID-19 pneumonia in pregnant women has been reported to be similar to that in non-pregnant women. In addition, pregnant women do not appear to be at a higher risk of catching COVID-19 or suffering from more severe disease than other adults of similar age. Moreover, there is currently no evidence that the virus can be transmitted to the fetus during pregnancy or during childbirth. Babies and young children are also known to only experience mild forms of COVID-19. The aims of this systematic review were to summarize the possible symptoms, treatments, and pregnancy outcomes of women infected with COVID-19 during pregnancy.

Proximity-dependent biotinylation detects associations between SARS coronavirus nonstructural protein 1 and stress granule-associated proteins.

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

Interspecies Jumping of Bat Coronaviruses.

In the last two decades, several coronavirus (CoV) interspecies jumping events have occurred between bats and other animals/humans, leading to major epidemics/pandemics and high fatalities. The SARS epidemic in 2002/2003 had a ~10% fatality. The discovery of SARS-related CoVs in horseshoe bats and civets and genomic studies have confirmed bat-to-civet-to-human transmission. The MERS epidemic that emerged in 2012 had a ~35% mortality, with dromedaries as the reservoir. Although CoVs with the same genome organization (e.g., Tylonycteris BatCoV HKU4 and Pipistrellus BatCoV HKU5) were also detected in bats, there is still a phylogenetic gap between these bat CoVs and MERS-CoV. In 2016, 10 years after the discovery of Rhinolophus BatCoV HKU2 in Chinese horseshoe bats, fatal swine disease outbreaks caused by this virus were reported in southern China. In late 2019, an outbreak of pneumonia emerged in Wuhan, China, and rapidly spread globally, leading to >4,000,000 fatalities so far. Although the genome of SARS-CoV-2 is highly similar to that of SARS-CoV, patient zero and the original source of the pandemic are still unknown. To protect humans from future public health threats, measures should be taken to monitor and reduce the chance of interspecies jumping events, either occurring naturally or through recombineering experiments.

Quantitative evaluation of aerosol generation during manual facemask ventilation.

Manual facemask ventilation, a core component of elective and emergency airway management, is classified as an aerosol-generating procedure. This designation is based on one epidemiological study suggesting an association between facemask ventilation and transmission during the SARS-CoV-1 outbreak in 2003. There is no direct evidence to indicate whether facemask ventilation is a high-risk procedure for aerosol generation. We conducted aerosol monitoring during routine facemask ventilation and facemask ventilation with an intentionally generated leak in anaesthetised patients. Recordings were made in ultraclean operating theatres and compared against the aerosol generated by tidal breathing and cough manoeuvres. Respiratory aerosol from tidal breathing in 11 patients was reliably detected above the very low background particle concentrations with median [IQR (range)] particle counts of 191 (77-486 [4-1313]) and 2 (1-5 [0-13]) particles.l-1 , respectively, p = 0.002. The median (IQR [range]) aerosol concentration detected during facemask ventilation without a leak (3 (0-9 [0-43]) particles.l-1 ) and with an intentional leak (11 (7-26 [1-62]) particles.l-1 ) was 64-fold (p = 0.001) and 17-fold (p = 0.002) lower than that of tidal breathing, respectively. Median (IQR [range]) peak particle concentration during facemask ventilation both without a leak (60 (0-60 [0-120]) particles.l-1 ) and with a leak (120 (60-180 [60-480]) particles.l-1 ) were 20-fold (p = 0.002) and 10-fold (0.001) lower than a cough (1260 (800-3242 [100-3682]) particles.l-1 ), respectively. This study demonstrates that facemask ventilation, even when performed with an intentional leak, does not generate high levels of bioaerosol. On the basis of this evidence, we argue facemask ventilation should not be considered an aerosol-generating procedure.

In vitro virucidal activity of Echinaforce®, an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2.

BACKGROUND: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro. METHODS: To evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium. RESULTS: In the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 µg/ml IC50. Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 µg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50µg/ml Echinaforce®. CONCLUSIONS: These results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.