Comparative study of the propagation and plaque titration conditions for human coronavirus OC43 as a surrogate for SARS-CoV-2.

The ongoing COVID-19 pandemic is threatening human health globally. The development of effective drugs and vaccines against SARS-CoV-2 is hindered by the limited access to high-biosafety-level facilities. Although human coronavirus (HCoV) OC43, a low-pathogenic endemic human coronavirus, has been used as a surrogate virus for SARS-CoV-2 research, a standard technique for HCoV-OC43 culture and plaque titration has not been established. Our objective was to establish optimized culture and titration protocols for HCoV-OC43. The growth kinetics and permissibility to HCoV-OC43 infection of seven different cell lines were examined concurrently at two different temperatures, 33°C and 37°C. Cell lines exhibiting a cytopathic effect (CPE) were selected for plaque titration. No significant difference in the rate of cell growth was observed at the two temperatures tested. Interestingly, HCoV-OC43 was found not to be a high-temperature-sensitive virus, since it grew well at 37°C. Although RD, LLC-MK2, MRC-5, and HCT-8 cell lines supported virus growth with an obvious cytopathic effect and a high yield of virus after two days of infection, only RD cells were suitable for producing countable plaques. The incubation of the cells with 1.2% low-viscosity Avicel as an overlay medium at 37°C for 4 days appeared to promote clearer and sharper plaque morphology. However, further optimization of the plaque titration protocol is still required due to the continued observation of plaque size variation and hazy zones. We propose a cost-effective protocol for HCoV-OC43 culture and plaque titration that can be implemented at a standard conventional temperature without the need for additional special equipment.

Persistence of two coronaviruses and efficacy of steam vapor disinfection on two types of carpet.

BACKGROUND: Coronaviruses, a group of highly transmissible and potentially pathogenic viruses, can be transmitted indirectly to humans via fomites. To date, no study has investigated their persistence on carpet fibers. Establishing persistence is essential before testing the efficacy of a disinfectant. METHODS: The persistence of BCoV and HCoV OC43 on polyethylene terephthalate (PET) and nylon carpet was first determined using infectivity and RT-qPCR assays. Then, the disinfectant efficacy of steam vapor was evaluated against both coronaviruses on nylon carpet. RESULTS: Immediately after inoculation of carpet coupons, 32.50% of BCoV and 3.87% of HCoV OC43 were recovered from PET carpet, compared to 34.86% of BCoV and 24.37% of HCoV OC43 recovered from nylon carpet. After incubation at room temperature for 1 h, BCoV and HCoV OC43 showed a 3.6 and > 2.8 log10 TCID50 reduction on PET carpet, and a 0.6 and 1.8 log10 TCID50 reduction on nylon carpet. Based on first-order decay kinetics, the whole gRNA of BCoV and HCoV OC43 were stable with k values of 1.19 and 0.67 h- 1 on PET carpet and 0.86 and 0.27 h- 1 on nylon carpet, respectively. A 15-s steam vapor treatment achieved a > 3.0 log10 TCID50 reduction of BCoV and > 3.2 log10 TCID50 reduction of HCoV OC43 on nylon carpet. CONCLUSION: BCoV was more resistant to desiccation on both carpet types than HCoV OC43. Both viruses lost infectivity quicker on PET carpet than on nylon carpet. Steam vapor inactivated both coronaviruses on nylon carpet within 15 s.

Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication.

Organisms respond to proteotoxic-stress by activating the heat-shock response, a cellular defense mechanism regulated by a family of heat-shock factors (HSFs); among six human HSFs, HSF1 acts as a proteostasis guardian regulating severe stress-driven transcriptional responses. Herein we show that human coronaviruses (HCoV), both low-pathogenic seasonal-HCoVs and highly-pathogenic SARS-CoV-2 variants, are potent inducers of HSF1, promoting HSF1 serine-326 phosphorylation and triggering a powerful and distinct HSF1-driven transcriptional-translational response in infected cells. Despite the coronavirus-mediated shut-down of the host translational machinery, selected HSF1-target gene products, including HSP70, HSPA6 and AIRAP, are highly expressed in HCoV-infected cells. Using silencing experiments and a direct HSF1 small-molecule inhibitor we show that, intriguingly, HCoV-mediated activation of the HSF1-pathway, rather than representing a host defense response to infection, is hijacked by the pathogen and is essential for efficient progeny particles production. The results open new scenarios for the search of innovative antiviral strategies against coronavirus infections.

Neurotoxic effects of coronavirus: Potential implications in Alzheimer's onset and progression.

The COVID-19, caused by SARS-CoV-2, first affects the respiratory tract but evidence is emerging that the virus, reaching the central nervous system (CNS), can lead to severe neurological disorders. In particular, CoV infection could cause an acceleration of the neurodegenerative process. On the other hand, patients diagnosed with Alzheimer's disease (AD) develop more serious forms of COVID-19 with worse relapses. Therefore, understanding the connection between the two pathologies, AD and infection by coronavirus, could help in the development of new therapeutic approaches to counter them. We used the SH-SY5Y cell line differentiated into neurons, as widely used in studies of AD if supplemented with exogenous fibrillary ß-amyloid (Aß). As a glial counterpart, human microglia (HMC3) and astrocytic (D54MG) cell lines were used to create co-cultures with neurons via transwell systems. In these experimental models, we generated infection with the Human Coronavirus OC43 (HCoV-OC43), a low-risk model of SARS-CoV-2. Our results suggest that the infection by HCoV-OC43 leads to a neurotoxic effect not depending on an already present event of Aß deposition. Indeed, unlike microglia, neurons and even more astrocytes are susceptible to CoV infection and, although the infection does not show a cytotoxic effect in the neurons in the first few days, significant alterations at a biochemical and morphological level have been observed, suggesting that the neurons are reacting to a stressful condition, including the prodromal and neurodegenerative features of AD. Interestingly, the interaction of infected astrocytes with the neurons resulted in the manifestation of signs of neurodegeneration, such as amyloid-beta deposition. By using exogenous fibrillary Aß, as an AD in vitro model, our data suggest that there is an aggravating effect both on the infection itself and on the neurological disease progression. In conclusion, the results of this study suggest a causal interplay between HCoV-OC43 and neurological diseases and demonstrate that the co-presence of different CNS cell populations is the necessary condition to study the pathogenic effects in vitro as a whole.

4-(3-Phenylsulfonylindol-2-yl)-1-(pyridin-2-yl)piperazinyl-methanones as Potent Inhibitors of both SARS-CoV-2 and HCoV-OC43 Viruses.

SARS-CoV-2 and HCoV-OC43 belong to the same ß genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two ß-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 µM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

Zanamivir and baloxavir combination to cure persistent influenza and coronavirus infections after hematopoietic stem cell transplant.

OBJECTIVES: Immunocompromised patients may experience prolonged shedding of influenza virus potentially leading to severe infections. Alternatives to monotherapy with neuraminidase inhibitors should be evaluated to entirely suppress viral replication and prevent drug-resistant mutations. METHODS: We investigated the clinical and virological evolution in a case of persistent influenza A and human coronavirus OC43 (HCoV-OC43) coinfection in a hematopoietic stem cell transplant recipient after different therapeutic strategies. RESULTS: Successive oseltamivir and zanamivir monotherapies failed to control both infections, with positive results persisting for over 110 days each. This led to the emergence of highly resistant oseltamivir strains due to neuraminidase mutations (E119V and R292K) followed by a deletion (del245-248), while maintaining sensitivity to zanamivir. The intra-host viral diversity data showed that the treatments impacted viral diversity of influenza virus, but not of HCoV-OC43. Considering the patient's underlying condition and the impact of prolonged viral shedding on pulmonary function, eradicating the influenza virus was necessary. A 10-day regimen combining zanamivir and baloxavir-marboxil effectively controlled influenza virus replication and was associated with the clearance of HCoV-OC43, finally resulting in comprehensive respiratory recovery. CONCLUSION: These observations underscore the importance of further investigating combination treatments as the primary approach to achieve influenza eradication in immunocompromised patients.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

The Novel A-Type Proanthocyanidin-Rich Phytocomplex SP4™ Acts as a Broad-Spectrum Antiviral Agent against Human Respiratory Viruses.

The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections.

Recombinant OC43 SARS-CoV-2 spike replacement virus: An improved BSL-2 proxy virus for SARS-CoV-2 neutralization assays.

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

Efficacy of Three Antimicrobials Against two SARS-COV-2 Surrogates, Bovine Coronavirus and Human Coronavirus OC43, on Hard or Soft Nonporous Materials.

The efficacy of three antimicrobials was evaluated against two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogates - bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 - on hard and soft nonporous materials. Three antimicrobials with three different active ingredients (chlorine, hydrogen peroxide, and quaternary ammonium compound + alcohol) were studied. Initially, a neutralization method was optimized for each antimicrobial. Then, we determined their efficacy against BCoV and HCoV OC43 in both suspension and on surfaces made with polyethylene terephthalate (PET) plastic and vinyl upholstery fabric. All tests were conducted under ambient environmental conditions with a soil load of 5% fetal bovine serum. After a 2-min exposure, all three antimicrobials achieved a >3.0 log10 reduction in viral titers in suspension. All three also reduced virus infectivity on both surface materials below the detection limit (0.6 log10 TCID50/carrier). Treatments in which the reduction in virus titer was <3.0 log10 were attributed to a decreased dynamic range on the carrier during drying prior to disinfection. The carrier data revealed that both surrogates were inactivated more rapidly (p <0.05) on vinyl or under conditions of high relative humidity. Three classes of antimicrobials were efficacious against both SARS-CoV-2 surrogate viruses, with BCoV demonstrating slightly less sensitivity compared to HCoV OC43. These findings also illustrate the importance of (1) optimizing the neutralization method and (2) considering relative humidity as a key factor for efficacy testing.

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.

Anticoronavirus activity of rhizome of Dryopteris crassirhizoma via multistage targeting of virus entry and viral proteases, Mpro and PLpro.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities. AIM OF THE STUDY: This study investigates the 30% ethanolic extract of RDC's antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections. MATERIALS AND METHODS: A 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action. RESULTS: RDC exhibited dose-dependent inhibition of HCoV-OC43-induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro). CONCLUSIONS: These findings underscore RDC's multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.

Development of novel antivrial agents that induce the degradation of the main protease of human-infecting coronaviruses.

The family of human-infecting coronaviruses (HCoVs) poses a serious threat to global health and includes several highly pathogenic strains that cause severe respiratory illnesses. It is essential that we develop effective broad-spectrum anti-HCoV agents to prepare for future outbreaks. In this study, we used PROteolysis TArgeting Chimera (PROTAC) technology focused on degradation of the HCoV main protease (Mpro), a conserved enzyme essential for viral replication and pathogenicity. By adapting the Mpro inhibitor GC376, we produced two novel PROTACs, P2 and P3, which showed relatively broad-spectrum activity against the human-infecting CoVs HCoV-229E, HCoV-OC43, and SARS-CoV-2. The concentrations of these PROTACs that reduced virus replication by 50 % ranged from 0.71 to 4.6 µM, and neither showed cytotoxicity at 100 µM. Furthermore, mechanistic binding studies demonstrated that P2 and P3 effectively targeted HCoV-229E, HCoV-OC43, and SARS-CoV-2 by degrading Mpro within cells in vitro. This study highlights the potential of PROTAC technology in the development of broad-spectrum anti-HCoVs agents, presenting a novel approach for dealing with future viral outbreaks, particularly those stemming from CoVs.

LC/MS-guided isolation and GNPS procedures to identify flavonoid of HCoV-OC43 inhibitors.

The screening of based target compounds supported by LC/MS, MS/MS and Global Natural Products Social (GNPS) used to identify the compounds 1-10 of Butea monsperma. They were evaluated in human malignant embryonic rhabdomyoma cells (RD cells) infected with Human coronavirus OC43 (HCoV-OC43) and showed significant inhibitory activity. Target inhibition tests showed that compounds 6 and 8 inhibited the proteolytic enzyme 3CLpro, which is widely present in coronavirus and plays an important role in the replication process, with an effective IC50 value. The study confirmed that dioxymethylene of compound 8 may be a key active fragment in inhibiting coronavirus (EC50 7.2 µM, SI > 139.1). The results have led to identifying natural bioactive compounds for possible inhibiting HCoV-OC43 and developing drug for Traditional Chinese Medicine (TCM).

Human coronavirus OC43 infection remodels connexin 43-mediated gap junction intercellular communication in vitro.

ß-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human ß-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding ß-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human ß-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also shows a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post-OC43 infection in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Impairment of Cx43 trafficking and the ability to form hemichannels and functional GJIC are hampered by virus-induced Golgi apparatus disruption. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human ß-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human ß-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human ß-coronavirus OC43 can be considered an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the changes in Cx43-mediated cell-to-cell communication during human ß-coronavirus OC43 infection. In vitro studies showed downregulation of the gap junction protein Cx43 and upregulation of the endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, HCoV-OC43 infection causes reduced Cx43 trafficking, causing impairment of functional hemichannel and GJIC formation by virus-mediated Golgi apparatus disruption. Overall, this study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.

Anti-coronavirus and anti-pulmonary inflammation effects of iridoids, the common component from Chinese herbal medicines for the treatment of COVID-19.

The practice of Chinese herbal medicines for the treatment of COVID-19 in China played an essential role for the control of mortality rate and reduction of recovery time. The iridoids is one of the main constituents of many heat-clearing and detoxifying Chinese medicines that were largely planted and frequently used in clinical practice. Twenty-three representative high content iridoids from several staple Chinese medicines were obtained and tested by a SARS-CoV-2 pseudo-virus entry-inhibition assay on HEK-293 T/ACE2 cells, a live HCoV-OC43 virus infection assay on HRT-18 cells, and a SARS-CoV-2 3CL protease inhibitory FRET assay followed by molecular docking simulation. The anti-pulmonary inflammation activities were further evaluated on a TNF-α induced inflammation model in A549 cells and preliminary SARs were concluded. The results showed that specnuezhenide (7), cornuside (12), neonuezhenide (15), and picroside III (21) exhibited promising antiviral activities, and neonuezhenide (15) could inhibit 3CL protease with an IC50 of 14.3 µM. Docking computation showed that compound 15 could bind to 3CL protease through a variety of hydrogen bonding and hydrophobic interactions. In the anti-pulmonary inflammation test, cornuside (12), aucubin (16), monotropein (17), and shanzhiside methyl ester (18) could strongly decrease the content of IL-1ß and IL-8 at 10 µM. Compound 17 could also upregulate the expression of the anti-inflammatory cytokine IL-10 significantly. The iridoids exhibited both anti-coronavirus and anti-pulmonary inflammation activities for their significance of existence in Chinese herbal medicines, which also provided a theoretical basis for their potential utilization in the pharmaceutical and food industries.

Human coronavirus OC43 nanobody neutralizes virus and protects mice from infection.

Human coronavirus (hCoV) OC43 is endemic to global populations and usually causes asymptomatic or mild upper respiratory tract illness. Here, we demonstrate the neutralization efficacy of isolated nanobodies from alpacas immunized with the S1B and S1C domain of the hCoV-OC43 spike glycoprotein. A total of 40 nanobodies bound to recombinant OC43 protein with affinities ranging from 1 to 149 nM. Two nanobodies WNb 293 and WNb 294 neutralized virus at 0.21 and 1.79 nM, respectively. Intranasal and intraperitoneal delivery of WNb 293 fused to an Fc domain significantly reduced nasal viral load in a mouse model of hCoV-OC43 infection. Using X-ray crystallography, we observed that WNb 293 bound to an epitope on the OC43 S1B domain, distal from the sialoglycan-binding site involved in host cell entry. This result suggests that neutralization mechanism of this nanobody does not involve disruption of glycan binding. Our work provides characterization of nanobodies against hCoV-OC43 that blocks virus entry and reduces viral loads in vivo and may contribute to future nanobody-based therapies for hCoV-OC43 infections. IMPORTANCE: The pandemic potential presented by coronaviruses has been demonstrated by the ongoing COVID-19 pandemic and previous epidemics caused by severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Outside of these major pathogenic coronaviruses, there are four endemic coronaviruses that infect humans: hCoV-OC43, hCoV-229E, hCoV-HKU1, and hCoV-NL63. We identified a collection of nanobodies against human coronavirus OC43 (hCoV-OC43) and found that two high-affinity nanobodies potently neutralized hCoV-OC43 at low nanomolar concentrations. Prophylactic administration of one neutralizing nanobody reduced viral loads in mice infected with hCoV-OC43, showing the potential for nanobody-based therapies for hCoV-OC43 infections.

Transcriptome dynamics of the BHK21 cell line in response to human coronavirus OC43 infection.

The progress of viral infection involves numerous transcriptional regulatory events. The identification of the newly synthesized transcripts helps us to understand the replication mechanisms and pathogenesis of the virus. Here, we utilized a time-resolved technique called metabolic RNA labeling approach called thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) to differentially elucidate the levels of steady-state and newly synthesized RNAs of BHK21 cell line in response to human coronavirus OC43 (HCoV-OC43) infection. Our results showed that the Wnt/ß-catenin signaling pathway was significantly enriched with the newly synthesized transcripts of BHK21 cell line in response to HCoV-OC43 infection. Moreover, inhibition of the Wnt pathway promoted viral replication in the early stage of infection, but inhibited it in the later stage of infection. Furthermore, remdesivir inhibits the upregulation of the Wnt/ß-catenin signaling pathway induced by early infection with HCoV-OC43. Collectively, our study showed the diverse roles of Wnt/ß-catenin pathway at different stages of HCoV-OC43 infection, suggesting a potential target for the antiviral treatment. In addition, although infection with HCoV-OC43 induces cytopathic effects in BHK21 cells, inhibiting apoptosis does not affect the intracellular replication of the virus. Monitoring newly synthesized RNA based on such time-resolved approach is a highly promising method for studying the mechanism of viral infections.

Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids.

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.

Polyoxometalate exerts broad-spectrum activity against human respiratory viruses hampering viral entry.

Human respiratory viruses have an enormous impact on national health systems, societies, and economy due to the rapid airborne transmission and epidemic spread of such pathogens, while effective specific antiviral drugs to counteract infections are still lacking. Here, we identified two Keggin-type polyoxometalates (POMs), [TiW11CoO40]8- (TiW11Co) and [Ti2PW10O40]7- (Ti2PW10), endowed with broad-spectrum activity against enveloped and non-enveloped human respiratory viruses, i.e., coronavirus (HCoV-OC43), rhinovirus (HRV-A1), respiratory syncytial virus (RSV-A2), and adenovirus (AdV-5). Ti2PW10 showed highly favorable selectivity indexes against all tested viruses (SIs >700), and its antiviral potential was further investigated against human coronaviruses and rhinoviruses. This POM was found to inhibit replication of multiple HCoV and HRV strains, in different cell systems. Ti2PW10 did not affect virus binding or intracellular viral replication, but selectively inhibited the viral entry. Serial passaging of virus in presence of the POM revealed a high barrier to development of Ti2PW10-resistant variants of HRV-A1 or HCoV-OC43. Moreover, Ti2PW10 was able to inhibit HRV-A1 production in a 3D model of the human nasal epithelium and, importantly, the antiviral treatment did not determine cytotoxicity or tissue damage. A mucoadhesive thermosensitive in situ hydrogel formulation for nasal delivery was also developed for Ti2PW10. Overall, good biocompatibility on cell lines and human nasal epithelia, broad-spectrum activity, and absence of antiviral resistance development reveal the potential of Ti2PW10 as an antiviral candidate for the development of a treatment of acute respiratory viral diseases, warranting further studies to identify the specific target/s of the polyanion and assess its clinical potential.