Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

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.

Effect of surgical mask on fMRI signals during task and rest.

Wearing a face mask has become essential to contain the spread of COVID-19 and has become mandatory when collecting fMRI data at most research institutions. Here, we investigate the effects of wearing a surgical mask on fMRI data in n = 37 healthy participants. Activations during finger tapping, emotional face matching, working memory tasks, and rest were examined. Preliminary fMRI analyses show that despite the different mask states, resting-state signals and task activations were relatively similar. Resting-state functional connectivity showed negligible attenuation patterns in mask-on compared with mask-off. Task-based ROI analysis also demonstrated no significant difference between the two mask states under each contrast investigated. Notwithstanding the overall insignificant effects, these results indicate that wearing a face mask during fMRI has little to no significant effect on resting-state and task activations.

Inhibition of SARS-CoV-2 replication by zinc gluconate in combination with hinokitiol.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is currently the major challenge to global public health. Two proteases, papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro or Mpro), are indispensable for SARS-CoV-2 replication, making them attractive targets for antiviral therapy development. Here we screened a panel of essential metal ions using a proteolytic assay and identified that zinc gluconate, a widely-used zinc supplement, strongly inhibited the proteolytic activities of the two proteases in vitro. Biochemical and crystallographic data reveal that zinc gluconate exhibited the inhibitory function via binding to the protease catalytic site residues. We further show that treatment of zinc gluconate in combination with a small molecule ionophore hinokitiol, could lead to elevated intracellular Zn2+ level and thereby significantly impaired the two protease activities in cellulo. Particularly, this approach could also be applied to rescue SARS-CoV-2 infected mammalian cells, indicative of potential application to combat coronavirus infections. Our studies provide the direct experimental evidence that elevated intracellular zinc concentration directly inhibits SARS-CoV-2 replication and suggest the potential benefits to use the zinc supplements for coronavirus disease 2019 (COVID-19) treatment.

Network pharmacology-based predictions of active components and pharmacological mechanisms of Artemisia annua L. for the treatment of the novel Corona virus disease 2019 (COVID-19).

BACKGROUND: Novel Corona Virus Disease 2019 (COVID-19) is closely associated with cytokines storms. The Chinese medicinal herb Artemisia annua L. (A. annua) has been traditionally used to control many inflammatory diseases, such as malaria and rheumatoid arthritis. We performed network analysis and employed molecular docking and network analysis to elucidate active components or targets and the underlying mechanisms of A. annua for the treatment of COVID-19. METHODS: Active components of A. annua were identified through the TCMSP database according to their oral bioavailability (OB) and drug-likeness (DL). Moreover, target genes associated with COVID-19 were mined from GeneCards, OMIM, and TTD. A compound-target (C-T) network was constructed to predict the relationship of active components with the targets. A Compound-disease-target (C-D-T) network has been built to reveal the direct therapeutic target for COVID-19. Molecular docking, molecular dynamics simulation studies (MD), and MM-GBSA binding free energy calculations were used to the closest molecules and targets between A. annua and COVID-19. RESULTS: In our network, GO, and KEGG analysis indicated that A. annua acted in response to COVID-19 by regulating inflammatory response, proliferation, differentiation, and apoptosis. The molecular docking results manifested excellent results to verify the binding capacity between the hub components and hub targets in COVID-19. MD and MM-GBSA data showed quercetin to be the more effective candidate against the virus by target MAPK1, and kaempferol to be the other more effective candidate against the virus by target TP53. We identified A. annua's potentially active compounds and targets associated with them that act against COVID-19. CONCLUSIONS: These findings suggest that A. annua may prevent and inhibit the inflammatory processes related to COVID-19.

Liushen Capsules, a promising clinical candidate for COVID-19, alleviates SARS-CoV-2-induced pulmonary in vivo and inhibits the proliferation of the variant virus strains in vitro.

BACKGROUND: Coronavirus disease 2019 (COVID-19) causes a global pandemic and has devastating effects around the world, however, there are no specific antiviral drugs and vaccines for the constant mutation of SARS-CoV-2. PURPOSE: In this study, we evaluted the antiviral and anti-inflammatory activities of Liushen Capsules (LS) on different novel coronavirus in vitro, studied its therapeutic effects on novel SARS-CoV-2 infected mice and observed the LS's clinical efficacy and safety in COVID-19. METHODS: The antiviral and aiti-inflammatory effects of LS on the 501Y.V2/B.1.35 and G/478K.V1/ B.1.617.2 strains were determined in vitro. A hACE2 mouse model of novel SARS-CoV-2 pneumonia was established. Survival rates, histological changes, inflammatory markers, lung virus titers and the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blotting and immumohistochemical staining in the lungs were measured. Subsequently, the disease duration, prognosis of disease, time of negative nucleic acid and the cytokines levels in serum were used to assess the efficacy of treatment with LS in patients. RESULTS: The results showed that LS (2, 1, 0.5 µg/mL) could significantly inhibit the replication of the two SARS-CoV-2 variants and the expression of pro-inflammatory cytokines (IL-6, IL-8, IP-10, CCL-5, MIP-1α, IL-1α) induced by the virus in vitro. As for the survival experiment in mice, the survival rate of virus group was 20%, while LS-treatment groups (40, 80, 160 mg/kg) could increase the survival rate to 60, 100 and 100%, respectively. LS (40, 80, 160 mg/kg) could significantly decrease the lung titers in mice and it could improve the pathological changes, inhibit the excessive inflammatory mediators (IFN-α, IFN-γ, IP-10, MCP-1) and the protein expression of p-NF-κB p65 in mice. Moreover, LS could significantly decrease SARS-CoV-2-induced activation of p-NF-κB p65, p-IκBα, and p-p38 MAPK and increase the protein expression of the IκBα. In addition, the patient got complete relief of symptoms after being treated with LS for 6 days and was proven with negative PCR test after being treated for 23 days. Finally, treatment with LS could reduce the release of inflammatory cytokines (IL-6, PDGF-AA/BB, Eotaxin, MCP-1, MIP-1α, MIP-1ß, GRO, CCL-5, MCP-3, IP-10, IL-1α). CONCLUSION: LS effectively alleviated novel SARS-CoV-2 or variants induced pneumonia in vitro and in vivo, and improved the prognosis of COVID-19. In light of the efficacy and safety profiles, LS could be considered for the treatment of COVID-19 with a broad-spectrum antiviral and anti-inflammatory agent.

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed. METHODS: A rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests. RESULTS: The new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05). CONCLUSIONS: We had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.

Two novel human coronavirus OC43 genotypes circulating in hospitalized children with pneumonia in China.

HCoV-OC43 is one of the mildly pathogenic coronaviruses with high infection rates in common population. Here, 43 HCoV-OC43 related cases with pneumonia were reported, corresponding genomes of HCoV-OC43 were obtained. Phylogenetic analyses based on complete genome, orf1ab and spike genes revealed that two novel genotypes of HCoV-OC43 have emerged in China. Obvious recombinant events also can be detected in the analysis of the evolutionary dynamics of novel HCoV-OC43 genotypes. Estimated divergence time analysis indicated that the two novel genotypes had apparently independent evolutionary routes. Efforts should be conducted for further investigation of genomic diversity and evolution analysis of mildly pathogenic coronaviruses.

Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo.

The current COVID-19 pandemic, caused by SARS-CoV-2, poses a serious public health threat. Effective therapeutic and prophylactic treatments are urgently needed. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. Here, we developed recombinant human ACE2-Fc fusion protein (hACE2-Fc) and a hACE2-Fc mutant with reduced catalytic activity. hACE2-Fc and the hACE2-Fc mutant both efficiently blocked entry of SARS-CoV-2, SARS-CoV, and HCoV-NL63 into hACE2-expressing cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion. hACE2-Fc also neutralized various SARS-CoV-2 strains with enhanced infectivity including D614G and V367F mutations, as well as the emerging SARS-CoV-2 variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.1 (Kappa), and B.1.617.2 (Delta), demonstrating its potent and broad-spectrum antiviral effects. In addition, hACE2-Fc proteins protected HBE from SARS-CoV-2 infection. Unlike RBD-targeting neutralizing antibodies, hACE2-Fc treatment did not induce the development of escape mutants. Furthermore, both prophylactic and therapeutic hACE2-Fc treatments effectively protected mice from SARS-CoV-2 infection, as determined by reduced viral replication, weight loss, histological changes, and inflammation in the lungs. The protection provided by hACE2 showed obvious dose-dependent efficacy in vivo. Pharmacokinetic data indicated that hACE2-Fc has a relative long half-life in vivo compared to soluble ACE2, which makes it an excellent candidate for prophylaxis and therapy for COVID-19 as well as for SARS-CoV and HCoV-NL63 infections.

Effect of Jinzhen granule on two coronaviruses: The novel SARS-CoV-2 and the HCoV-229E and the evidences for their mechanisms of action.

BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) pose a huge threat to human public health, no specific treatment is available. Jinzhen granule (JZ) is a traditional eight ingredients-Chinese medicine with prominent efficacy for treating viral-induced diseases. However, little is known about the antiviral effect and mechanism of JZ against SARS-CoV-2 and HCoV-229E. PURPOSE: This study aimed to reveal the antiviral effects of JZ against SARS-CoV-2 and HCoV-229E, and to further explore the underlying mechanisms regulating the host immune response. METHODS: The chromatographic separation of JZ was performed using a Shimadzu analytical high-performance liquid chromatograph with UV detection and Alltech ELSD 2000ES. We conducted cytopathic effect (CPE) and plaque reduction assays to evaluate the antiviral effect of JZ. A lethal human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 was established to determine the protective effect of JZ on mortality and lung virus titers. Real-time quantitative PCR assays were used to analyze the expression of proinflammatory cytokines in vitro and in vivo. Western blotting was further performed to determine the activities on regulating the nuclear factor kappa B (NF-κB)/MAPK pathway. Finally, mitochondrial membrane potential assays, flow cytometry analysis and western blotting were used to assess the anti-apoptotic potency toward HCoV-229E infection. RESULTS: The results showed that 13 chemical components were identified and five peaks were determined and quantitated (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g). We found that JZ exerted inhibitory potency against SARS-CoV-2 and HCoV-229E in vitro by using CPE and plaque reduction assays, and it was further found that JZ protected mice infected by SARS-CoV-2 from death and inhibited lung virus titers. JZ also significantly decreased the induction of inflammatory cytokines (IL-1α, IL-6, CCL-5 and MIP-1ß), similar to the observed in vitro effect. Moreover, JZ suppressed the release of inflammatory cytokines in vitro and it decreased the protein expression of p-p38 MAPK, p-JNK, p-NF-κB p65 and p-IκBα induced by HCoV-229E and increased the expression of IκBα. Notably, JZ significantly protected HCoV-229E-infected Huh-7 cells from mitochondrial damage and decreased apoptotic cells. The activation of the mitochondria-mediated apoptotic pathway was inhibited by JZ, as shown by the reduced expression of cleaved caspase-9, caspase-3 and p-PARP. CONCLUSIONS: In conclusion, JZ (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g) exhibited antiviral activities against SARS-CoV-2 and HCoV-229E by regulating the NF-κB/MAPK pathway and the mitochondria-mediated apoptotic pathway. These findings demonstrated the efficacy of JZ against CoVs and suggested JZ treatment as a novel clinical therapeutic strategy for COVID-19.

Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

Mapping and role of T cell response in SARS-CoV-2-infected mice.

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.

Increased Pathogenicity and Virulence of Middle East Respiratory Syndrome Coronavirus Clade B In Vitro and In Vivo

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Isolation of infectious SARS-CoV-2 from urine of a COVID-19 patient.

SARS-CoV-2 caused a major outbreak of severe pneumonia (COVID-19) in humans. Viral RNA was detected in multiple organs in COVID-19 patients. However, infectious SARS-CoV-2 was only isolated from respiratory specimens. Here, infectious SARS-CoV-2 was successfully isolated from urine of a COVID-19 patient. The virus isolated could infect new susceptible cells and was recognized by its' own patient sera. Appropriate precautions should be taken to avoid transmission from urine.