Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics.

The emergence of the COVID-19 pandemic prompted an increased interest in seasonal human coronaviruses. OC43, 229E, NL63, and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8, and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses; however, the abundance and the proportion of vRNA copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63 and OC43 leads to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans.IMPORTANCESeasonal human coronavirus is an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up- or down-modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Airborne Influenza Virus in Daycare Centers.

In this study, we investigated the concentration of airborne influenza virus in daycare centers and influencing factors, such as common cold prevalence, air pollutants, and meteorological factors. A total of 209 air samples were collected from daycare centers in Kaohsiung and the influenza virus was analyzed using real-time quantitative polymerase chain reaction. Air pollutants and metrological factors were measured using real-time monitoring equipment. Winter had the highest positive rates of airborne influenza virus and the highest prevalence of the common cold, followed by summer and autumn. The concentration of CO was significantly positively correlated with airborne influenza virus. Daycare center A, with natural ventilation and air condition systems, had a higher concentration of airborne influenza A virus, airborne fungi, and airborne bacteria, as well as a higher prevalence of the common cold, than daycare center B, with a mechanical ventilation system and air purifiers, while the concentrations of CO2, CO, and UFPs in daycare center A were lower than those in daycare center B. We successfully detected airborne influenza virus in daycare centers, demonstrating that aerosol sampling for influenza can provide novel epidemiological insights and inform the management of influenza in daycare centers.

Phytochemicals: Promising Inhibitors of Human Rhinovirus Type 14 3C Protease as a Strategy to Fight the Common Cold.

BACKGROUND: Human rhinovirus 3C protease (HRV-3Cpro) plays a crucial role in viral proliferation, establishing it as a prime target for antiviral therapy. However, research on identifying HRV-3Cpro inhibitors is still limited. OBJECTIVE: This study had two primary objectives: first, to validate the efficacy of an end-point colorimetric assay, previously developed by our team, for identifying potential inhibitors of HRV-3Cpro; and second, to discover phytochemicals in medicinal plants that inhibit the enzyme's activity. METHODS: Rupintrivir, a well-known inhibitor of HRV-3Cpro, was used to validate the colorimetric assay. Following this, we conducted a two-step in silico screening of 2532 phytochemicals, which led to the identification of eight active compounds: apigenin, carnosol, chlorogenic acid, kaempferol, luteolin, quercetin, rosmarinic acid, and rutin. We subsequently evaluated these candidates in vitro. To further investigate the inhibitory potential of the most promising candidates, namely, carnosol and rosmarinic acid, molecular docking studies were performed to analyze their binding interactions with HRV-3Cpro. RESULTS: The colorimetric assay we previously developed is effective in identifying compounds that selectively inhibit HRV-3Cpro. Carnosol and rosmarinic acid emerged as potent inhibitors, inhibiting HRV-3Cpro activity in vitro by over 55%. Our analysis indicated that carnosol and rosmarinic acid exert their inhibitory effects through a competitive mechanism. Molecular docking confirmed their competitive binding to the enzyme's active site. CONCLUSION: Carnosol and rosmarinic acid warrant additional investigation for their potential in the development of common cold treatment. By highlighting these compounds as effective HRV-3Cpro inhibitors, our study presents a promising approach for discovering phytochemical inhibitors against proteases from similar pathogens.

Common cold viruses circulating in children threaten wild chimpanzees through asymptomatic adult carriers.

Reverse zoonotic respiratory diseases threaten great apes across Sub-Saharan Africa. Studies of wild chimpanzees have identified the causative agents of most respiratory disease outbreaks as "common cold" paediatric human pathogens, but reverse zoonotic transmission pathways have remained unclear. Between May 2019 and August 2021, we conducted a prospective cohort study of 234 children aged 3-11 years in communities bordering Kibale National Park, Uganda, and 30 adults who were forest workers and regularly entered the park. We collected 2047 respiratory symptoms surveys to quantify clinical severity and simultaneously collected 1989 nasopharyngeal swabs approximately monthly for multiplex viral diagnostics. Throughout the course of the study, we also collected 445 faecal samples from 55 wild chimpanzees living nearby in Kibale in social groups that have experienced repeated, and sometimes lethal, epidemics of human-origin respiratory viral disease. We characterized respiratory pathogens in each cohort and examined statistical associations between PCR positivity for detected pathogens and potential risk factors. Children exhibited high incidence rates of respiratory infections, whereas incidence rates in adults were far lower. COVID-19 lockdown in 2020-2021 significantly decreased respiratory disease incidence in both people and chimpanzees. Human respiratory infections peaked in June and September, corresponding to when children returned to school. Rhinovirus, which caused a 2013 outbreak that killed 10% of chimpanzees in a Kibale community, was the most prevalent human pathogen throughout the study and the only pathogen present at each monthly sampling, even during COVID-19 lockdown. Rhinovirus was also most likely to be carried asymptomatically by adults. Although we did not detect human respiratory pathogens in the chimpanzees during the cohort study, we detected human metapneumovirus in two chimpanzees from a February 2023 outbreak that were genetically similar to viruses detected in study participants in 2019. Our data suggest that respiratory pathogens circulate in children and that adults become asymptomatically infected during high-transmission times of year. These asymptomatic adults may then unknowingly carry the pathogens into forest and infect chimpanzees. This conclusion, in turn, implies that intervention strategies based on respiratory symptoms in adults are unlikely to be effective for reducing reverse zoonotic transmission of respiratory viruses to chimpanzees.

Characteristics of upper respiratory tract rhinovirus in children with allergic rhinitis and its role in disease severity.

Allergic rhinitis (AR) is a global health challenge that particularly affects the quality of life of children. Human rhinovirus (HRV) infection usually causes common cold in the upper respiratory tract (URT) and can also affect airway allergy development, such as asthma exacerbation, but its relationship with AR is poorly understood. The study aimed to gain insight into the characteristics of HRV that is prevalent in AR children and its role in AR severity. A total of 362 children with symptomatic AR were enrolled from southwestern China during 2022-2023, and nasal lavage samples were collected for HRV molecular characterization and cytokine measurement. HRV was detected in 40% of the AR children, with peak detection in autumn. The positive rate was not correlated with whether the subjects were under allergen-specific immunotherapy (AIT). Among the detected HRVs, 42% were species A, 36% were species B, and 22% were species C, involving 21 A genotypes, 6 B genotypes, and 7 C genotypes. HRV positivity was significantly associated with symptom severity (visual analog scale [VAS] score) and elevated levels of local nasal IgE, interleukin-25 (IL-25), IL-4, and CXCL13 in AR children who did not receive antiallergic treatment. All three species of HRV strains (A1B, A21, B27, B70, and C17) had been isolated and were able to infect respiratory epithelial tissue in vitro. Complete genome sequencing showed that the antigenic epitopes of the isolated HRVs had certain variations. Our work reveals the etiological characteristics of URT-HRV in AR children and suggests a role of HRV infection in the pathogenesis of childhood AR. IMPORTANCE: Our study revealed high human rhinovirus (HRV) detection rate in children with allergic rhinitis (AR), and HRV infection (A, B, or C species) is positively associated with the symptom severity in AR children. Elevated nasal IgE, interleukin-25 (IL-25), IL-4, and CXCL13 levels suggest a potential pathogenic mechanism by which HRV infection induces nasal type 2 immune/inflammation responses and local IgE production in AR patients. In addition, etiological analysis found that the main prevalent HRV species in AR children are A and B (~80%), which is different from acute respiratory infection and asthma exacerbation, where species A and C are dominant. The data reveal the distinct species prevalence characteristics of HRV infection in AR. Finally, we isolated all three species of HRV strains from nasal cavity of AR children with varying degrees of antigenic epitope mutations and in vitro infectivity, highlighting the importance of strengthening monitoring and intervention for respiratory HRV infection in AR children.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Sialoglycan binding triggers spike opening in a human coronavirus.

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

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.

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.

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.

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.

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).

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.

Air Pollution Exposure Impairs Airway Epithelium IFN-ß Expression in Pre-School Children.

Introduction: Air pollution is a risk factor for respiratory infections and asthma exacerbations. We previously reported impaired Type-I and Type-III interferons (IFN-ß/λ) from airway epithelial cells of preschool children with asthma and/or atopy. In this study we analyzed the association between rhinovirus-induced IFN-ß/λ epithelial expression and acute exposure to the principal outdoor air pollutants in the same cohort. Methods: We studied 34 children (17asthmatics/17non-asthmatics) undergoing fiberoptic bronchoscopy for clinical indications. Bronchial epithelial cells were harvested by brushing, cultured and experimentally infected with Rhinovirus Type 16 (RV16). RV16-induced IFN-ß and λ expression was measured by quantitative real time PCR, as was RV16vRNA. The association between IFNs and the mean exposure to PM10, SO2 and NO2 in the day preceding bronchoscopy was evaluated using a Generalized Linear Model (GLM) with Gamma distribution. Results: Acute exposure to PM10 and NO2 was negatively associated to RV16-induced IFNß mRNA. For each increase of 1ug/m3 of NO2 we found a significative decrease of 2.3x103 IFN-ß mRNA copies and for each increase of 1ug/m3 of PM10 a significative decrease of 1x103 IFN-ß mRNA copies. No significant associations were detected between IFN-λ mRNA and NO2 nor PM10. Increasing levels of NO2 (but not PM10) were found to be associated to increased RV16 replication. Conclusions: Short-term exposure to high levels of NO2 and PM10 is associated to a reduced IFN-ß expression by the airway epithelium, which may lead to increased viral replication. These findings suggest a potential mechanism underlying the link between air pollution, viral infections and asthma exacerbations.