Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

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.

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2.

BACKGROUND: Antibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoVs exposure and SARS-CoV-2 correlates of protection are not clearly identified. METHODS: We performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic sera from four different groups: pediatrics and adolescents, individuals 21 to 70 years of age, older than 70 years of age, and individuals living with HCV or HIV. Data was then further analysed using machine learning to identify predictive patterns of neutralization based on sCoVs serology. FINDINGS: Antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) with median inhibition ranging from 17.6 % to 23.3 % in serum that interferes with SARS-CoV-2 spike attachment to ACE2 independently of age group. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. INTERPRETATION: Our data support the concept that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, which may potentially impact COVID-19 disease severity through other latent variables. FUNDING: This study was supported by a grant by the CIHR (VR2 -172722) and by a grant supplement by the CITF, and by a NRC Collaborative R&D Initiative Grant (PR031-1).

Immune dysregulation and immunopathology induced by SARS-CoV-2 and related coronaviruses - are we our own worst enemy?

Human coronaviruses cause a wide spectrum of disease, ranging from mild common colds to acute respiratory distress syndrome and death. Three highly pathogenic human coronaviruses - severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and SARS-CoV-2 - have illustrated the epidemic and pandemic potential of human coronaviruses, and a better understanding of their disease-causing mechanisms is urgently needed for the rational design of therapeutics. Analyses of patients have revealed marked dysregulation of the immune system in severe cases of human coronavirus infection, and there is ample evidence that aberrant immune responses to human coronaviruses are typified by impaired induction of interferons, exuberant inflammatory responses and delayed adaptive immune responses. In addition, various viral proteins have been shown to impair interferon induction and signalling and to induce inflammasome activation. This suggests that severe disease associated with human coronaviruses is mediated by both dysregulated host immune responses and active viral interference. Here we discuss our current understanding of the mechanisms involved in each of these scenarios.

Comparative genomic analysis reveals varying levels of mammalian adaptation to coronavirus infections.

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.

Methods to Measure Antibody Neutralization of Live Human Coronavirus OC43.

The human Betacoronavirus OC43 is a common cause of respiratory viral infections in adults and children. Lung infections with OC43 are associated with mortality, especially in hematopoietic stem cell transplant recipients. Neutralizing antibodies play a major role in protection against many respiratory viral infections, but to date a live viral neutralization assay for OC43 has not been described. We isolated a human monoclonal antibody (OC2) that binds to the spike protein of OC43 and neutralizes the live virus derived from the original isolate of OC43. We used this monoclonal antibody to develop and test the performance of two readily accessible in vitro assays for measuring antibody neutralization, one utilizing cytopathic effect and another utilizing an ELISA of infected cells. We used both methods to measure the neutralizing activity of the OC2 monoclonal antibody and of human plasma. These assays could prove useful for studying humoral responses to OC43 and cross-neutralization with other medically important betacoronaviruses.

Assessment of the Feasibility of Using Noninvasive Wearable Biometric Monitoring Sensors to Detect Influenza and the Common Cold Before Symptom Onset.

Importance: Currently, there are no presymptomatic screening methods to identify individuals infected with a respiratory virus to prevent disease spread and to predict their trajectory for resource allocation. Objective: To evaluate the feasibility of using noninvasive, wrist-worn wearable biometric monitoring sensors to detect presymptomatic viral infection after exposure and predict infection severity in patients exposed to H1N1 influenza or human rhinovirus. Design, Setting, and Participants: The cohort H1N1 viral challenge study was conducted during 2018; data were collected from September 11, 2017, to May 4, 2018. The cohort rhinovirus challenge study was conducted during 2015; data were collected from September 14 to 21, 2015. A total of 39 adult participants were recruited for the H1N1 challenge study, and 24 adult participants were recruited for the rhinovirus challenge study. Exclusion criteria for both challenges included chronic respiratory illness and high levels of serum antibodies. Participants in the H1N1 challenge study were isolated in a clinic for a minimum of 8 days after inoculation. The rhinovirus challenge took place on a college campus, and participants were not isolated. Exposures: Participants in the H1N1 challenge study were inoculated via intranasal drops of diluted influenza A/California/03/09 (H1N1) virus with a mean count of 106 using the median tissue culture infectious dose (TCID50) assay. Participants in the rhinovirus challenge study were inoculated via intranasal drops of diluted human rhinovirus strain type 16 with a count of 100 using the TCID50 assay. Main Outcomes and Measures: The primary outcome measures included cross-validated performance metrics of random forest models to screen for presymptomatic infection and predict infection severity, including accuracy, precision, sensitivity, specificity, F1 score, and area under the receiver operating characteristic curve (AUC). Results: A total of 31 participants with H1N1 (24 men [77.4%]; mean [SD] age, 34.7 [12.3] years) and 18 participants with rhinovirus (11 men [61.1%]; mean [SD] age, 21.7 [3.1] years) were included in the analysis after data preprocessing. Separate H1N1 and rhinovirus detection models, using only data on wearble devices as input, were able to distinguish between infection and noninfection with accuracies of up to 92% for H1N1 (90% precision, 90% sensitivity, 93% specificity, and 90% F1 score, 0.85 [95% CI, 0.70-1.00] AUC) and 88% for rhinovirus (100% precision, 78% sensitivity, 100% specificity, 88% F1 score, and 0.96 [95% CI, 0.85-1.00] AUC). The infection severity prediction model was able to distinguish between mild and moderate infection 24 hours prior to symptom onset with an accuracy of 90% for H1N1 (88% precision, 88% sensitivity, 92% specificity, 88% F1 score, and 0.88 [95% CI, 0.72-1.00] AUC) and 89% for rhinovirus (100% precision, 75% sensitivity, 100% specificity, 86% F1 score, and 0.95 [95% CI, 0.79-1.00] AUC). Conclusions and Relevance: This cohort study suggests that the use of a noninvasive, wrist-worn wearable device to predict an individual's response to viral exposure prior to symptoms is feasible. Harnessing this technology would support early interventions to limit presymptomatic spread of viral respiratory infections, which is timely in the era of COVID-19.

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.

Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

Carrageenan nasal spray may double the rate of recovery from coronavirus and influenza virus infections: Re-analysis of randomized trial data.

In this individual patient data meta-analysis we examined datasets of two randomized placebo-controlled trials which investigated the effect of nasal carrageenan separately on children and adults. In both trials, iota-carrageenan was administered nasally three times per day for 7 days for patients with the common cold and follow-up lasted for 21 days. We used Cox regression to estimate the effect of carrageenan on recovery rate. We also used quantile regression to calculate the effect of carrageenan on colds of differing lengths. Nasal carrageenan increased the recovery rate from all colds by 54% (95% CI 15%-105%; p = .003). The increase in recovery rate was 139% for coronavirus infections, 119% for influenza A infections, and 70% for rhinovirus infections. The mean duration of all colds in the placebo groups of the first four quintiles were 4.0, 6.8, 8.8, and 13.7 days, respectively. The fifth quintile contained patients with censored data. The 13.7-day colds were shortened by 3.8 days (28% reduction), and 8.8-day colds by 1.3 days (15% reduction). Carrageenan had no meaningful effect on shorter colds. In the placebo group, 21 patients had colds lasting over 20 days, compared with six patients in the carrageenan group, which corresponds to a 71% (p = .003) reduction in the risk of longer colds. Given that carrageenan has an effect on diverse virus groups, and effects at the clinical level on two old coronaviruses, it seems plausible that carrageenan may have an effect on COVID-19. Further research on nasal iota-carrageenan is warranted.

A severe case of human coronavirus 229E pneumonia in an elderly man with diabetes mellitus: a case report.

BACKGROUND: With pandemic of coronavirus disease 2019 (COVID-19), human coronaviruses (HCoVs) have recently attached worldwide attention as essential pathogens in respiratory infection. HCoV-229E has been described as a rare cause of lower respiratory infection in immunocompetent adults. CASE PRESENTATION: We reported a 72-year-old man infected by HCoV-229E with rapid progression to acute respiratory distress syndrome, in conjunction with new onset atrial fibrillation, intensive care unit acquired weakness, and recurrent hospital acquired pneumonia. Clinical and radiological data were continuously collected. The absolute number of peripheral T cells and the level of complement components diminished initially and recovered after 2 months. The patient was successfully treated under intensive support care and discharged from the hospital after 3 months and followed. CONCLUSION: HCoV-229E might an essential causative agent of pulmonary inflammation and extensive lung damage. Supportive treatment was essential to HCoVs infection on account of a long duration of immunological recovery in critical HCoV-229E infection.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

More than just a common cold: Endemic coronaviruses OC43, HKU1, NL63, and 229E associated with severe acute respiratory infection and fatality cases among healthy adults.

Respiratory viral infection can cause severe disease and hospitalization, especially among children, the elderly, and patients with comorbidities. In Brazil, the official surveillance system of severe acute respiratory infection (SARI) investigates influenza A (IAV) and B (IBV) viruses, respiratory syncytial virus (RSV), adenovirus (HAdV), and parainfluenza viruses (hPIV 1-3). In Rio Grande do Sul (RS), Brazil, many fatalities associated with SARI between 2013 and 2017 occurred among patients without underlying diseases and for whom the causative agent had not been identified using official protocols. This cross-sectional study analyzed the presence of coronaviruses (HCoV), bocavirus (HBoV), metapneumovirus (hMPV), and rhinovirus in patients who died of SARI despite not having comorbidities, and that were negative for IAV, IBV, RSV, HAdV, and hPIV. Nasopharyngeal aspirates/swabs from patients were used for nucleic acid extraction. The presence of HCoVs OC43, HKU1, NL63, and 229E; HBoV; hMPV; and rhinovirus was assessed by quantitative reverse transcription-polymerase chain reaction. Clinical data were also analyzed. Between 2013 and 2017, 16 225 cases of SARI were reported in RS; 9.8% of the patients died; 20% of all fatal cases were patients without comorbidities and for whom no pathogen was detected using standard protocols. Analysis of 271 of these cases identified HCoV in nine cases; HBoV, hMPV, and rhinovirus were detected in 3, 3, and 10 cases, respectively. Of note, patients infected with HCoV were adults. Results reinforce the importance of including coronaviruses in diagnostic panels used by official surveillance systems because besides their pandemic potential, endemic HCoVs are associated to severe disease in healthy adults.

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.