RESUMO
Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.
Assuntos
Regiões 3' não Traduzidas , Flavivirus , Replicação Viral , Vírus do Nilo Ocidental , Regiões 3' não Traduzidas/genética , Flavivirus/genética , Flavivirus/fisiologia , Humanos , Animais , Replicação Viral/genética , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/fisiologia , Infecções por Flavivirus/virologia , Exonucleases/metabolismo , Exonucleases/genética , Chlorocebus aethiops , Exorribonucleases/metabolismo , Exorribonucleases/genética , Células HEK293 , Células Vero , Linhagem Celular , Interferon Tipo I/metabolismo , Genoma ViralRESUMO
It is thought to be risk-free, environmentally benign, and safe for biological processes to produce zinc oxide nanoparticles from renewable resources. This study examined Cassia javanica's ability to create ZnONPs. The generated ZnONPs were analyzed using a variety of techniques, such as TEM, FTIR spectroscopy, UV-Vis spectroscopy, and XRD analysis. The antibacterial potential of ZnONPs has been investigated using both Agar well diffusion and microtitreplate (MTP) methods. One method used to evaluate ZnONPs' capacity to scavenge free radicals at different concentrations was the DPPH method. The permanent zinc oxide (ZnO) shape and the naturally occurring crystal structure of ZnONPs were validated by the XRD data. ZnONPs showed antibacterial activity with MICs of 31.7 µg/mL toward Bacillus subtilis, 62.5 µg/mL for Salmonella typhimurium, Escherichia coli while Clostridium sporogenes and Bacillus pumilus was 125µg/mL. Furthermore, ZnONPs demonstrated a range of antibiofilm activities toward Staphylococcus aureus (MRSA). ZnONPs showed an intriguing antioxidant capacity, achieving IC50 of 109.3 µg/ml µg/mL. Additionally, ZnONPs demonstrated low toxic effect on Vero cell with IC50 154.01 µg/mL as well as possible anticancer action when applied to the carcinoma cell lines HepG2 with IC50 of 47.48 µg/mL. Furthermore, ZnONPs at 62.5 µg/mL had a promising antiviral impact against HSV1 and COX B4, with antiviral activities of 75.4% and 65.8%, respectively.
Assuntos
Antibacterianos , Antineoplásicos , Antioxidantes , Antivirais , Biofilmes , Cassia , Testes de Sensibilidade Microbiana , Óxido de Zinco , Óxido de Zinco/farmacologia , Óxido de Zinco/química , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Biofilmes/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/química , Antivirais/farmacologia , Antivirais/química , Animais , Cassia/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Chlorocebus aethiops , Células Vero , Nanopartículas Metálicas/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Nanopartículas/químicaRESUMO
The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses.
Assuntos
Endossomos , Canais de Cátion TRPM , Internalização do Vírus , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Endossomos/metabolismo , Endossomos/virologia , Concentração de Íons de Hidrogênio , Humanos , Animais , Células HEK293 , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ebolavirus/fisiologia , Ebolavirus/metabolismo , Vírus da Coriomeningite Linfocítica/fisiologia , Chlorocebus aethiops , Envelope Viral/metabolismo , Vírus Lassa/metabolismo , Vírus Lassa/fisiologiaRESUMO
Nanoscale research is gaining interest in the biomedical, engineering, and environmental fields. Current expensive traditional chemical methods for synthesizing nanoparticles (NPs) inevitably lead to the synthesis of NPs with potentially less or no toxic effects on living cells. To overcome these challenges, in this study, we use a simple, inexpensive, and less toxic one-pot green chemistry approach instead of a chemical method to synthesize alumina nanoparticles (AlNPs) from Carica papaya extract. Nano-alumina has been widely studied due to its remarkable biological and physiochemical properties at nanoscale. However, to date, its biomedical application is limited due to the lack of sufficient data on cytotoxicity in living cells. The physicochemical properties of nano-alumina were determined by FT-IR, DLS, SEM and HRTEM. The cytotoxic effects of the synthesized nano-alumina were studied in cell lines LT and VERO at concentrations of 10-480 µg/mL in vitro. The cell viability of nano-alumina was evaluated using the MTT assay and the AO /EB double staining technique. Our results based on DLS and HRTEM analyzes confirmed spherical AlNPs with a zeta potential and average particle size of - 25 to 5 mV and 52 nm, respectively. The nano-alumina tested showed low toxicity to both cell lines after 28- and 48-h exposure. Furthermore, cell viability statistically decreased with increasing incubation time and concentration of AlNPs up to 480 µg/mL (p < 0.001). However, a minimal increase in cytotoxicity was observed at threshold levels in the range of 120-480 µg/mL. The half-maximal inhibitory concentration (IC50) of AlNPs in the VERO and LT cell lines were 153.3, 252.0 µg/mL and 186.6, 395.3 µg/mL, respectively, after 24- and 48-h exposure to AlNPs. Thus, we conclude that the cytotoxic effect of AlNPs depends on the concentration, exposure time and cell type. The result suggests that the concentration used in this study may be useful for biomedical applications.
Assuntos
Óxido de Alumínio , Sobrevivência Celular , Química Verde , Óxido de Alumínio/química , Animais , Chlorocebus aethiops , Células Vero , Química Verde/métodos , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , Tamanho da Partícula , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Carica/química , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: In early 2020, COVID-19 pandemic has mobilized researchers in finding new remedies including repurposing of medicinal plant products focusing on direct-acting antiviral and host-directed therapies. In this study, we performed an in vitro investigation on the standardized Marantodes pumilum extract (SKF7®) focusing on anti-SARS-CoV-2 and anti-inflammatory activities. METHODS: Anti-SARS-CoV-2 potential of the SKF7® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays. RESULTS: The SKF7® inhibited wild-type Wuhan (EC50 of 21.99 µg/mL) and omicron (EC50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC50 value of 6.31 µg/mL). The SKF7® prominently inhibited 3CL protease activity. The SKF7® inhibited the LPS induced-TLR4 response with the EC50 of 16.19 µg/mL. CONCLUSIONS: In conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7®. Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7® in affecting the COVID-19 pathogenesis are warranted.
Assuntos
Antivirais , Extratos Vegetais , SARS-CoV-2 , Animais , Humanos , Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células Vero , Chlorocebus aethiops , Extratos Vegetais/farmacologia , Células A549 , Plantas Medicinais/química , Tratamento Farmacológico da COVID-19 , Anti-Inflamatórios/farmacologia , Malásia , COVID-19 , Proteases 3C de CoronavírusRESUMO
BACKGROUND: This in vitro study aimed to investigate the effect of several phenolic compounds, including doxorubicin, quercetin, and resveratrol, on HSV-1 infection. METHODS: The cytotoxicity of the drugs was assessed on Vero cells using the MTT assay. HSV-1 was treated with the drugs, and the supernatants were collected at various time points. TCID50% and qPCR tests were conducted on the supernatants to determine viral titration post-inoculation. RESULTS: The TCID50% assay showed significant changes in viral titration for acyclovir, doxorubicin, and quercetin at most concentrations (p-value < .05), while no significant changes were observed for resveratrol. The qPCR results demonstrated that drug-treated HSV-1 exhibited a significant reduction in DNA titers at various time points compared to non-treated HSV-1 infected Vero cells, except doxorubicin (0.2 µM) and acyclovir (5 µm). However, over time, DNA virus levels gradually increased in the drug-treated groups. Notably, at certain concentrations of doxorubicin and quercetin-treated groups, virus titer significantly declined, similar to acyclovir. CONCLUSIONS: Our findings suggest that quercetin at concentrations of 62 and 125 µM significantly reduced HSV-1 infectivity, as well as these two concentrations of quercetin showed a significant difference in virus reduction compared with acyclovir (10 µM) at certain time points. The anti-inflammatory properties of quercetin, in contrast to acyclovir, make it a potential candidate for anti HSV-1 treatment in life-threatening conditions such as Herpes encephalitis. Additionally, doxorubicin, an anticancer drug, showed meaningful inhibition of HSV-1 at non-toxic concentrations of 2 and 8 µM, suggesting its potential interference with HSV-1 in viral-oncolytic therapy in cancer treatment.
Assuntos
Aciclovir , Antivirais , Herpesvirus Humano 1 , Quercetina , Herpesvirus Humano 1/efeitos dos fármacos , Antivirais/farmacologia , Chlorocebus aethiops , Células Vero , Animais , Quercetina/farmacologia , Aciclovir/farmacologia , Fenóis/farmacologia , Doxorrubicina/farmacologia , Resveratrol/farmacologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Herpes Simples/tratamento farmacológico , Herpes Simples/virologiaRESUMO
Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the Journal of Extracellular Vesicles, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.
Assuntos
Lesão Pulmonar Aguda , COVID-19 , Vesículas Extracelulares , Vírus da Influenza A Subtipo H1N1 , Células-Tronco Mesenquimais , SARS-CoV-2 , Animais , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , SARS-CoV-2/fisiologia , COVID-19/terapia , Lesão Pulmonar Aguda/terapia , Lesão Pulmonar Aguda/virologia , Camundongos , Células Vero , Humanos , Chlorocebus aethiops , Infecções por Orthomyxoviridae/terapia , Replicação Viral , Mesocricetus , Modelos Animais de Doenças , Masculino , Influenza Humana/terapia , FemininoRESUMO
BACKGROUND: Japanese encephalitis (JE) is a severe infectious disease of the central nervous system. Vaccination with Vero cell culture-derived vaccines may effectively reduce JE incidence. RESEARCH DESIGN AND METHODS: In this single-center, randomized, blinded, positive-controlled clinical trial in China involving 600 healthy infants aged 6-11 months, participants were divided into experimental and control groups administered JEV-PI and JEV-LI, respectively. Antibody titers were determined after 0- and 7-day immunization schedules. A booster dose followed 12 months later. RESULTS: After primary vaccination and before booster vaccination, the positive conversion rate, geometric mean titer (GMT), and geometric mean increase (GMI) of JEV-PI-neutralizing antibodies exceeded those of JEV-LI. After booster immunization, the GMT and GMI of JEV-PI were higher than those of JEV-LI. After primary immunization, the local, systemic, and overall adverse reactions were of grades 1 and 2, with a low incidence of grade 3. After booster immunization, these differences were mainly grades 1 and 2, with no differences between JEV-PI and JEV-LI. CONCLUSION: JEV-PI is a promising vaccine as infants acquired long-lasting and highly neutralizing immune antibodies after inoculation with JEV-PI. TRIAL REGISTRATION: The trial was registered in the Chinese Clinical Trial Registry (https://www.chictr.org.cn/showproj.html?proj = 203130; registration number: ChiCTR2300074692; registration date: 14/08/2023).
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Encefalite Japonesa , Imunização Secundária , Vacinas contra Encefalite Japonesa , Humanos , Vacinas contra Encefalite Japonesa/imunologia , Vacinas contra Encefalite Japonesa/administração & dosagem , Vacinas contra Encefalite Japonesa/efeitos adversos , Lactente , Encefalite Japonesa/prevenção & controle , Encefalite Japonesa/imunologia , China , Masculino , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Células Vero , Anticorpos Neutralizantes/sangue , Animais , Feminino , Imunização Secundária/métodos , Imunogenicidade da Vacina , Esquemas de Imunização , Vacinação/métodosRESUMO
SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Células Gigantes , Hospedeiro Imunocomprometido , SARS-CoV-2 , Replicação Viral , Humanos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Células Gigantes/virologia , Evasão da Resposta Imune , Temperatura , Masculino , Feminino , Pessoa de Meia-Idade , RNA Viral/genética , Chlorocebus aethiops , Células VeroRESUMO
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has filled a gap in our knowledge regarding the prevention of CoVs. Swine coronavirus (CoV) is a significant pathogen that causes huge economic losses to the global swine industry. Until now, anti-CoV prevention and control have been challenging due to the rapidly generated variants. Silver nanoparticles (AgNPs) with excellent antimicrobial activity have attracted great interest for biosafety prevention and control applications. In this study, we synthesized chitosan-modified AgNPs (Chi-AgNPs) with good biocompatibility to investigate their antiviral effects on swine CoVs. In vitro assays showed that Chi-AgNPs could significantly impaired viral entry. The direct interaction between Chi-AgNPs and CoVs can destroy the viral surface spike (S) protein secondary structure associated with viral membrane fusion, which is caused by the cleavage of disulfide bonds in the S protein. Moreover, the mechanism showed that Chi-AgNPs reduced the virus-induced apoptosis of Vero cells via the ROS/p53 signaling activation pathway. Our data suggest that Chi-AgNPs can serve as a preventive strategy for CoVs infection and provide a molecular basis for the viricidal effect of Chi-AgNPs on CoVs.
Assuntos
Antivirais , Quitosana , Nanopartículas Metálicas , Prata , Glicoproteína da Espícula de Coronavírus , Animais , Quitosana/química , Quitosana/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Nanopartículas Metálicas/química , Chlorocebus aethiops , Prata/química , Prata/farmacologia , Células Vero , Antivirais/farmacologia , Antivirais/química , Suínos , Apoptose/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , COVID-19/virologia , COVID-19/prevenção & controleRESUMO
The African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal disease in swine, for which no antiviral drugs or vaccines are currently available. Studying viral-host protein-protein interactions advances our understanding of the molecular mechanisms underlying viral replication and pathogenesis and can facilitate the discovery of antiviral therapeutics. In this study, we employed affinity tagging and purification mass spectrometry to characterize the interactome of VPS39, an important cellular factor during the early phase of ASFV replication. The interaction network of VPS39 revealed associations with mitochondrial proteins involved in membrane contact sites formation and cellular respiration. We show that the ASFV proteins CP204L and A137R target VPS39 by interacting with its clathrin heavy-chain functional domain. Furthermore, we elaborate on the potential mechanisms by which VPS39 may contribute to ASFV replication and prioritize interactions for further investigation into mitochondrial protein function in the context of ASFV infection.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Replicação Viral , Vírus da Febre Suína Africana/metabolismo , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Animais , Suínos , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Interações Hospedeiro-Patógeno , Chlorocebus aethiops , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Células Vero , Humanos , Ligação ProteicaRESUMO
Toxoplasma gondii is a coccidian protozoan of zoonotic importance that causes toxoplasmosis. Although the current treatments for toxoplasmosis may be associated with adverse effects and limited efficacy for different biological forms of the parasite, evidence suggests that alkaloid molecules such as harmaline and piperine exhibit antiparasitic effects against protozoa parasites. This investigation aimed to evaluate the in vitro effect of harmaline and piperine against T. gondii tachyzoites in infected Vero cell cultures. After 24 hours of host cell infection, the cultures were treated with harmaline or piperine (0.49 to 15.63 µg/mL). Negative and positive controls were RPMI/DMSO (0.1%) and sulfadiazine (200 µg/mL). Harmaline significantly reduced parasite multiplication by 20% compared to the negative control, while piperine decreased between 55.56% and 88.89% in a dose-dependent manner. According to an intracellular parasite proportion scale, it was observed that the Vero cells with low or moderate parasitic proliferation were more prevalent after the alkaloid treatment. The study demonstrated that the alkaloids had antiparasitic effects on T. gondii, with piperine being the most effective. Additional studies must be carried out to clarify other aspects of the action of the alkaloids on parasites.
Assuntos
Alcaloides , Benzodioxóis , Harmalina , Piperidinas , Alcamidas Poli-Insaturadas , Toxoplasma , Benzodioxóis/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Alcaloides/farmacologia , Toxoplasma/efeitos dos fármacos , Piperidinas/farmacologia , Animais , Chlorocebus aethiops , Células Vero , Harmalina/farmacologia , Testes de Sensibilidade ParasitáriaRESUMO
Mayaro virus (MAYV) is the causative agent of Mayaro fever, which is characterized mainly by acute fever and long-term severe arthralgia, common manifestations of other arbovirus infections, making the correct diagnosis a challenge. Besides, MAYV infections have been reported in South America, especially in Brazil. However, the lack of vaccines or specific antiviral drugs to control these infections makes the search for new antivirals an urgent need. Herein, we evaluated the antiviral potential of synthetic ß-enaminoesters derivatives against MAYV replication and their pharmacokinetic and toxicological (ADMET) properties using in vitro and in silico strategies. For this purpose, Vero cells were infected with MAYV at an MOI of 0.1, treated with compounds (50 µM) for 24 h, and virus titers were quantified by plaque reduction assays. Compounds 2b (83.33%) and 2d (77.53%) exhibited the highest activity with inhibition rates of 83.33% and 77.53%, respectively. The most active compounds 2b (EC50 = 18.92 µM; SI > 52.85), and 2d (EC50 = 14.52 µM; SI > 68.87) exhibited higher potency and selectivity than the control drug suramin (EC50 = 38.97 µM; SI > 25.66). Then, we investigated the mechanism of action of the most active compounds. None of the compounds showed virucidal activity, neither inhibited virus adsorption, but compound 2b inhibited virus entry (62.64%). Also, compounds 2b and 2d inhibited some processes involved with the release of new virus particles. Finally, in silico results indicated good ADMET parameters of the most active compounds and reinforced their promising profile as drug candidates against MAYV.
Assuntos
Alphavirus , Antivirais , Ésteres , Replicação Viral , Antivirais/farmacologia , Antivirais/química , Chlorocebus aethiops , Animais , Células Vero , Ésteres/farmacologia , Ésteres/química , Alphavirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Simulação por Computador , Brasil , Infecções por Alphavirus/tratamento farmacológico , Infecções por Alphavirus/virologiaRESUMO
Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.
Assuntos
Antivirais , Febre de Chikungunya , Vírus Chikungunya , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Vírus Chikungunya/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/química , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Humanos , Animais , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Avaliação Pré-Clínica de Medicamentos , Replicação Viral/efeitos dos fármacos , Descoberta de Drogas , Chlorocebus aethiops , Células VeroRESUMO
Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172-554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Doenças dos Suínos/virologia , Virulência , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Células Vero , Chlorocebus aethiops , Variação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Graphene nanoplatelets (UGZ-1004) are emerging as a promising biomaterial in regenerative medicine. This study comprehensively evaluates UGZ-1004, focusing on its physical properties, cytotoxicity, intracellular interactions, and, notably, its effects on mesenchymal stem cells (MSCs). UGZ-1004 was characterized by lateral dimensions and layer counts consistent with ISO standards and demonstrated a high carbon purity of 0.08%. Cytotoxicity assessments revealed that UGZ-1004 is non-toxic to various cell lines, including 3T3 fibroblasts, VERO kidney epithelial cells, BV-2 microglia, and MSCs, in accordance with ISO 10993-5:2020/2023 guidelines. The study focused on MSCs and revealed that UGZ-1004 supports their gene expression alterations related to self-renewal and proliferation. MSCs exposed to UGZ-1004 maintained their characteristic surface markers. Importantly, UGZ-1004 promoted significant upregulation of genes crucial for cell cycle regulation and DNA repair, such as CDK1, CDK2, and MDM2. This gene expression profile suggests that UGZ-1004 can enhance MSC self-renewal capabilities, ensuring robust cellular function and longevity. Moreover, UGZ-1004 exposure led to the downregulation of genes associated with tumor development, including CCND1 and TFDP1, mitigating potential tumorigenic risks. These findings underscore the potential of UGZ-1004 to not only bolster MSC proliferation but also enhance their self-renewal processes, which are critical for effective regenerative therapies. The study highlights the need for continued research into the long-term impacts of graphene nanoplatelets and their application in MSC-based regenerative medicine.
Assuntos
Proliferação de Células , Grafite , Células-Tronco Mesenquimais , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Grafite/química , Grafite/farmacologia , Camundongos , Chlorocebus aethiops , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células Vero , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas/química , Linhagem Celular , Nanoestruturas/químicaRESUMO
The development of serum-free media (SFM) is critical to advance cell culture techniques used in viral vaccine production and address the ethical concerns and contamination risks associated with fetal bovine serum (FBS). This study evaluated the effects of marine microalgal extracts and growth factor cocktails on the activity of Madin-Darby canine kidney (MDCK) and Vero cells. Five marine microalgal species were used: Spirulina platensis (SP), Dunaliella salina (DS), Haematococcus pluvialis (HP), Nannochloropsis salina (NS), and Tetraselmis sp. (TS). DS and SP extracts significantly increased the proliferation rate of both MDCK and Vero cells. DS had a proliferation rate of 149.56% and 195.50% in MDCK and Vero cells, respectively, compared with that in serum-free medium (SFM). Notably, DS and SP extracts significantly increased superoxide dismutase (SOD) activity, which was 118.61% in MDCK cells and 130.08% in Vero cells for DS, and 108.72% in MDCK cells and 125.63% in Vero cells for SP, indicating a reduction in intracellular oxidative stress. Marine microalgal extracts, especially DS and SP, are feasible alternatives to FBS in cell culture as they promote cell proliferation, ensure safety, and supply essential nutrients while reducing oxidative stress.
Assuntos
Proliferação de Células , Microalgas , Animais , Cães , Microalgas/química , Células Vero , Chlorocebus aethiops , Meios de Cultura Livres de Soro/química , Proliferação de Células/efeitos dos fármacos , Células Madin Darby de Rim Canino , Técnicas de Cultura de Células/métodos , Superóxido Dismutase/metabolismoRESUMO
African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.
Assuntos
Fator 6 Ativador da Transcrição , Vírus da Febre Suína Africana , Febre Suína Africana , Cálcio , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Replicação Viral , Animais , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/genética , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Suínos , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Células Vero , Chlorocebus aethiopsRESUMO
Recent advancements in the large-scale cultivation of Tetraselmis sp. in Korea have enabled year-round production of this marine microalgae. This study explores the potential industrial applications of Tetraselmis sp. biomass by investigating the antiviral properties of its extracts and primary components. The antiviral effects of Tetraselmis sp. extracts were evaluated in Zika virus (ZIKV)-infected cells. Following extensive isolation and purification, the main compounds were characterized using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses. Their antiviral activities were confirmed using in vitro and in silico tests. Tetraselmis sp. extracts reduced infectious viral particles and non-structural protein 1 messenger RNA levels in ZIKV-infected cells without inducing cytotoxicity. Additionally, they modulated the interferon-mediated immune system responses. Tetraselmis sp. extracts are composed of four main chlorophylls: chlorophyll a, chlorin e6-131-152-dimethyl-173-phytyl ester, hydroxychlorophyll a, and hydroxypheophytin a. Among them, chlorophyll a, chlorin e6-131-152-dimethyl-173-phytyl ester, and hydroxypheophytin showed the antiviral activities in ZIKV-infected cells and molecular docking simulations predicted interactions between these chlorophylls and ZIKV. Our findings suggest that Tetraselmis sp. chlorophyll extracts exert antiviral effects against ZIKV and could serve as potential therapeutic candidates against ZIKV infection.
Assuntos
Antivirais , Clorofila , Microalgas , Simulação de Acoplamento Molecular , Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Zika virus/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Microalgas/química , Clorofila/farmacologia , Clorofila/análogos & derivados , Humanos , Animais , Chlorocebus aethiops , Clorófitas/química , Células Vero , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log10 TCID50 as the limit of detection.