RESUMO
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.
Assuntos
Comunicação Celular , Conexina 43 , Coronavirus Humano OC43 , Retículo Endoplasmático , Junções Comunicantes , Humanos , Junções Comunicantes/metabolismo , Conexina 43/metabolismo , Células A549 , Coronavirus Humano OC43/fisiologia , Coronavirus Humano OC43/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Complexo de Golgi/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Infecções por Coronavirus/patologia , Estresse OxidativoRESUMO
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.
Assuntos
Antivirais , Compostos de Tungstênio , Internalização do Vírus , Replicação Viral , Humanos , Internalização do Vírus/efeitos dos fármacos , Antivirais/farmacologia , Replicação Viral/efeitos dos fármacos , Compostos de Tungstênio/farmacologia , Rhinovirus/efeitos dos fármacos , Rhinovirus/fisiologia , Linhagem Celular , Infecções Respiratórias/virologia , Infecções Respiratórias/tratamento farmacológico , Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus Humano OC43/fisiologia , AnimaisRESUMO
Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV-OC43 p65-like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP-derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV-OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception-, immune-, and cellular energy-related activities. Affinity capture shows the association of OC43p with an Na+ /K+ -transporting ATPase, providing a potential direct target and mechanistic insight into virus-induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV-OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus-induced neuropathic pain.
Assuntos
Coronavirus Humano OC43 , Neuralgia , Sequência de Aminoácidos , Animais , Coronavirus Humano OC43/fisiologia , Feminino , Humanos , Peptídeos , Ratos , Medula EspinalRESUMO
The COVID-19 pandemic has resulted in a huge number of deaths from 2020 to 2021; however, effective antiviral drugs against SARS-CoV-2 are currently under development. Recent studies have demonstrated that green tea polyphenols, particularly EGCG, inhibit coronavirus enzymes as well as coronavirus replication in vitro. Herein, we examined the inhibitory effect of green tea polyphenols on coronavirus replication in a mouse model. We used epigallocatechin gallate (EGCG) and green tea polyphenols containing more than 60% catechin (GTP60) and human coronavirus OC43 (HCoV-OC43) as a surrogate for SARS-CoV-2. Scanning electron microscopy analysis results showed that HCoV-OC43 infection resulted in virion particle production in infected cells. EGCG and GTP60 treatment reduced coronavirus protein and virus production in the cells. Finally, EGCG- and GTP60-fed mice exhibited reduced levels of coronavirus RNA in mouse lungs. These results demonstrate that green tea polyphenol treatment is effective in decreasing the level of coronavirus in vivo.
Assuntos
Antivirais/farmacologia , Catequina/análogos & derivados , Infecções por Coronavirus/tratamento farmacológico , Polifenóis/farmacologia , Chá/química , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/uso terapêutico , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus Humano OC43/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos , Polifenóis/química , Polifenóis/uso terapêuticoRESUMO
Transmission of SARS-CoV-2 by aerosols has played a significant role in the rapid spread of COVID-19 across the globe. Indoor environments with inadequate ventilation pose a serious infection risk. Whilst vaccines suppress transmission, they are not 100% effective and the risk from variants and new viruses always remains. Consequently, many efforts have focused on ways to disinfect air. One such method involves use of minimally hazardous 222 nm far-UVC light. Whilst a small number of controlled experimental studies have been conducted, determining the efficacy of this approach is difficult because chamber or room geometry, and the air flow within them, influences both far-UVC illumination and aerosol dwell times. Fortunately, computational multiphysics modelling allows the inadequacy of dose-averaged assessment of viral inactivation to be overcome in these complex situations. This article presents the first validation of the WYVERN radiation-CFD code for far-UVC air-disinfection against survival fraction measurements, and the first measurement-informed modelling approach to estimating far-UVC susceptibility of viruses in air. As well as demonstrating the reliability of the code, at circa 70% higher, our findings indicate that aerosolized human coronaviruses are significantly more susceptible to far-UVC than previously thought.
Assuntos
Coronavirus Humano 229E/efeitos da radiação , Infecções por Coronavirus/prevenção & controle , Coronavirus Humano OC43/efeitos da radiação , Desinfecção/métodos , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Aerossóis/isolamento & purificação , Microbiologia do Ar , COVID-19/prevenção & controle , Simulação por Computador , Coronavirus Humano 229E/isolamento & purificação , Coronavirus Humano 229E/fisiologia , Coronavirus Humano OC43/isolamento & purificação , Coronavirus Humano OC43/fisiologia , Desinfecção/instrumentação , Desenho de Equipamento , Humanos , Modelos BiológicosRESUMO
Emerging evidence suggests that blue light has the potential to inactivate viruses. Therefore, we investigated the effect of 405 nm, 410 nm, 425 nm and 450 nm pulsed blue light (PBL) on human alpha coronavirus HCoV-229 E and human beta coronavirus HCoV-OC43, using Qubit fluorometry and RT-LAMP to quantitate the amount of nucleic acid in irradiated and control samples. Like SARS-CoV-2, HCoV-229E and HCoV-OC43 are single stranded RNA viruses transmitted by air and direct contact; they have similar genomic sizes as SARS-CoV-2, and are used as surrogates for SARS-CoV-2. Irradiation was carried out either at 32.4 J cm-2 using 3 mW cm-2 irradiance or at 130 J cm-2 using 12 mW cm-2 irradiance. Results: (1) At each wavelength tested, PBL was antiviral against both coronaviruses. (2) 405 nm light gave the best result, yielding 52.3% (2.37 log10) inactivation against HCoV-OC43 (p < .0001), and a significant 1.46 log 10 (44%) inactivation of HCoV-229E (p < .01). HCoV-OC43, which like SARS-CoV-2 is a beta coronavirus, was more susceptible to PBL irradiation than alpha coronavirus HCoV-229E. The latter finding suggests that PBL is potentially antiviral against multiple coronavirus strains, and that, while its potency may vary from one virus to another, it seems more antiviral against beta coronaviruses, such as HCoV-OC43. (3) Further, the antiviral effect of PBL was better at a higher irradiance than a lower irradiance, and this indicates that with further refinement, a protocol capable of yielding 100% inactivation of viruses is attainable.
Assuntos
Coronavirus Humano 229E/efeitos da radiação , Coronavirus Humano OC43/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , SARS-CoV-2/efeitos da radiação , Coronavirus Humano 229E/fisiologia , Coronavirus Humano OC43/fisiologia , Relação Dose-Resposta à Radiação , Humanos , SARS-CoV-2/fisiologiaRESUMO
There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Células A549 , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Benzamidas , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Coronavirus Humano OC43/fisiologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Piperidinas , Piridinas , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Resfriado Comum/prevenção & controle , Coronavirus Humano OC43/fisiologia , Proteína DEAD-box 58/genética , Infecções por Rhabdoviridae/prevenção & controle , Vírus da Estomatite Vesicular Indiana/fisiologia , Células A549 , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Chlorocebus aethiops , Resfriado Comum/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/prevenção & controle , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Células RAW 264.7 , Infecções por Rhabdoviridae/metabolismo , Células Vero , Estomatite Vesicular/metabolismo , Estomatite Vesicular/prevenção & controleRESUMO
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.
Assuntos
Coronavirus Humano OC43/fisiologia , Efeito Citopatogênico Viral , Receptores Virais/metabolismo , Serina Endopeptidases/metabolismo , Carga Viral/métodos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Coronavirus Humano OC43/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Receptores Virais/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Células Vero/virologia , Cultura de Vírus , Internalização do Vírus , Replicação ViralRESUMO
COVID-19 pandemic results in record high deaths in many countries. Although a vaccine for SARS-CoV-2 is now available, effective antiviral drugs to treat coronavirus diseases are not available yet. Recently, EGCG, a green tea polyphenol, was reported to inhibit SARS-CoV-2 3CL-protease, however the effect of EGCG on coronavirus replication is unknown. In this report, human coronavirus HCoV-OC43 (beta coronavirus) and HCoV-229E (alpha coronavirus) were used to examine the effect of EGCG on coronavirus. EGCG treatment decreases 3CL-protease activity of HCoV-OC43 and HCoV-229E. Moreover, EGCG treatment decreased HCoV-OC43-induced cytotoxicity. Finally, we found that EGCG treatment decreased the levels of coronavirus RNA and protein in infected cell media. These results indicate that EGCG inhibits coronavirus replication.
Assuntos
Coronavirus Humano 229E/efeitos dos fármacos , Coronavirus Humano OC43/efeitos dos fármacos , Polifenóis/farmacologia , Chá/química , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Coronavirus Humano 229E/fisiologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/fisiologia , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologiaRESUMO
The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG's antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Coronavirus Humano OC43/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Tapsigargina/farmacologia , Animais , Antivirais/uso terapêutico , Betacoronavirus/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Coronavirus Humano OC43/fisiologia , Estresse do Retículo Endoplasmático , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Ribavirina/farmacologia , SARS-CoV-2/fisiologia , Tapsigargina/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.
Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de VírusRESUMO
A child with pre-B acute lymphoblastic leukaemia (ALL) developed fatal encephalitis associated with human coronavirus OC43 (HCoV-OC43). During chemotherapy the child had a persistent HCoV-OC43 respiratory infection and later developed progressive encephalitis. Cerebrospinal fluid was negative for pathogens including HCoV-OC43, but a brain biopsy was HCoV-OC43-positive by metagenomic next-generation sequencing.
Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano OC43/fisiologia , Encefalite/virologia , Encéfalo/patologia , Encéfalo/virologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/patologia , Coronavirus Humano OC43/isolamento & purificação , Encefalite/imunologia , Encefalite/mortalidade , Encefalite/patologia , Evolução Fatal , Humanos , Hospedeiro Imunocomprometido , LactenteRESUMO
BACKGROUND: While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV-OC43 infections have rarely been investigated as healthcare-associated infections in this population. OBJECTIVES: In this report, HCoV-OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. STUDY DESIGN: Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient's sequences. Amino acids substitutions were analysed. RESULTS: Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. CONCLUSIONS: HCoV-OC43 may be implicated in healthcare-associated infections.
Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Infecção Hospitalar/virologia , Genoma Viral/genética , Adulto , Idoso , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Coronavirus Humano OC43/isolamento & purificação , Coronavirus Humano OC43/fisiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , Europa (Continente)/epidemiologia , Feminino , Genótipo , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , Estudos Retrospectivos , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Stephaniatetrandra and other related species of Menispermaceae are the major sources of the bis-benzylisoquinoline alkaloids tetrandrine (TET), fangchinoline (FAN), and cepharanthine (CEP). Although the pharmacological properties of these compounds include anticancer and anti-inflammatory activities, the antiviral effects of these compounds against human coronavirus (HCoV) remain unclear. Hence, the aims of the current study were to assess the antiviral activities of TET, FAN, and CEP and to elucidate the underlying mechanisms in HCoV-OC43-infected MRC-5 human lung cells. These compounds significantly inhibited virus-induced cell death at the early stage of virus infection. TET, FAN, and CEP treatment dramatically suppressed the replication of HCoV-OC43 as well as inhibited viral S and N protein expression. The virus-induced host response was reduced by compound treatment as compared with the vehicle control. Taken together, these findings demonstrate that TET, FAN, and CEP are potential natural antiviral agents for the prevention and treatment of HCoV-OC43 infection.
Assuntos
Antivirais/farmacologia , Benzilisoquinolinas/farmacologia , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus Humano OC43/fisiologia , Extratos Vegetais/farmacologia , Stephania tetrandra/química , Benzilisoquinolinas/química , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/metabolismo , Coronavirus Humano OC43/genética , Citocinas/genética , Citocinas/metabolismo , Humanos , Extratos Vegetais/químicaRESUMO
The OC43 strain of human coronavirus (HCoV-OC43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. Coronavirus structural envelope (E) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. Making use of recombinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ-binding motif, we demonstrated that a fully functional HCoV-OC43 E protein is first needed for optimal production of recombinant infectious viruses. Furthermore, HCoV-OC43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence.
Assuntos
Sistema Nervoso Central/virologia , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/fisiologia , Neurônios/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Sistema Nervoso Central/patologia , Infecções por Coronavirus/patologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidade , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Proteínas do Envelope Viral/genética , VirulênciaRESUMO
Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.
Assuntos
Adaptação Biológica , Coronavirus Humano OC43/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Lectinas/genética , Lectinas/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Animais , Coronavirus Humano OC43/fisiologia , Humanos , Mutação , Ligação Proteica , Receptores Virais/metabolismoRESUMO
Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43(FL). Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.
Assuntos
Coronavirus Humano OC43/enzimologia , Coronavirus Humano OC43/fisiologia , Hemaglutininas Virais/metabolismo , Proteínas Virais de Fusão/metabolismo , Replicação Viral , Animais , Linhagem Celular , Coronavirus Humano OC43/genética , Cricetinae , Técnicas de Inativação de Genes , Teste de Complementação Genética , Hemaglutininas Virais/genética , Proteínas Virais de Fusão/genéticaRESUMO
The complete genome sequences of the human coronavirus OC43 (HCoV-OC43) laboratory strain from the American Type Culture Collection (ATCC), and a HCoV-OC43 clinical isolate, designated Paris, were obtained. Both genomes are 30,713 nucleotides long, excluding the poly(A) tail, and only differ by 6 nucleotides. These six mutations are scattered throughout the genome and give rise to only two amino acid substitutions: one in the spike protein gene (I958F) and the other in the nucleocapsid protein gene (V81A). Furthermore, the two variants were shown to reach the central nervous system (CNS) after intranasal inoculation in BALB/c mice, demonstrating neuroinvasive properties. Even though the ATCC strain could penetrate the CNS more effectively than the Paris 2001 isolate, these results suggest that intrinsic neuroinvasive properties already existed for the HCoV-OC43 ATCC human respiratory isolate from the 1960s before it was propagated in newborn mouse brains. It also demonstrates that the molecular structure of HCoV-OC43 is very stable in the environment (the two variants were isolated ca. 40 years apart) despite virus shedding and chances of persistence in the host. The genomes of the two HCoV-OC43 variants display 71, 53.1, and 51.2% identity with those of mouse hepatitis virus A59, severe acute respiratory syndrome human coronavirus Tor2 strain (SARS-HCoV Tor2), and human coronavirus 229E (HCoV-229E), respectively. HCoV-OC43 also possesses well-conserved motifs with regard to the genome sequence of the SARS-HCoV Tor2, especially in open reading frame 1b. These results suggest that HCoV-OC43 and SARS-HCoV may share several important functional properties and that HCoV-OC43 may be used as a model to study the biology of SARS-HCoV without the need for level three biological facilities.