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.

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.

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.
OBJECTIVE: 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 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.

β-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human β-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 β-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 β-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 β-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human β-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human β-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 β-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.

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.
OBJECTIVE: 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.

Virus-encoded replicases often generate aberrant RNA genomes, known as defective viral genomes (DVGs). When co-infected with a helper virus providing necessary proteins, DVGs can multiply and spread. While DVGs depend on the helper virus for propagation, they can in some cases disrupt infectious virus replication, impact immune responses, and affect viral persistence or evolution. Understanding the dynamics of DVGs alongside standard viral genomes during infection remains unclear. To address this, we conducted a long-term experimental evolution of two betacoronaviruses, the human coronavirus OC43 (HCoV-OC43) and the murine hepatitis virus (MHV), in cell culture at both high and low multiplicities of infection (MOI). We then performed RNA-seq at regular time intervals, reconstructed DVGs, and analyzed their accumulation dynamics. Our findings indicate that DVGs evolved to exhibit greater diversity and abundance, with deletions and insertions being the most common types. Notably, some high MOI deletions showed very limited temporary existence, while others became prevalent over time. We observed differences in DVG abundance between high and low MOI conditions in HCoV-OC43 samples. The size distribution of HCoV-OC43 genomes with deletions differed between high and low MOI passages. In low MOI lineages, short and long DVGs were the most common, with an additional cluster in high MOI lineages which became more prevalent along evolutionary time. MHV also showed variations in DVG size distribution at different MOI conditions, though they were less pronounced compared to HCoV-OC43, suggesting a more random distribution of DVG sizes. We identified hotspot regions for deletions that evolved at a high MOI, primarily within cistrons encoding structural and accessory proteins. In conclusion, our study illustrates the widespread formation of DVGs during betacoronavirus evolution, influenced by MOI and cell- and virus-specific factors.

With the lifting of COVID-19 non-pharmaceutical interventions, the resurgence of common viral respiratory infections was recorded in several countries worldwide. It facilitates viral co-infection, further burdens the already over-stretched healthcare systems. Racing to find co-infection-associated efficacy therapeutic agents need to be rapidly established. However, it has encountered numerous challenges that necessitate careful investigation. Here, we introduce a potential recombinant minibody-associated treatment, 3D8 single chain variable fragment (scFv), which has been developed as a broad-spectrum antiviral drug that acts via its nucleic acid catalytic and cell penetration abilities. In this research, we demonstrated that 3D8 scFv exerted antiviral activity simultaneously against both influenza A viruses (IAVs) and coronaviruses in three established co-infection models comprising two types of coronaviruses [beta coronavirus-human coronavirus OC43 (hCoV-OC43) and alpha coronavirus-porcine epidemic diarrhea virus (PEDV)] in Vero E6 cells, two IAVs [A/Puerto Rico/8/1934 H1N1 (H1N1/PR8) and A/X-31 (H3N2/X-31)] in MDCK cells, and a combination of coronavirus and IAV (hCoV-OC43 and adapted-H1N1) in Vero E6 cells by a statistically significant reduction in viral gene expression, proteins level, and approximately around 85%, 65%, and 80% of the progeny of \'hCoV-OC43-PEDV\', \'H1N1/PR8-H3N2/X-31\', and \'hCoV-OC43-adapted-H1N1\', respectively, were decimated in the presence of 3D8 scFv. Taken together, we propose that 3D8 scFv is a promising broad-spectrum drug for treatment against RNA viruses in co-infection.

The incessant mutations of viruses, variable immune responses, and likely emergence of new viral threats necessitate multiple approaches to novel antiviral therapeutics. Furthermore, the new antiviral agents should have broad-spectrum activity and be environmentally stable. Here, we show that biocompatible tapered CuS nanoparticles (NPs) efficiently agglutinate coronaviruses with binding affinity dependent on the chirality of surface ligands and particle shape. L-penicillamine-stabilized NPs with left-handed curved apexes display half-maximal inhibitory concentrations (IC50) as low as 0.66 pM (1.4 ng/mL) and 0.57 pM (1.2 ng/mL) for pseudo-type SARS-CoV-2 viruses and wild-type Wuhan-1 SARS-CoV-2 viruses, respectively, which are about 1,100 times lower than those for antibodies (0.73 nM). Benefiting from strong NPs-protein interactions, the same particles are also effective against other strains of coronaviruses, such as HCoV-HKU1, HCoV-OC43, HCoV-NL63, and SARS-CoV-2 Omicron variants with IC50 values below 10 pM (21.8 ng/mL). Considering rapid response to outbreaks, exposure to elevated temperatures causes no change in the antiviral activity of NPs while antibodies are completely deactivated. Testing in mice indicates that the chirality-optimized NPs can serve as thermally stable analogs of antiviral biologics complementing the current spectrum of treatments.

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, \"one-protein-at-a-time\" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.